Biotechnology Exam #3 Ch. 6, CHEM 527 Exams, Biotechnology Exam #2 Ch.18 Plant Biotechnology, Biotechnology Ch. 3 Exam 1, Biotechnology Exam #2 Ch. 5 (Genomics), Biotechnology Ch. 2 Exam #1, Biotechnology Ch. 1 Exam 1, Biotech Test 3, Biotechnology E...
What kind of ligations require 10 to 100 times more T4 DNA ligase than do ligations of DNA molecules with extensions (sticky ends)?
blunt end
What kind of molecule is formed when the 4-nucleotide extension of the BsmFI-cut DNA is filled in?
blunt end
What regulates plant cell growth and development?
auxin and cytokinins
What do spectinomycin, streptomycin, and kanamycin do to plants?
inhibit greening, faster proliferation, shoot formation
What is the objective of the genetic engineering protocol of bacteriophages?
replace middle I/E segment with cloned DNA by size fractionation
What does the pBR322 selection scheme for identifying transformed cells with insert DNA vector constructs rely on?
replica plating
What two major approaches have been used in peptide engineering of deptomycin.Chemoenzymatic synthesis for fatty acid may involve what enzyme
Combinatorial biosynthesis and chemoenzymatic synthesis; ECB deacylase
What is lac operon repressed by? What else does this affect?
-By the binding of repressor lacI -RNA polymerase can't sit down, so can't transcribe the region
Amplification of MYC and MDM2 in Neuroblastoma cells
-Cancer cells have genomic instability -increase in chromosome number -amplification of specific bits of the chromosome -in this picture the 2 cancer genes are amplified as small DNA fragments that remain separate from the chromosomal DNA within the nucleus -MYC is red and MDM2 is green -Normal chromosomes are blue -With MYC, you have pieces of DNA that have gotten off the chromosome and replicated in left -in the right there is amplification on the chromosome increasing the protein product from these cancer causing cells
In mink, brown fur color is dominant to silver-blue fur color. If a homozygous brown mink is mated with a silver-blue mink and 8 offspring are produced. how many would be expected to be silver-blue?
0
About what percentage of total bacterial DNA is plasmid DNA?
0.1% to 5%
What average does N1aIII cut?
1 in 256 bp
What is the transformation frequency in plants?
1 to 5%
What is metagenomics? What was used as the taxonomic marker? What was the goal of the metagenomic project?
Collection of specific genomes in an environmental sample 16S ribosomal rRNA in bacteria To construct a comprehensive DNA library from all microorganisms of a particular ecosystem
What increases the affinity of the promoter for RNA polymerase, thereby increasing transcription of genes downstream from promoter?
CAP binding to CAP box
What do both adaptors in SAGE have?
CATG extension complementary to extension produced by N1aIII digest, 5 bp recognition site for RE BsmFI, sequence for priming a PCR
What sequence does N1aIII recognize and where does it cut?
CATG, outside GC base pairs leaving 3' GTAC extension
What is good about mass spec?
Can run it through, separate it, activate it again, and run it through
Kinases are involved in at least what two diseases?
Cancer and immune disorder
Provide industrial and two pharmaceutical applications for carotenoid
Cardiovascular disease and cancer; industrial: nutritional supplement
What are some types of molecular databases? Who usually lives in the extreme places?
Cellular regulation Chromosome Genes Genomics Intermolecular interactions Metabolic pathways Protein motifs Proteome RNA Transgenic Archaea extremophiles
Metabolic engineering was successfully realized in what 2 pharmaceutical application for Cephalosporin biosynthesis?
Cephalosporin C yield improvement and DAOC production
Provide four advantage of using alternative host
Choice of host dictates the precursors, sigma factors, rare tRNAs, and cofactors
What is used in the synthesis of the 2nd DNA strand that results in double stranded cDNA and what does this result contain?
DNA polymerase, T7 RNA polymerase promoter
What are the spherical gold or tungsten particles in microprojectile bombardment coated with?
DNA precipitated with CaCl2, spermidine, or PEG
What happens once DNA is inside the cell in microprojectile bombardment?
DNA removed from particles and integrates into plant DNA
What are the extensions of the BsmFI fragments filled in by?
DNA synthesis
The metabolism and regulation of what 3 major pathways are related to cancer
Glycolysis, Pentose phosphate pathway, TCA cycle. Deregulation is related to cancer
For chemoenzymatic design heparan sulfate, provide four different types of enzyme
Glycosyltransferases, heparan sulfate c5 epimerase, sulfotransferase, n deacetylase
which of the following significantly contriubtes to the specificity of bp in double stranded DNA
H bonding b/w bases
Name carrier for H, P, and C:
H carrier: NAD, NADP, FAD, FMN P carrier: ATP, GTP C carrier: Tetrahydrofolate, adenosylmethionine
For one resulting catalyst, the substitution of what amino acid by what amino acid lead to what substrate specificity
H to N, altered substrate specificity.
What enzyme is a negative regulator of progenic (?) differentiation?
HDAC3
What enzyme and protein promote neuronal death
HDAC3, HDAC1
What enzyme and protein promote neuronal survival
HDRP, HDAC1
What are some ways to sequence the genome of an uncultured organism?
HE cloning, robotics, next gen sequencing, bioinformatics
Why is it necessary to have an origin of DNA replication in Ti plasmid-based vectors?
allow plasmid to replicate in e coli
What specific effector molecule attaches to an inactive repressor and causes a conformational change that enables the specific complex to bind to its corresponding operator region and turn off transcription of the operon?
corepressor
What happens to the cos ends after injection of lambda DNA into E. coli?
cos ends base pair to form circular DNA molecule
Before mRNA can be cloned into a vector what must happen?
converted to double stranded DNA by reverse transcriptase and DNA polymerase I
What are the cloning vectors called in which they can carry about 45 kb of cloned DNA and are maintained as plasmids in E. coli?
cosmids
what is the catalytic mechanism of MOMT
cosubstrate binds to substrate first and SAH is released from the enzyme last
What is an experimental approach to produce markerless transgenic plants?
cotransformation of plants with two separate DNAs, one with marker gene and other with target foreign gene
What is an advantage of the system for which researchers can transform tomato plastids and obtain high level expression of foreign proteins both in green leaves and in tomato fruit?
edible vaccines
Watson and Crick, with the help of Rosalind Franklin, developed the ___ model of DNA.
double helix
How can phenotypes of plants be modified?
downregulation of expression of certain genes
Does the transriptional terminator signal upstream or downstream of the coding sequence?
downstream
What is the term for purification of the desired compound from either the cell medium or the cell mass?
downstream processing
What is an advantage of using RAD54-transgenic plants?
dramatically increase probability of targeting specific changes in plant genes
After transcription of the operon, where are the proteins of the operon produced for bacteria?
during translation
What is another name for reverse labeling?
dye swapping
What organisms do Ti plasmids not replicate in?
e coli
What is one of the most popular reporter gene products?
e coli GUS gene
Where are all of the cloning steps carried out in the binary cloning vector approach?
e coli before vector introduced to a tumefaciens
What components are included in a binary cloning vector?
e coli or a tumefacions origin of DNA replication, shuttle vector, or single broad host range origin of DNA replication
In eukaryotes, what is responsible for the transcription of all types of RNA?
each type of RNA has a different RNA polymerase
study of how living things relate to each other and to their environment
ecology
In ecological classification, the next smallest level after the biosphere is the ___.
ecosystem
Organism, population, and community make up the ___.
ecosytem
How is the formation of the single stranded form of T-DNA initiated?
enzyme encoded by vir gene at both borders of intact T DNA region
What is the term used for tracking the simultaneous transcription of thousands of genes of either a cell or a tissue sample?
gene expression profiling
Which one of the following nucleotide pair bonds would be found in a DNA molecule?
guanine-cytosine
Sea stars live in saltwater ecosystems. Some species live in shallow tidal pools, while others live in the deepest parts of the oceans. This is a description of the ___ of sea stars.
habitat
place where an organism spends its life
habitat
A cell that contains one member of each chromosome pair
haploid
How is mRNA different from DNA?
has 2' and 3' hydroxyl
What are isocaudomers?
have same nucleotide extensions but different binding sites
Energy that passes through a food chain is lost to the environment as ___.
heat
What is the most dominant biotech crop?
herbicide tolerant soybean
The passing of characteristics from parents to offspring
heredity
The passing on of traits from parents to offspring is called ___.
heredity
What are the regions of chromosomes that are tightly packed?
heterochromatin
What type of protein are a set of different protein chains or subunits assembled to form a functional protein?
heteromeric
Omnivores, carnivores, herbivores, scavengers, and decomposers are all
heterotroph
obtains energy and nutrients from autotrophs
heterotroph
What is the genotype of the unknown rabbit in Figure 13-4?
heterozygous
What is the genotype of the unknown rabbits
heterozygous
Which of the following describes an organism that has the genotype Bb?
heterozygous
What is the probability that each cDNA in SAGE will have atleast one N1aIII recognition site?
high
What plant cell DNA-delivery methods are limited to use only with plant cell protoplasts that can be regenerated into viable plants?
liposome fusion, electroporation, direct gene transfer
What does a DNA microarray experiment consist of?
hybridizing nucleic acid sample from mRNA of cell to ssDNA sequence bound in ordered arrangement to platform
many crop plants such as wheat and corn have been developed as _______in order to develop larger and stronger plants
hybrids
How are the two polynucleotide chains of DNA held together?
hydrogen bonds between bases
How do oleosins make it possible for the water soluble target protein to be exposed to the aqueous environment?
hydrophilic terminal regions
What is used to encapsulate foreign DNA into liposomes where the protoplasts then engulf the liposomes containing the foreign DNA in the presence of PEG by endocytosis?
liposomes
What do the small molecules acetosyringone and hydroxysyringone do?
induce virulence genes carried on Ti plasmid
What happens to the E. coli lac promoter when there is an addition of either lactose or IPTG to the medium?
induction (turned on) of promoter
Is transformation usually an efficient or inefficient process? What are the odds?
inefficient, 1/1000
What is an advantage that transgenic plants have over bacteria?
inexpensive to grow on a large scale
What is the phenotype of generation 1 in Figure 10-5?
inflated
What must a ligated construst contain to be useful?
information for cellular maintanence
What can happen with too much plasmid DNA?
inhibitory
What proteins facilitate the binding of the initiator tRNA to the mRNA-small ribosomal subunit complex?
initiation factors
What are the raw data of dye emissions of each probe cell of a microarray often converted to for effective comparisons?
log
A dog's phenotype can be determined by ___.
looking at the dog
What plasmids are represented by 1 to 4 copies per cell?
low copy number plasmids
What are the main experimental approaches for determining gene expression profiles?
microarray and SAGE
What kind of communities are metagenomic projects especially effective with and why?
microbial, few species
What method involves foreign DNA being injected into plant protoplasts using glass needles?
microinjection
What plant cell DNA-delivery method has limited usefulness because only one cell can be injected at a time, requiring the service of a highly skilled individual?
microinjection
What is the most important alternative to Ti plasmid DNA delivery systems for plants?
microprojectile bombardment
What method can be used to deliver genes into chloroplasts and mitochondria?
microprojectile bombardment
How is foreign DNA typically introduced into the chloroplast genome?
microprojectile bombardment on plasmid vector with both foreign DNA and selectable marker
Which of the following monitors a cell's progress from phase to phase during the cell cycle?
microtubules
relationship between organisms in which one organism benefits at the expense of another
parasitism
How are most geniomic libraries created?
partial digestions of genomic DNA
If cells are placed in a strong sugar solution, water will ___.
pass from the cells to the sugar solution.
What recognizes the promoter which contains the Pribnow box and TTGACA sequence?
sigma factor RpoD or sigma 70
What does the transcritpional terminator do?
signal RNA polymerase to stop RNA synthesis
The historical method used to assign genes to particular human chromosome was to ___.
study linkage data from human pedigrees
How do you know if a functional enzyme is produced by a colony that carries a cloned gene encoding the enzyme?
substrate converted to colored product
What does SIGEX stand for?
substrate-induced gene expression
Which segment in fig 13-6 is not a palidrome
B
Which segment in fig 13-6 will attach to genetic material with the sequence TCGA
B
Which structure shown in Figure 11-3 is a pyrimidine?
B
8. Which of the following is/are true for protein synthesis in eukaryotes? A) All proteins are initially synthesized with methionine at their C-terminus. B) All proteins are initially synthesized with methionine at their N-terminus. C) All proteins are initially synthesized with tryptophan at their C-terminus. D) All proteins are initially synthesized with a multiple of 3 amino acids in their sequence. E) None of the above
B) All proteins are initially synthesized with methionine at their N-terminus.
What enzyme digests agarose and is used to retrieve separated DNA molecules from agarose gels?
B-Agarase I
What enzyme is primarily involved in the cleavage of lactose into glucose and galactose?
B-galactosidase
The diagram in Figure 10-2 shoes a diploid cell with two homologous pairs of chromosomes. Due to independent assortment, the possible allelic combinations that could be found in gametes produced by the meiotic division of this cell are ___.
BD, bD, Bd, and bd
Different Restriction Enzymes cut different DNA sequences
Bacteria that make a restriction enzyme also make a corresponding methylase. This corresponding methylase stops the bacteria from cutting its own DNA at sites that the restriction enzyme would otherwise recognize in the bacteria.
What 2 bacterial enzymes were required in metabolic engineering for DAOC production in P. crysogenum?
Bacterial-Sc Epimerase and Expandase
DNA Markers for Genetic Screening -- RFLPs Restriction Fragment Length Polymorphisms
Base changes or small insertions and deletions that vary between individuals can create or eliminate restriction enzyme sites. (as seen in sickle cell anemia testing)
From BC to lipid pathway, provide one key upregulating enzyme, one key metabolite, one key downregulating biomarker, provide one decrease specific lipid
Bcmo-1, RA, PPARgamma, triacylglycerol
There are some inherited predispositions to cancer
Breast Cancer - genes affected are BRCA1, BRCA2 - recombination repair Hereditary nonpolyposis colon cancer - due to mismatch repair XP (xeroderma pigmentosa) - nucleotide excision repair -if you have the mutated gene you are at a greater risk for developing that cancer -Many of these genes affected are involved in DNA repair, causing the mutations and cancer WHICH ties mutation to cancer
Name 1 buffer and 1 salt in stabilizing
Buffer: Potassium phosphate Salt: ammonium sulfate Organic alcohols: ethanol, glycerol, polyethylene glycol, inositol
Metabolic engineering can be applied using what 4 different type of industrial valuable molecules?
Bulk chemicals, fine chemicals, drugs, fuels
9. The Meselson-Stahl experiment established that: A) DNA polymerase has a crucial role in DNA synthesis. B) DNA synthesis in E. coli proceeds by a conservative mechanism. C) DNA synthesis in E. coli proceeds by a semiconservative mechanism. D) DNA synthesis requires dATP, dCTP, dGTP, and dTTP. E) newly synthesized DNA in E. coli has a different base composition than the preexisting DNA.
C
In Figure 10-8, what gametes will result in each chromatid crossed with a nonsister chromatid?
C
In base-excision repair, the first enzyme to act is: A) AP endonuclease. B) Dam methylase. C) DNA glycosylase. D) DNA ligase. E) DNA polymerase.
C
The 5'-terminal cap structure of eukaryotic mRNAs is a(n): A) 7-methylcytosine joined to the mRNA via a 2',3'-cyclic linkage. B) 7-methylguanosine joined to the mRNA via a 5' - 3' diphosphate linkage. C) 7-methylguanosine joined to the mRNA via a 5' - 5' triphosphate linkage. D) N6 -methyladenosine joined to the mRNA via a 5' - 5' phosphodiester bond. E) O6 -methylguanosine joined to the mRNA via a 5' - 5' triphosphate linkage.
C
The PCR reaction mixture does not include: A) all four deoxynucleoside triphosphates. B) DNA containing the sequence to be amplified. C) DNA ligase. D) heat-stable DNA polymerase. E) oligonucleotide primer(s)
C
The repair of cyclobutane pyrimidine dimers by bacterial DNA photolyase involves the cofactor: A) coenzyme A. B) coenzyme Q. C) FADH- D) pyridoxal phosphate (PLP). E) thiamine pyrophosphate (TPP).
C
Which of the following is false about transposition of DNA? A) The diversity of immunoglobins is in part due to DNA recombination by transposition. B) Transposition occurs in both prokaryotes and eukaryotes. C) Enzymes are not required for transposition. D) The first step of transposition can be single- or double-stranded DNA cleavage. E) Transposition can lead to simple movement of a DNA region or duplication of that region in a new location.
C
Which segment in Figure 13-6 will attach to genetic material with the sequence TCGA?
C
Listed below are procedure involved in the production of a transgenic organism. From the choices provided, select the sequence that represents the proper order of events.
C - 2,4,3,1
14. Which of the following is correct with respect to the amino acid composition of proteins? A) Larger proteins have a more uniform distribution of amino acids than smaller proteins. B) Proteins contain at least one each of the 20 different standard amino acids. C) Proteins with different functions usually differ significantly in their amino acid composition. D) Proteins with the same molecular weight have the same amino acid composition. E) The average molecular weight of an amino acid in a protein increases with the size of the protein.
C) Proteins with different functions usually differ significantly in their amino acid composition.
What is the law of screening? What is the first law of directed evolution?
"You get what you screen for"
What stereoisomer is used as modulator for what
Chemokine receptor
When are introns removed from the primary transcript?
after entire gene is transcribed
Watson and Crick called the three-dimensional shape of DNA a
double helix
What is the Michaelis-Menten Equation
V=Vmax[S]/(Km+ [S])
layer of Earth that supports life
biosphere
a segment of DNA that controls the production of a protein
gene
According to Figure 10-5, the constricted pod shape is ___.
recessive
DNA is to RNA as double stranded is to
single stranded
What is maize though to be derived from?
teosinte
The uniting of the male and female gametes
fertilization
Genomics
the study of genomes
You can do Locus-specific PCR of a VNTR or a VNTR southern blot to analyze someones DNA profile (VNTR with a southern blot requires a bigger sample of DNA)
***Colin Pitchfork convicted in 1987 of rape and murder based on DNA evidence
Knockout Mice: Targeted DNA Integration Via recombination In Embryonic Stem Cells
*Don't need to be worry about this but be aware -direct DNA into specific sites in mice and only in mice -done by homologous recombination -Create plasmid w seqeunces that are homologous to sites in chromosome and look where the DNA entered chromosome through double crossover * produce a plasmid that has sequences homologous to a target site in the chromosome* -used to turn genes off by making a frameshift which inactivates that particular gene -put plasmid growing in cells in tissue culture that s in an antibiotic medium > this shuts down native gene because only the cells containing the plasmid are antibiotic resistant this is called a KNOCKOUT -you can do this w eukaryotes in tissue culture but you can't get animals back from it but only in mice - use embryonic stem cells and grow them in tissue culture and put it into an already grown embryo and these stem cells will participate in making a embryo -used for studies for models for human disease like muscular dystrophy and cystic fibrosis
Two types of Gene Libraries
*Genomic DNA libraries -- cloned fragments of DNA isolated from chromosomes; represents all DNA sequences -used for cloning regulatory sequences (promoters), studying genome organization, sequencing of whole genomes, if you want to find introns *cDNA libraries -- cloned DNA copies of mRNA isolated from cells or tissues -used for isolating expressed genes -used to study genes expressed in certain cells and tissues under certain conditions
Analysis of cloned genes - Blotting - Use blotting to see how the gene is put together inside the cell or use blotting to measure expression
*Southern blotting is where DNA is cut with a restriction enzyme, run on a gel, and probed for a specific sequence, used to study structure of a gene or chromosome -used for which clones in a library contain a given DNA sequence *Northern blotting isolates RNA (takes specific mRNA from a certain type of cell/tissue, separated by gel electrophoresis, then transferred to a membrane to be probed) and then is run on a gel, and probed for a specific sequence - to study gene expression ***probes for the presence of mRNA complementary to a cloned gene
Because of genetically engineered foods...
*we've had genetically modified crops approved for human consumption since 1990, improvements in agriculture and yields has plateaued probably -Crop yields have more than quadrupled in the last century (developed countries) -Half of the increase has been due to improvements in agricultural practices, half to plant breeding -In the future genetic engineering is going to play an Increasingly important role
Where is the site of initiation of transcription in bacteria?
+1 nucleotide
What are the limitations for pBR322?
few cloning sites, time consuming
What is the yeast two hybrid system?
- A known "bait" protein is used to mine for an unknown "prey" protein. System for detecting pairwise protein interaction in which a bait protein is fused to the DNA-binding domain and a prey protein is fused to the activation domain of a specific transcription factor
How is derepression (turning on) of the trp promoter achieved?
- removing tryptophan - adding 3-indoleacrylic acid to growth media
What are the various means used to keep regulatable systems under complete control?
- repressor protein gene and promoter placed on two different plasmids that maintain different numbers of copies per cell which helps maintain the appropriate ratio between repressor protein and promoter. the repressor gene is placed on low-copy-number plasmid that maintains 1-8 copies per cell and cloned gene with promoter inserted into high-copy-number plasmid that maintains 30-100 copies per cell - repressor protein gene may be carried as a single gene in chromosomal DNA which keeps repressor protein levels low
Two competing Groups Sequenced the Human Genome: Celera Inc. (Craig Venter) and International Human Genome Consortium (Francis Collins)
-"Draft" Sequence Published Feb 2001 -"Finished" Sequence 2003 -human genome project was an international project - worked collaboratively - Celera company helped in the late 1990's and sequenced faster and cheaper -these 2 groups were competing against each other, in 2000, clinton got them to negotiate and publish there data together at the same time in the same month ***Celera just wanted to identify as many genes as possible so they dropped out of the project, and human genome consortium finished the job
Where does RNA polymerase bind in bacterial DNA?
-10 & -35 region
What is the tac promoter constructed from?
-10 region of lac, -35 region of trp, leftward pt promoter from bacteriophage lambda, and gene 10 promoter from bacteriophage T7
Where is the TATA box located?
-25 bp
Where do the componenets of the eukaryotic promoter lie?
-25, -75, -90 bp from +1 site
Genetic and Fossil Evidence Suggests Modern Humans Migrated out of Africa in Two Waves
-70,000 years ago to India, Southern Asia and Australia -50,000 years ago to Europe and Northern Asia -Europe and Asia were already occupied -- Neanderthals
Where is the CCAAT box located?
-75 bp
Where is the GC box located?
-90 bp
Genetic Modification Of Plants Using Agrobacterium tumefaciens Crown Gall Disease
-Agrobacterium carries a plasmid and transfers a piece of that plasmid into plant cells upon infection (transfer T-DNA) -the T-DNA contains genes that stimulate auxin and cytokinin synthesis in infected plant tissues -Best way of introduction of new DNA uses natural soil bacteria that is a cellular overgrowth - tumor induced plasmid and transfers it into plant chromosome and the piece of DNA that goes into plant cell contains genes that messes up plant hormone production causing the mass of cells -people found that you can take out whatever DNA you want and insert it into the Tumor induced plasmid (Ti) which will become part of the original plant chromosome
Cloning by PCR - Polymerase Chain Reaction
-Allows amplification of specific DNA sequences -Used to clone genes without making a library -Wide range of applications in genetics, molecular biology, evolution, medicine, criminal cases
Retroviral Oncogenes are altered versions of normal cellular genes
-Altered activity or over expression of oncogenes results in cancer (gain of function mutations) -Only one allele of a proto-oncogene needs to be mutated or misexpressed in order to trigger uncontrolled growth SO oncogenes are a dominant cancer phenotype *viral oncogene is an oncogene in a virus
How to calculate microarrays: How is the ratio described? What does positive log value mean? What does negative log value mean? What does 0 log value mean? What determines the DNA?
-An n-fold change -Positive means more expression in test sample -Negative means lower expression in test sample -No different in expression between test and control. There is 0 change in transcription between those -The amount of mRNA you have
Gene Therapy for ADA/SCID
-Bacterium carrying plasmid with cloned normal human ADA gene and a genetically disabled retrovirus are coupled so the cloned ADA gene is incorporated into the virus -bone marrow cells are removed and cultured from SCID patient, and the engineered retrovirus is added to cell cultures -the retrovirus infects blood cells, transfers ADA gene to cells -genetically modified bone marrow cells are bulked up and returned to patient and see if you get a therapeutic response
Applications of Gene cloning: Recombinant DNA
-Biotechnology: the genetic alteration of animals, plants, and bacteria **Transgenic organisms: In eukaryotes the introduced DNA must insert into a chromosome to be retained and expressed.
Herbicide Resistance --- Glyphosate and others
-Broad spectrum herbicide -Blocks EPSP synthase, an enzyme in the pathway for synthesis of aromatic amino acids. -Animals lack this enzyme, so toxicity of glyphosate is low -Breaks down quickly in soil (1-2 weeks) -Cheap -Transgenic plants expressing an EPSP synthase gene from bacteria are resistant to glyphosate -Herbicide resistance - grow plants without chemicals is not realistic to support our whole population - spraying with organically synthesized compounds is always a risk, this particular compound is the least toxic we know of but not completely nontoxic, this kills all plants by blocking EPSP which blocks amino acid synthesis (all animals get amino acids from diet) glyphosate has low toxicity to all animals and it degrades quickly in soil -FDA put this on the carcinogen list SO it has the potential to cause cancer but it is not known to cause cancer, only people at risk are people who make or use this compound -need to spray less with a more toxic herbicide because it persists longer in the soil
Bacillus thuringiensis - BT toxin Insect Resistance
-Bt is a naturally occurring soil bacterium that produces a protein that is toxic to insects -BT has been used as an insecticide for over 100 years, without evidence of environmental problems or human or animal toxicity -Organic farmers use BT dust for insect control *BT causes the digestive tract of insects that ingest it to lyse -effective against caterpillar/mosquitoes/beetles - used in agriculture for over 100 years -Comes in the form of powder which is a dry bacterial culture -Genetically modified plants that makes BT makes its usage easier
Carcinogens cause cancer by causing mutations
-Cancer is a genetic disease - what we know is that the association between cancer and mutations is tight ALL OF THE FOLLOWING ARE CARCINOGENS THAT CAUSE CANCER: -Radiation -Polycyclic hydrocarbons -benzopyrene -Nitrosamines -Aflatoxin
Cancer cells exhibit: *loss of growth control - cells continue to divide *altered cell appearance - partially dedifferentiated *ability to move to new site - metastasis
-Cancer is due to accumulation of mutations in somatic cells of genes involved in growth control -Cancer is a diverse set of diseases, many types -general features of all cancers: loss of growth control so cells continue to divide when they shouldn't, there is altered cellular appearance because they're expressing a different set of genes which alters their phenotype so they look different, cells lose their identity and move to different sites in the body, usually they stay in the same specific area (metastasis) We know that cancer is due to the accumulation of genes in growth control MITOSIS
Chapter 16 -- Cancer
-Cancer results from interaction of genes and environment -Today, the most frequent is skin, then lung -if you looked at the same list of cancer frequencies from the1900s, the list would be the same except lung cancer would be number 10 instead of 2
Evidence that Cancer is a Genetic Disease
-Carcinogens are mutagens -Predisposition to cancer is inherited in some families: Mutations in genes encoding DNA repair enzymes result in predisposition to cancer -Specific chromosome rearrangements are seen in some cancers: CML and the "Philadelphia chromosome" -Cancer cells show genomic instability: Translocations, deletions, gene amplifications *Cancer causing chemicals are mutagenic - you can measure how frequently chemicals cause damage to DNA by measuring the rate at which you treat salmonella with the chemical and measure how much mutations you get -predisposition to cancer can be inherited, in particular the enzymes for DNA repair promotes cancer -in some cancers you have very specific chromosome rearrangements which suggests that typical genetic rearrangements are causing cancer -cancer cells show wide scale genomic instability: insertions, deletions, amplifications, translocations - creating abnormal cells with weird genetic expression
Philadelphia Chromosome - Chronic Myelogenous Leukemia
-Caused by a reciprocal translocation between chromosome 9 and 22 - placing BCR and ABL gene next to each other, now cell division regulator ABL is under control of promoter of BCR and then you'll have over production of ABL which kicks off the cancer -presence of this translocation causes the cancer 95% of the time -IT IS SPECIFICALLY THE TRANSLOCATED 22 CHROMOSOME WITH A SMALL PIECE OF CHROMOSOME 9 ON THE BOTTOM OF IT (9 has the ABL gene)
What do you do with a soil,mud, intestine sample?
-Concentrate microorganims by centrifugation -Extract DNA -Clone DNA fragments into a vector -Transform E. coli with a metagenomic library -Screen libraries fro novel genes or operons
What do you do with a marine/freshwater sample?
-Concentrate microorganisms by filtration -Extract DNA -Clone DNA fragments into a vector -Transform E. coli with a metagenomic library -Shotgun sequencing to obtain whole or partial genomic sequences
Next Gen Sequencing the quest for the $1000 genome
-Current work focuses on sequencing different individuals to see how we differ and how these differences correlate with health and disease --- We are all 99.9% the same -The search for SNPs - single nucleotide polymorphisms - single base changes in the genome that usually cause disease -Sequencing has be revolutionized: cost came way down, and so this opens new avenues for new things, start looking at what the differences are between humans and how does this relate to disease -Look for snaps that correlate loss of functionality (SNP) - even at this cost it would take 30,000$ to sequence the genome 8 times to be accurate - only need to sequence coding part which you can do for less than 1000$
Southern blotting
-DNA can come from several sources -DNA samples are cut into fragments using 1 or more restriction enzymes, fragments are loaded onto agarose gel and separated by gel electrophoresis -The DNA in the gel is denatured with alkaline treatment to form single stranded fragments -Gel is then overlaid with DNA binding membrane (usually nylon) -To get the DNA fragments to the membrane by placing this on top of a wick (sponge) in buffer solution (gel is directly on top of sponge with membrane on top of it), with paper towels and weight on top of it - this draws buffer up through the gel, transferring the DNA fragments to the membrane -Membrane (filter) is placed in a heat sealed food bag with solution containing single stranded radioactive DNA probe; probe hybridizes with complementary seqeunces -Membrane is washed to removed unbound probe, dried, x-ray film is applied
Human Genome Consortium
-DNA from 8 males was sequenced - 23.1 billion bp sequenced -Human genome size - 3.2 Gb only 2.9 Gb was sequenced: centromeres and telomeres were skipped because they have very little DNA and are very repeated, they are difficult to sequence because of so many repeats so the areas around them are ignored -Gaps - about 5% of the sequence was in gaps between clones, Sequencing accuracy about 99.9% - "draft genome" -"Complete Sequence" 2003: 99.99% accuracy, some 300 gaps still remained -Human genome project doesn't say where the DNA came from - did it 8 fold coverage: didn't sequence females because they're missing the Y -In 2001 when the published they had 95% of the sequence, 5% had gaps, accuracy of 99.9% - this is draft sequence -2003 human genome project is done - they got it to 99.99% accuracy - about 300 gaps remaining, can't have it all but have most of it
Celera
-DNA from five subjects was selected for genomic DNA sequencing: two males and three females -one African-American, one Asian-Chinese, one Hispanic-Mexican, and two Caucasians -27.3 million sequence reads; 14.9 billion bp of sequence -when they published, Celera said they had DNA from 5 different people, different ethnic mix, human genome is only 3 billion base pairs but you have to sequence it multiple times to get an accurate reading with no gaps, sequenced each genome 5 times, which is barely enough to get a draft sequence
Genomic DNA Cut with a Restriction Enzyme Individual DNA Bands are not usually seen Because there are so many different fragments
-DNA has been cut with different restriction enzymes then run on different lanes, and the DNA has been stained w fluorescent dye, the right shows bands containing DNA sequences complementary to probe SHOWING hybridization -No bands on left because when you take DNA and cut w ECOR1 you expect to get a lot of fragments of different lengths, so the bands are in there but theres so many they appear as a smear ****SIZE OF A BAND SHOES THE SIZE OF A RESTRICTION SITE
You can also use Allele-Specific Oligonucleotides for genetic testing to find single nucleotide differences, but that are not limited to just the restriction enzyme cutting sites (like with RFLPs)
-DNA is extracted and the B globin gene is amplified using PCR, this is then denatured and spotted onto strips of DNA binding membrane and then probed with an ASO for either the normal B globin gene or mutant. If hybridized with the normal and they are homozygous dominant (normal allele, not sickle) then they will have the strongest hybridization, if heterozygous then they will have some hybridization, if homozygous recessive they will have no hybridization to the probe *only identical nucleotide sequences can hybridize to ASO -Way to detect single base pair substitution: using hybridization using specific short pieces of DNA -This is for sickle cell -Used to detect single base seqeunces so you can distinguish these 2 molecules binding to the DNA
Process of analyzing DNA with STRs using PCR
-Each primer set is tagged with 1 fluorescent dyes - red, blue, green, yellow -Each primer is designed to amplify DNA fragments from specific loci, so different loci can have the same color -primers are specific for flanking the STR locus and labeled with blue dye -the double stranded DNA s denatured, primers annealed, and each allele (repeat sequence) is amplified by PCR in the presence of dNTPS and Taq DNA polymerase > then separated by cap electro -the sizes of amplified DNA fragments from one color can help differentiate the loci products that have the same color -after amplification, the sizes of amplified fragments are measured by capillary electrophoresis - uses a capillary tube with gel to run a current through it, smaller fragments travel further -a laser detects the fluorescent fragments through the tube -after this the information is translated using computer system finding the size and quantities of each fragment -this DNA profile is then compared to that of other peoples
What can you do with your environmental sample, down to using ITPG?
-Fragment them to smaller pieces. -Clone into multi cloning site in plasmid -Induce all clones with IPTG -Cell sort to see which turn green -Then analyze inserted DNA fragments for catabolic operons and regulatory elements
Genetic Testing for Point Mutations
-Genetic detection of Sickle cell anemia -MstII RFLP -Use this to detect individuals that are heterozygous, homozygous recessive, or homozygous dominant for sickle cell anemia -Use the region where the point mutation can occur in the restriction site as a probe, for the first exon of the gene - if it is normal - you get 2 bands (GAG) mutant is (GTG) *This only happens if the single base change is right in the restrction enzyme site which is unlikely to be the case but it can happen
Transgenic Organisms
-Genetic modification of bacteria, animals, and plants -Introduction of genes from other organisms -clone gene from donor organism into a plasmid -modify gene sequence to permit expression in target organism -introduce plasmid into cells of target organism
What are advantages of robotics? Limitations of robotics?
-Helpful in generating and screening clones -Quicker process -Depend on transcription and translation features of E. coli -Need a different method -Only 40% of heterlogous genes are expressed in E. coli
Chapter 19: Applications of Gene cloning Recombinant DNA
-Identification and Isolation of Important Genes in Humans and other Organisms -Screening for genetic disorders -DNA fingerprinting -- identification of individuals -Biotechnology -- genetic alteration of animals, plants, and bacteria -Gene Therapy -- correcting genetic disorders
RFLPs allow detection of inheritance of chromosome segments in families that are linked to a disease gene
-Inheritance of RFLPs is Codominant -RFLP allows u to detect the inheritance for genetic variance - imagine you have an RFLP that's inherited with association with an inherited disorder, take DNA from diff people cut with a restriction enzyme and hybridize with a probe > detect diff in where the enzyme cuts bc you'll have slightly different sequences in each -probe needs to be close or linked to gene you're seeing you might have, nearby will work too -Detecting genotype of people with respect to disorder is easy -Use southern blot to see the difference in fragments that are cut using restriction enzymes - DNA fragments are separated with gel electrophoresis, transferred to a nylon membrane, visualized with southern blot hybridization, and probed for mutation region
Gene Sequence Comparisons Help Identify Gene Function: Human NF1 and Yeast Ira Genes
-Ira and NF1 proteins both regulate the Ras protein -More and more genes are cloned and studied from different organisms, and knowing the functions of some of these genes in one organism can help predict the function of these genes in a different organism ***this is a comparison at the amino acid level with a human NF1 gene and the yeast ira gene, the NF1 gene was cloned years ago but no one knew what this protein did, when the yeast ira sequence was cloned, it turned out that it was similar to NF1 and ira gene has a known function: SO we now know about NF1 gene too -the yellow boxes are identical matches between the 2 genes, the lines are chemically similar but not identical -20-30% matching is actually significant, 5% is random similarities, you expect similarities because of a lot of mutations have occured since we evolved from this yeast ***organisms are used as model systems to be informative about the human genome
Is the promoter for the lac operon inducible? What does this mean?
-It is off until we turn it on. -We turn it on by adding lactose -IPTG is an analog that binds and removes the repressor and turns on the gene
To explain ^
-Limitations in PCR: not specific in finding a sequence - primers are hard to design, make sure primers don't hit multiple sites in genome, the temperature where you anneal is really important -Make sure primers don't bind to off target sites and if they do, after 1 round of replication, it will bind perfectly to non perfect site -Anneal temperate based on MP of primers, run 3 degrees cooler than what you find it to be -You can amplify something unintended -enzymes can make errors: taq polymerase doesn't have proof reading function, will introduce errors into DNA that you're copying - when it puts in wrong base it stops replicating and reaction terminates, can't copy long pieces of DNA with taq polymerase bc of error rate - to get around this they do a long pcr with an enzyme w proof reading function
What are some limitations of 2D gel separations?
-Low abundance proteins lost -Membrane bound proteins underrepresented -Alkaline proteins not easily detected -Long run times (2 gels) -Staining needed -Quantification needed
The difficulties with human gene therapy primarily have to do with delivery of the DNA to sufficient numbers of target cells in the patient to get a therapeutic response, generally less than 10% of treated cells
-MLV - only infects dividing cells, tends to integrate in transcriptionally active regions - cancer prone -Lentiviruses (HIV) - infect both dividing and nondividing cells, less likely to integrate in transcriptionally active regions -Adenovirus and AAV - DNA genomes, do not integrate into the chromosomes but will infect nondividing and dividing cells -Viruses can trigger immune responses in the patient particularly if the patient has been exposed to the virus before (adenovirus particularly bad in this way)
What are sources of metagenomic libraries?
-Metal/coal mines -Arctic plankton -Freshwater bodies -High temp springs -Intestinal microbial communities from insects, humans, mice -Sediments -Soils
Microsatellites (STRs/ short tandem repeats) are used for DNA Fingerprinting
-Microsatellites - 2 to 7 base sequences repeated less than 50 times -Different individuals can differ in the number of copies at a given site. -"STRs" - short tandem repeats -known locus STR D8S1179 is made up of a 4-base pair sequence, TCTA, repeated 7-20 times depending on the allele (number of copies of STR)
Cloning the gene for Huntington's Disease -- "Positional Cloning"
-Nancy Wexler cloned the Huntington's gene in 1991 -You look for linkage between inheritance of the disorder and DNA markers that have been mapped to specific sites on chromosomes -5000 related individuals in Venezula who were segregating for HD - including 100 who had the disease -FIRST SUCCESS -Nothing was known about Huntington's disease except that it was inherited - she looked for the disorder and inheritance of RFLPs, map it -connection between the dominant allele for a disease and how that RFLP in the dominant allele, recessive allele is cut differently and is not linked to the disease
Many important genes have been cloned using mapped RFLPs
-Neurofibromatosis -Muscular Dystrophy -Cystic Fibrosis -Huntingtons disease -Proof that the correct gene has been cloned?? -individuals with the disorder will have mutations in the candidate gene -Proof would be that individuals with the disorder have some sequence that people that don't have it don't have
GENETIC SCREENING
-Once a Gene for a disorder has been cloned the cloned gene can be used to identify individuals with the disorder using RFLPs *Detection of b-thalassemia - for the beta globin gene -Deletion or insertion mutations are easiest to detect In this version, there is a deletion that removes exon 3 > detected because there is an enzyme that cuts on either side of beta globin gene, and people with the mutation to cause sickle cell, like here, there band is shorter than with people with the normal allele, aka it'll be closer to the bottom -use pattern matching to detect mutant -All grey means you have 2 normal alleles -This method works well if mutant allele has big insertion or deletion in it, which is the case for this beta knot (mutant allele) -A single base change would have to be right in the restriction site to detect it
Analysis of cloned genes - restriction mapping
-Once you clone a gene you can make a map of it using restriction enzymes -Once you clone a gene you break it up into smaller pieces to take a closer look at it > and that is done with restriction maps -Clone DNA into a plasmid > cut plasmid with different restriction enzymes so you can make a restriction map and see where they are cut within the plasmid -If you get 2 size markers, that means the enzyme cut twice -The size markers indicate how far the cuts are from each other: EcoRI cuts twice at 7 and 3, so the distance between the 2 cuts in 1 direction will add up to 7, and the other add up to 3
To identify genes the computers look for ORFs (open reading frames) and other features of genes
-Once you have the sequence the job is finding the genes, you can use computers algorithms to find sequences consistent w genes, look for open reading frames with no stop codons -Open reading frames have no stop codons aka its probably exons, and identify exons this way, look for promoter sequences/splicing seqeunces -train computers to find this, and find the likely genes, this is annotating the genes, if an annotated gene maps back to an EST then you really have the right gene, verifies the annotation GOAL: FIND ALL THE GENES
Most oncogenes are involved in the hormone signal pathways leading to expression of genes that stimulate cell division
-Oncogenes are genes whose normal function is to promote cell division (under appropriate conditions) -Most oncogenes are genes that code for proteins that code for hormone response -all cell division is regulated by these hormone responses that promote or inhibit cell division -Ras proteins transmit signals from the cell membrane to the nucleus, stimulating the cell to divide in response to growth factors -Ras protein switches off from active (GTP) and inactive form when growth factors aren't binded (GDP) -growth factors bind to the growth factor receptors on the cell membrane, activating Ras -the active GTP bound form of Ras then sends its signals through cascades of protein phosphorylations in the cytoplasm -the end point of these cascades activates activate nuclear transcription factors that stimulate expression of genes whose products start the cell into the cell cycle -once Ras sends its signal to nucleus, it transforms active GTP to GDP, to inactive it -mutations of proto-oncogene Ras into an oncogene will prevent it from turning GTP into GDP, so the cell thinks there is always a growth factor present and always divides **mutations in any of the Ras signaling pathway phosphorylations make them stimulated all the time in the absence of Ras, making them potential Oncogenes
Disadvantages of PCR
-PCR is not necessarily specific for the intended sequence -The primers and annealing temperature determine the specificity of PCR -Short versus Long PCR - PCR is not necessarily accurate -Taq Polymerase lacks 3'-5' exonuclease activity
PCR is a rapid method of DNA cloning > eliminates the use for host cells for cloning
-PCR used to amplify target DNA seqeunces that are initially present in small quantities -double stranded target DNA + DNA polymerase + and 4 DNA bases (A/T/C/G) are added to test tube -some info must be known about target DNA in order to synthesize 2 oligonucleotide primers (single stranded) -these primers bind to complementary DNA after they have been denatured, and are extended by DNA polymerase at the 3' end to make a clone -each cycle doubles the number of DNA molecules in the reaction -Specific DNA polymerase is used bc not all of them can handle the temperature change of annealing and cooling - Taq polymerase -DNA used can come from many samples - even used for forensics like hair samples and dried blood
Virus Resistance: transgenic plants expressing a piece of the viral genome are often resistant to the virus
-Particularly effective with RNA viruses and the mechanism of resistance involves RNA interference ***Papayas and Squash -In the 1990's Hawaiian Papaya production dropped by more than 50% due to Papaya ringspot virus. -Resistant varieties began to be planted in 2000 and the Papaya industry has recovered -this was a success in papayas -RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of siRNA mRNA molecules. -siRNA comes from the binding of the virus with the RNA and then the dicer cuts it up and the RITS inactivates it -Use genetic modifications to build RNA response to a virus so the plant is ready for the virus before it comes - this is done with RNA interference
DNA Fingerprinting -- Identification of Individuals
-RFLP differences between individuals -VNTRs - Variable Number Tandem Repeats "Minisatellites" short sequences (2-50 bases long) multiple copies at many different sites individuals vary in the number of copies 2-100 copies per site at ~ 40 sites -FE, if you examined 4 different VNTR loci, and each had 20 possible alleles, there would be 4^20 possible genotypes in this profile -someones unique DNA profile can be found for each person if 5-6 loci are analyzed - this is due to the large number of possible VNTRs and alleles -different alleles > from different repeat lengths
Restriction Enzymes
-Restriction endonucleases, cut DNA in a sequence specific manner -Recognition sites are Palindromic sequences: nucleotide sequence reads the same on both strands of the DNA when read in the 5' to 3' direction. -Each restriction enzyme recognizes a specific restriction site and cuts the DNA in a specific way **G/A cut: enzyme does this. Then theres a complementary overhanging ends called sticky ends, and these will be used for reassociation (different pieces from different sources) and they will stick to each other using complementary base pairing. -EcoRI have sticky ends, the vector must also been cut by the same restriction enzyme
New methods of DNA Sequencing are Driving down the cost and time required. The hope is that we will soon be able to sequence individual genomes for $1000
-Revolutions in DNA sequencing - ways to make DNA sequencing cheaper and faster > goal is to bring the cost of sequencing genomes down -Human genome sequencing itself took 10 years and took 3 or 5 billion dollars > now it can be done in a matter of days -You can target specific spots in genome and get a lot of info into medical background
Human Gene Therapy - aims to transfer normal genes into a patients cells
-SCID -- ADA deficiency, SCIDX -The Bubble-boy died of SCID at age 11 Good Candidate Disorders for Gene Therapy Trials? *Simple recessive disorder due to a single gene *Life threatening with little or no other treatment available *Disorder of the circulatory system - SCID - people born w recessive disorders that eliminates their immune system -People with SCID are sensitive to any infection that comes their way -There are 2 different mutations than can cause SCID -This seemed like good candidate for gene therapy in humans - it is a simple recessive disorder for 1 gene (put in correct gene), the disorder is life threatening with little or no other treatment, and this is a disorder of the circulatory system - so you can take cells from the bone marrow and alter them and put them back -anything with the circulatory system should be fixable w gene therapy
Golden Rice - Increased Vitamin A content (carotenoid synthesis genes from daffodil)
-Soybeans and canola with monounsaturated Oleic acid instead of polyunsaturated oils -It is possible to change the nutritional value of things - like in rice. It is genetically modified to increase the expression of vitamin A content
Several Types of "Vectors" are used in cloning, they differ in the size of the "inserts" they can carry
-Standard Plasmids -- inserts < 10 kbp -Bacteriophage Vectors -- inserts 10-50 kbp -BAC Vectors -- inserts 100 kbp, ***All to know- different vectors are used to carry different size pieces of dna, so this makes them used for different purposes. -standard bacterial plasmids can hold about 10,000 bases of dna -Viruses-lambda- can hold a piece of DNA up to about 50,000 base pairs, typical size 25,000. this is a virus where its lysogenic, enters bacterial cell and becomes dormant. So to use bacteriophage as a vector, what has been done is to isolate the pieces at the end and you don't need the genes in the middle. Use these ends to clone DNA into it and the virus will package it. The f plasmid- only part is needed is the part for replication. ***lysogeny - viral DNA is integrated into bacterial chromosome, so every time the chromosome is replicated the viral DNA is replicated and passed down
Exome Sequencing - Personalized DNA Sequencing
-Take DNA from an individual and hybridize it to a gene chip containing sequences that match all human exons (protein coding genes). -After hybridization, recover the exons from the slide and sequence them. -Cost ~ $1,000 (1% of the genome but all the genes) -Hybridize DNA onto a slide and where u have spotted oligonucleotides that correspond to every exon, wash the slide to wash away anything that different hybridize, then wash it so the exons come off, take the exons and sequence it -Used to screen tumors to find an effective treatment
RFLPs Can be Mapped - Mapped RFLPs can be used to map and then clone genetic disorders
-Take a 3 point cross from 2 different people and you have different probes and fragment sizes and you can look to see if there is linkage between different alleles with these RFLPs -anything that says D1 is a RFLP site mapped to chromosome 1 - once they're mapped you can look at inheritance of RFLP and alleles of genes
Cloning DNA yields a "gene library" - collection of clones that contains all the DNA sequences of an organisms genome
-The "library" is a collection of clones 1. Isolated DNA from the organism 2. Cut DNA with Restriction Enzymes 3. Insert DNA fragments into a "Vector" (plasmid) 4. Introduce recombinant DNA into host cell E. coli) 5. Each bacterial colony contains a single recombinant DNA clone *restriction enzyme used for whole genomes is YAC Major challenge: how to identify the specific clone that has the gene you want? Screening Libraries
GWAS - Genome Wide Association Studies -The search for genes responsible for complex traits using SNPs FOR EXAMPLE - Type II diabetes
-genomes of thousands of unrelated people with a particular disease are analyzed, usually using microarray analysis -results are compared with people without the disease to identify genetic variations that may increase risk of getting the disease -utilize large scale use of SNP microarrays that probe for 500,000 SNPs to evaluate results from diff people -by looking at SNP changes that occur in people with the disease, scientists can calculate the disease risk associated with each genetic variation using statistics
Explain how DNA microarrays work.
-The 2 cDNA strands are labelled with different fluorescent dyes -Hybridization occurs -In the microarray, cDNA is prepared an array of either gene sequences or gene-specific oligos -Scanned for both fluorescent dyes -Testing if up or down regulated based on the conditions -Extract from each sample mRNA -Convert the mRNA into cDNA library -Label with a probe -Each probe represents a sequence of a gene -Put them on, allow to hybridize to DNA microarray chip -Design micro array chip from the genome because you know the specific genes' annotations Outcomes- Upregulated, down regulated, both expressed, neither were expressed under either condition The sensors read how much of mix/light there is of the two. Will give relative rate of both so you can see how upregulated or not
What does the repressor bind to? What happens now? When can you get transcription? When is the optimal time to induce the cells?
-The operator -RNA polymerase can't sit down and transcribe -Add IPTG. Binds to repressor and causes it to be removed -Induce (when many cells) the promotor to be turned on when ready
2009 - successful correction of X-linked adrenoleukodystrophy (ALD) in two young boys using a lentivirus (retrovirus that can infect non-dividing cells)
-Thousands of clinical trials are underway to treat cystic fibrosis hemophilia Parkinsons HIV and more -there is not a cancer problem with this, the problem is DNA delivery
How to make transgenic animals
-Transfection - cloned DNA Introduced into tissue culture cells -Add target DNA to cell culture in pea tree dish -DNA enters nucleus and is expressed but is not integrated into chromosome -1 colony forms/10^6 cells treated with 40 pg gene DNA *DNA must integrate into a chromosome to be maintained and stably expressed *The site of DNA integration is essentially random *DNA integration may cause a mutation *****Site where new DNA is integrated occurs via nonhomologous DNA repair mechanism, but it puts this piece of DNA anywhere, which is a problem that was only solved in mice
Making transgenic mice
-Transgenic Mice by injection of DNA into a fertilized egg -then the fertilized eggs are placed in the oviduct of a receptive (previously pregnant) female -RECOMBINANT DNA CAN BE PUT INTO A SPECIFIC SPOT IN MICE
*****Genetic corrections of cells in the germ line are ruled out because they would be heritable putting future generations at risk.
-Treatment is targeted to specific organs in the patient -Disorders of the circulatory system are most amenable because bone marrow stem cells can be removed and grown in tissue culture **in the circulatory system you can take cells out, make genetic modifications to them in a dish, and then put the cells back > the biggest problem is to get DNA into the target organism in big enough amounts to fix the problem - delivery mechanism is tailored to the organism
Predictions based on annotation and analysis of protein functional domains and motifs: genes in the human genome that have been assigned on the basis of similarity to proteins of known function (in other organisms) (slide 11 set 31)
-Use computers to predict what the unknown genes do using blast to compare predicted (unknown) gene to everything else that has ever been cloned that has info for what that gene does - if 2 genes are similar according to blast, they have proteins with similar function -Doesn't tell you which genes it codes for only tells us that it codes for a specific transcription factor, not which ones -molecular function unknown: 40% unknown, we don't know what is expressed and coded for by these genes
DNA is cut with a restriction enzyme that cuts outside of the VNTR
-VNTRs have codominant inheritance - so you'll see the bands for each allele at each loci they're testing -take a piece of DNA, cut it with a restriction enzyme that cleaves on either side of the VNTR repeat region -the digested DNA is separated by gel electrophoresis and then run on a southern blot -briefly, separated DNA is transferred from the gel to a membrane and hybridized with a radioactive probe that recognizes sequences within the VNTR region -expose membrane to x-ray film, the pattern of bands is measured, with larger VNTR repeat alleles remaining at the top of the gel, and smaller VNTRs which migrate more rapidly, being closer to the bottom ***THE LENGTH OF THE VNTR REPEATS REFLECTS A DIFFERENT ALLELE
Retrovirus Life Cycle
-Viral RNA enters host cell -DNA copy of Virus made -Viral DNA integrates into host chromosome -Viral RNA made by transcription -Release of new viral particles (PROTEINS)
Plants and Mammals are also used as "bioreactors" for production of human proteins because bacteria do not glycosylate proteins
-bioreactors - living factories - that will continuously make the product containing the desired therapeutic protein that can be isolated in a noninvasive way -regardless of the host, therapeutic proteins may then be purified from host cells
Overproduction of the Gene for Growth Hormone in Fish
-contain transgenic growth hormone genes -transgenic atlantic salmon have copies of chinook salmon growth hormone next to a constitutive expressed (unregulated, constantly active) promoter -no adverse health effects from added gene
Expression of b-globin genes during development
-different members of the beta gene family have slightly different functions, and are active in their own time in development -People with sickle cell > treat it to improve expression of delta because it will make a normal beta chain and increase the good beta in people with sick cell disease
slide 4 set 32 ????
-figured out
Retroviruses can acquire host genes
-first you have a nonacute retrovirus - the genome of the virus can infect the cell but not transform it -the retrovirus can then pick up a copy of the host proto-oncogene during its infection of the cell and integrate it into its genome -the cellular proto-oncogene may be mutated during the process of transfer into the virus, or it may be expressed at abnormal levels because it is now under the control of viral promoters -these new retroviruses can infect and transform normal cells into tumor cells, they are ACUTE TRANSFORMING RETROVIRUSES *the transfer of gene from host cell into the viral genome allows the virus to infect and transform cells MAKING IT AN ONCOGENIC RETROVIRUS -An acute retrovirus has an additional part - these are the genes that cause cancer, somehow the virus picked up a normal gene while infecting the host cell, which mutates normal gene and causes cancer -This can happen because the virus planks down into chromosome and gene is right next door, the neighboring piece of DNA can end up in the retrovirus during its replication
Transgenic plants are made using the soil bacterium Agrobacterium tumefaciens -bacterial EPSP synthase gene provides resistance to the broad spectrum herbicide Glyphosate
-herbicides kill plants by inhibiting the action of the enzyme EPSP sythase -to make herbicide resistant plants you ... -take the EPSP synthase gene from bacteria and fuse it to the right promoter and poly A signals CREATING a fusion gene -introduce fusion gene into TI plasmid that doesn't have tumor genes and then this is transformed into host plant cells in tissue culture -Plants expressing the EPSP synthase gene are grown from the tissue culture - you know they're expressing it if they are survive on glyphosphate medium *cells that acquire the EPSP synthase gene are able to synthesize large quantities of it, making them resistant to herbicide glyphosate -use a promoter that comes from a virus to obtain a high level of expression in all cells throughout the plant, use specific promoters if you want it to be specific placement like in leaves or stems, etc
-Genes are separated by regions of spacer DNA -Genes are on both strands, DNA on both strands can code -Genes on different strands can overlap -If there is the same gene running in the opposite direction on the opposite strand > it codes for a different protein
-human have fewer overlapping genes than simpler eukaryotes
Limitations of PCR
-info about DNA is needed to make primers -minor contamination of DNA can cause problems for accuracy (ex: skin cells from researcher contaminate samples from crime scene) -Usually, DNA poly in PCR only extends primers for short distances, not to the end of the strand, SO PCR is usually used to amplify relatively short pieces of DNA
Transgenic Animals -- mice, sheep, pigs, etc *Microinjection of DNA into a Fertilized Egg *Egg is implanted into uterus
-isolate newly fertilized eggs in host female animal -inject purified cloned DNA containing a vector and the transgene of interest into nucleus of egg -only a small amount of the transgenic eggs will contain the transgenic DNA into the egg cell genome -injected eggs are then placed into the oviduct of a animal previously impregnated by a male -once baby is born it is screened for the trans gene via PCR -the integrated DNA must be present in germ (gamete) cells so it is passed to all offspring -most F1 aren't homozygous for the transgene so the F1 siblings must mate to make homozygous transgenic animals
Eukaryotic genomes
-linear, genes less densely packed, -monocistronic with introns in the genes -amount of repeat DNA and intron size increase with evolutionary complexity -Amount of noncoding spacer DNA and the number and size of introns increases with evolutionary complexity in eukaryotes -the average gene has 12 introns in it -Duchenne Muscular Dystrophy gene -- 2 Mb, >70 introns! ***biggest gene
Gene Chips - Microarrays
-oligonucleotides specific for different genes are spotted onto a slide - 64,000 genes on an area the size of a microscope slide coverslip -used for looking at gene expression patterns in genetic diseases -may use probes for only a few specific genes thought to be expressed in different cell types OR may contain probes for each gene in the genome -On this chip, pieces of DNA that will hybridize to specific probes are used to look at different gene expression in different tissues -Take normal cells and tumor cells, isolate mRNA in both, change them into cDNA, and treat them with fluorescent dyes, amount of each cDNA is proportional to amount of mRNA - mix fluorescent cDNAs and wash and let them dry and hybridize and they'll find the oligonucleotide they correspond to - laser and measure the amount of fluorescence in each, which is the output *****the probes are already on the chip/microarray for certain genes you're looking at, so when you add the fluorescent cDNAs, the cDNAs hybridize to the corresponding field gene that encodes their mRNA -yellow means they are expressed, or present in the same probe gene -you have a gene as the probe, whatever hybridizes to it (the color) tells you if the mRNA is expressed in normal or cancer cells -More red > tumor -More green > normal -Yellow has equal amount of mRNA in normal and cancer cells - level of expression of the same gene in both normal and cancer cells -Used to see which genes have been turned on or off in tumor - red is the gene turned on in cancer cells -If an mRNA is present only in normal cells, the probe representing the gene encoding that mRNA will appear as a green dot because only the "green" cDNA have hybridized to it
In 2009 the FDA Approved transgenic Goats for the production Of anticoagulant
-put human anticoagulant gene next to promoter for beta caesin, a common protein in milk, they were able to target anticoagulant expression in the mammary gland, so this is highly expressed in the milk, and then isolate it from the milk
Some genes in eukaryotes are present in multiple copies - like for rRNA and tRNA -slide set 31 slide 19
-rRNAs 500-5000 copies in tandem arrays -tRNAs 2 to 20 copies in clusters -coding genes themselves are copies -We need multiple gene copies because there is no amplification process for making rRNA/tRNA SO to have enough rRNA for ribosomes we need to have multiple copies of the gene -normal genes can get amplified many times for more multiplication
PCR (powerpoint notes)
-repeated rounds of steps -target DNA and excess of primers and DNA polymerase and the 4 bases, heat up to denature, and then cool the mixture to temp so the primers will just manage to base pair w target and polymerase will start to copy, then heat back up and put it back through the cycle again -doubling of target sequence
Gene Families Evolve by Gene Duplication through Unequal Crossing Over Followed by Sequence Drift
-repeated sequences in the genome - transposable elements - increase the frequency of recombination errors like this -Gene families come from unequal crossing over, if chromosomes are misaligned and crossing over occurs, there will be a duplication on one chromosome and deletion on the other -Duplicating a gene doesn't harm anything so you retain the original gene with a second copy that can take on new function - important in evolution of new genes, let evolution shuffle the parts - this is where transposable elements become important because there presence in the genome creates the errors in crossing over leading to new families - when they align w repeat seqeunces it confuses meiosis mechanisms and forms misalignments when crossing over occurs SO VALUABLE FOR EVOLUTION OF THE GENOME
SNPs - single nucleotide polymorphisms (single nucleotide changes)
-resequencing of the human genome in different individuals has identified 10 million SNPs -SNPs can be used to identify gene variants associated with complex disorders (polygenic traits). -A polygenic trait is one whose phenotype is influenced by more than one gene. Traits that display a continuous distribution, such as height or skin color, are polygenic. -Use these as genetic markers and use these in screening too -May be associated or linked to alleles that cause different disorders -Used to find genes that are associated with complex disorders or polygenic traits, each gene having a tiny effect on increasing the disorder
Bacterial genomes
-small -circular -very little repeated DNA -closely packed protein coding genes -lacks introns -polycistronic genes common but do have monocistronic
RFLPs - only used when there is a mutation in the restriction site, this is the only way you'll see a difference if you run 2 different peoples DNA on a gel
-so you have a cloned DNA segment with an RFLP -if there is a mutant allele, in the presence of a restriction enzyme, it will not be able to cut it, so there will be a large band -for a normal allele there will be 2 smaller bands -so if someone is heterozygous, there is 1 large band for the mutant allele and 2 small bands for the normal -someone with sickle cell (that is homo recessive) will have just 1 large band for both mutant alleles -and homozygous dominant will be 2 smaller bands and thats it
Oligonucleotide synthesis
-ssDNA sequences (up to 40 - 50 bases) are easily synthesized. -Synthetic reactions: add one base at a time and you can make things up for a specific sequence. -short, single stranded fragments of DNA that can be used as a DNA probe for alleles that differ by even a single nucleotide, will hybridize only to complementary base sequence, not even to a sequence off by 1 base
Process of PCR
-start with double stranded DNA to be cloned in test tube with primer (present in excess), DNA polymerase, 4 DNA bases (A/G/C/T) and -first you denature the DNA (heat it up 92-95 deg) -Then the primers anneal to the single stranded DNA (when it is cooled down 45-65 deg) -the primers are extended by DNA polymerase (Taq) at the 3' end, the temperature is raised a little 65-75 deg -That is the end of the first cycle - number of DNA molecules is doubled
Northern Blot of Human RNA from Different Organs: Probed with cloned FMR1 gene (mutations in this gene result in mental retardation)
-take RNA from different cells, run it out on agarose gel, and transfer to a membrane paper, and run it on single stranded DNA probe derived from a cloned copy of a gene -In this case, we are probing for the fmr1 gene (fragile x) RNA from different tissues here and this northern blot tells us that mRNA that corresponds to fmr1 is 4.4 bases long, and that there is more in the brain, placentena, etc and very little in the liver -In the heart the message is small > this may be due to alternate splicing of the mRNA *****Bigger fatter band > more rna that corresponds
Screening a gene library by DNA hybridization using a cloned gene as the "probe"
-the probe is a DNA or RNA sequence that is complementary to the target gene/sequence to be identified in a library 1. colonies of the library are overlaid with a DNA binding membrane such as nylon > this transfers the pattern of bacterial colonies from the plate to the membrane 2. Colonies are transferred to membrane, then lysed and denatured with NaOH 3. Membrane is placed in a heat sealed bag with a solution containing the labeled probe; the probe hybridizes with complementary DNA from colonies 4. Membrane is removed from bag and rinsed to remove excess probe then dried; Xray film is placed over the membrane > hybridization of a clone to colony shows up by a spot on Xray film 5. Colonies containing the inserted probe are identified from the orientation of the spots on the original plate, and removed 6. cells that hybridized with the probe are transferred to a medium for growth and further analysis
How to use a retro virus for gene therapy
-use retrovirus to deliver new gene -the bacterium containing the plasmid with the cloned normal gene > the target gene is inserted into a genetically disabled retrovirus -the retrovirus infects target cells that you want to insert your target gene into and transfers the target gene into it -the infected cells are then cultured to ensure the target gene is active -and then re-implanted back into person -useful because they insert DNA into chromosome
Gene Families - many protein coding genes in eukaryotes are present in multiple copies - similar but not identical (as with alpha and beta)
-we also have multiple copies of protein coding genes, which tells us how genome functions -A gene family is a group of genes that code for similar but not identical proteins EX hemoglobin -We have alpha and beta gene families for hemoglobin, on different chromosomes, there are 5 copies of alpha that are similar but not identical -Chromosome 16 - alpha gene family -Chromosome 11 - beta gene family -On chromosome 11 (beta) - there are 6 copies of that gene all within 50,000 bp, the basic structure of all the genes are very similar, and the length for all beta chains are the same but have amino acid differences between them -Alpha and beta are so incredibly similar -look for surrounding genes to see if it is part of a gene family, lots of pseudo genes ***pseudogenes -- nonfunctional copies of coding genes - found in both alpha and beta -the number of copies includes the pseudo genes too
Provide 3 examples of misfolding peptide, disorder, treatment and mechanism?
1) Amyloid Aβ peptide, Alzheimer's disease, resveratrol, conformational change 2) α1-antitrypsin, liver fibrosis, CBZ with autophagy, and SAHA (or small molecule inhibitor) with epigenetic protein refolding 3) CFTR, cystic fibrosis, (kalydeco) small molecule drug from Vertex, conformational change
Provide 2 enzyme related reasons the efficacy of B-lactam antibiotic in treating gram positive bacteria.
1) B-lactam similar structure bind to enzyme and form acyl-enzyme to inactivate 2) All PBPs: serine DD-transpeptidases will stop cell wall synthesis, killing bacteria
What are the 3 major pharmaceutical applications, including desired technology and expected goal for enzyme biotechnology?
1) Biosynthesis→ product yield improvement and biotransformation 2) Biocatalysis→ bioprocess 3) Inhibitor-design and inhibitor-screen→ new product
Steps in evolution of enzymes:
1) Duplication 2) Amplification 3) Point mutations 4) Recombination.
Provide 3 types of epigenetic modification or regulation, then for each type provide 2 enzymes involved in modification and regulation.
1) Epigenetic DNA modification with methyltransferase and dehydroxymethylases 2) Epigenetic RNA methylation with Cfr and Rlmn (or methyltransferases) 3) Epigenetic enzyme modification regulation with histone acetyltransferase and histone methyltransferase
Name the 2 parts of the Retro-evolution Hypothesis:
1) Evolution initially selects the site that binds the substrate to the enzyme, and 2) Novel catalytic properties are acquired by mutations
What 4 strategies for improving stability and activity for tylosin reductase
1) High activity strain 2) Late-Log Phase Cell Harvesting 3) Buffer and Cofactor Optimization 4) Activity Optimization: DTT, Glycerol, and PMSF
Provide 2 mechanistic reason why B-lactam antibiotic is a suicide substrate of the enzyme
1) Hydrolyzed product (imipenem) 2) Form irreversible EI complex
For producing 4 types of molecules by metabolic engineering from DNA to compound, provide 5 critical common steps?
1) Identify DNA source 2) Enzyme metabolic pathways and cell design 3) Constructed chromosome 4) Place chromosome in ghost envelope to cells, if needed, or directly to cell to generate engineered microbial catalyst 5) Overall process optimization
What are 3 potential pharmaceutical applications of metabolic engineering of Cephalosporin biosynthesis?
1) Improve Cephalosporin C yield 2) Produce Deacetoxy-Cephalosporin C (DOAC) as important cephalosporin intermediate 3) Modify substrate specificity of expandase
Using enzyme as drug target, provide 2 pharmaceutical technology. What is the desirable business outcome?
1) Inhibitor-screening 2) Inhibitor-design New inhibitors as drugs
8. What are 2 molecular mechanisms in generating prion phenotypes? (
1) Loss of function: (PSI+) stop codon readthrough 2) Gain of function: (RNQ+) induction of (PSI+), first prion induction of second prion.
Provide 2 enzymatic reason why PBP2x is an excellent enzyme target for bacterial resistance?
1) Low affinity (to B-lactam) 2) Abnormal branch stem peptides (as a substrate for resistance PBP2x)
List three rules how enzyme specificity determined:
1) Lowers energy of activation for one particular reaction of the substrate 2) Depends on apo-enzyme, the protein component and not the co-enzyme/ cofactor 3) Follows "lock and key" model or "induced fit" model
Starting with mrsa, provide 4 major reasons as rationale of why PBP2a is an excellent enzyme target for antibiotic resistance.
1) MRSA is a typical example of a PBP-associated bacterial resistance and it kills more people in USA than HIV/AIDS 2) Resistance in S. aureus strains correlated with their acquisition of chromosomal mecA gene which codes for the penicillin-binding protein 2a (PBP2a) 3) PBP2a is believed in carrying out the transpeptidase activity (cross-linking) in the presence of B-lactam antibiotics 4) Low affinity of PBP2a to B-lactams is considered to be closely associated with the bacterial insensitivity toward the B-lactams, thus making PBP2a an excellent drug target for bacterial antibiotic resistance.
What are 2 major reasons or metabolic kinetic reasons, why momt used in metabolic engineering?
1) Metabolite accumulation 2) Independent metabolite inhibition.
How was macrocin o-methyltransferase was determined as a dimer with two identical subunits
1) Molecular weight by gel filtration 2) Subunit size by SDS-PAGE 3) N-Terminal sequencing for identical subunit
From glucose/glycerol, what are 4 major strategy for producing taxol precursor that is hydrozylated into taxodiene in E. coli?
1) Partitioned the taxadiene metabolic pathway into two modules: bacterial and plant enzyme 2) Systematic multivariate search 3) Bioreactor fermentation optimization 4) Hydroxylase engineering and bioreactor fermentation optimization
What are 4 steps enzymatic molecular strategy for momt metabolic engineering to improve tylosin yield?
1) Purification 2) Characterization (w/ Kinetic mechanism) 3) Reverse Genetics (enzyme to gene) 4) Enzyme engineering (activity increase)
Provide 3 critical steps in the purification, reactivation and structure activity evaluation of recombinant expandase/hydroxylase.
1) Single chromatographic purification: mono Q FPLC 2) Final refolding by dialysis, for enzyme activation 3) Urea-dependent structure-activity evaluation by circular dischroism, fluorescence and gel filtration HPLC.
What are the 2 main steps in direct and stop method activity and analysis of enzyme? 2 steps in direct activity assay
1) Stop reaction 2) Direct product analysis
Steps in Cloning a Gene
1. Isolated DNA from the organism 2. Cut DNA with Restriction Enzymes 3. Insert DNA fragments into a "Vector" (plasmid) 4. Introduce recombinant DNA (in the form of the plasmid) into host cell E. coli 5. Grow the host and reisolate recombinant DNA clones **to distinguish host cells that have taken up vectors, the vector should carry a slectable marker gene - usually an antiobiotic gene or an enzyme absent from host cell, because then will be obvious when plated on this antiobotic and show they are there
Some Strategies for Screening Libraries
1. Probing using an already cloned gene 2. Isolate the protein, sequence it and make a synthetic oligonucleotide 3. Expression Libraries - antibody detection used
What is catalyzed by a ribozyme
1. cleavage fo precurose RNAs to tRNAs 2. removal of introns during trasn-esterification rxns involved in centain mRNA splicing 3. formation of peptide bonds during protein synthesis
"Shotgun Cloning" approach used by Celera
1. genome cut into small pieces with Restriction Enzymes and cloned 2. clones are sequenced and contigs identified by overlapping end sequences 3. chromosome sequence assembled -Use multiple restriction enzymes, make multiple libraries, and sequence the ends of the pieces, and using this to identify which overlap each other and identify contigs this way instead of physically mapping it, "if 2 pieces have the same end they probably came from same chromosome" -Identified contigs by sequencing the ends then they sequenced the whole pieces and used the computer to put together final chromosome assembly **Computers assemble the sequence of individual contigs into one continuous sequence based on where they overlap/ align (meaning they have the same dna sequence there)
To generate an enzyme catalyst for a stereoselective biomolecular diels-alder reaction, provide three major steps. provide two enzymes each used as a scaffold. what is the design technology. the substitution of what amino acid to what amino acid led to what substrate specificity
1. theozymes ensemble to generate a huge number of structure with 2 key aa bound to the active site. 2. scaffold fitting. 3. design optimization. DIPFPase and ketosteroid isomerase. rosetta design. H to N led to altered substrate specificity.
Using double reciprocal plot to calculate v max, y inter is 0.02
1/.02=50 Km is x intercept is -.2 5=1/.
What fraction of this cross will be recessive for both traits?
1/16
Where is the TATAAT or Pribnow box located in bacteria?
10 bp upstream from site of initiation (-10 region)
Where does BsmFI cut?
10 nucleotides downstream from recognition site in one strand and 14 nucleotides in other
How many base pairs are T-DNA typically?
10 to 30 kb
For the microarray system that uses oligonucleotides, how long are the probes typically?
10 to 40 nucleotides, sometimes 100
5. For search algorithm, to show the power of gene recombination, a sequence space of 1014 can be effectively searched by examining only 4000 solutions. 1014/4000=
10 to the 10
How many transformed cells are estimated per bombardment?
10,000
Nature enzyme can accelerate a reaction to what number over that uncatalyzed reaction under theological conditions.
1017
What enzyme is involved in what selected oxidation of trihydroxysteroid colic acid. Substrate is colic acid. What is the cosubstrate:
12 alpha hydroxysteroid dehydrogenase in regio selective oxidation. acetone
How large has the E. coli virus lambda or bacteriophage been engineered to be a vector for?
15-20 kb
What size YACs have a good chance of being trasferred to plant cells?
150 kb
What are the -35 and -10 regions of tac and trc promoters separated by?
16 tac or 17 trc base pairs
How did researchers start to examine the sequence diversity of microorganisms?
16S RNA as a marker
In what year was the NSFnet replaced by the Internet?
1995
What is involved in 2D gel electrophoresis?
1st- isoelectric focusing Banding pattern is based on the charge. 2nd- SDS denaturing gel. Sort by size and charge. Then sort by size.
Tylosin biosynthesis involved in what 3 biological reactions and what 2 enzyme classes?
2 enzymes classes: oxidoreductases and transferases 3 biological reactions: oxidoreduction (hydroxylation, dehydrogenation, reduction), glycosyltransfer, methyltransfer.
What is the name of the RNA-DNA chimeric molecule?
2'-O-methyl RNA
Listed below are procedures involved in the production of a transgenic organism. From the choices provided, select the sequence that represents the proper order of events. 1. Recombinant DNA is transferred into a bacterial cell. 2. A specific gene is identified in a DNA sequence. 3. The DNA fragment is recombined into a vector. 4. The DNA fragment to be inserted is isolated.
2, 4, 3, 1
Oxidation of primary alcohol to aldehyde and then to carboxylic acid involve what two dehydrogenases using what copper enzyme with what copper substrate. couple enzymes and co-substrate
2- phenylethanol dehydrogenase, phenylacetaldehyde dehydrogenase, NADH and oxidase, oxygen
How large are Ti plasmids?
200 to 800 kb
Some Current Applications of Plant Biotechnology
2011 data: 90% of corn, soybean, and cotton crops are GMO -the method described above is used to make these crops
How many vir genes are on the plasmid?
25 in 7 operons
How many base pairs does 1 human chromosome contain?
263 Mb
How large should a genomic library be?
3 more times the amount of DNA in a genome
One mechansim used by DNA polymerase to ensure that the inserted base is correct is what? It allows polymerase to backtrackand remove the nuclotide that was most recently incoroporated in to the primer strand
3 to 5 exonucleasue
What does a polynucleotide consist of?
3' OH at one end and 5' phosphate at other
What is the sequenced gene in SAGE derived from?
3' end of mRNA
What is the name of the DNA segment that follows a gene?
3' flanking downstream region
What facilitates the interaction between the mRNA and the ribosome?
3' polyAtail
What anticodon binds to the start codon of mRNA?
3'-UAC-5' of initiator tRNA or fMet-tRNA
What size DNA fragments have plants been successfully transformed with?
30 to 150 kb
In cotransformation, what percentage of the transformed plants contain both genes?
30 to 80%
What in vitro assay and what cell based assay can be synergistically useful in cyclin D1/CDK4 anti-proliferative drug discovery?
32P-ATP Radioactive Assay and the Cell-Based Assay, where the activity of the fusion enzyme is needed for cell growth, can be synergistically useful in cyclin D1/CDK4 based anti-proliferative drug discovery.
What wavelength is commonly used in what enzymatic activity assay
340 nm for oxidoreductase
The effort to completely map and sequence the human genome will likely result in knowing the sequence of the approximately ___ genes on the 46 human chromosomes.
35 000 to 40 000
Where is the TTGACA sequence located in bacteria?
35 bp upstream from site of initiation (-35 region)
Where are the vir genes located on the Ti plasmid?
35 kb region outside of T-DNA region
The effort to completely map and sequence the human genome will likely result in knowing the sequence of the approximately____genes on the 46 chromosomes
35,000- 40,000
What directs the synthesis of a significant amount of the target protein in root tissue?
35S promoter and mas2' promoter
In sticky ends, how many nucleotides are included in each extension?
4
If vmax = 11 minutes per milligram and S is 40 and km is 4, what is velocity?
440/44= 10 nanomols/min and milligrams of enzyme----same as vmax unit.
Eukaryotic glycoprotein and glycolipid are derived from how many nucleotide sugars? Provide two enzymes involved in deoxygenation
9; dehydratase and c-4 aminotransferase
In Figure 10-8, what gametes will result if there is only a single crossover?
A
Where are the phosphate group and base attached on DNA and RNA?
5' and 1' carbon of sugar
Where does the alpha phosphate group link to the nucleotide?
5' carbon and 3' OH
What end of the T-DNA carries the right border?
5' end
Where is the Shine-Delgarno sequence located in prokaryotes?
5' end of mRNA with complementary sequence near 3' end of rRNA
What does DNA polymerase I do?
5' exonuclease activity which removes nucleotides
What is the name of the DNA segment that precedes a gene?
5' flanking upstream region
What direction does translation occur?
5' to 3' along mRNA
A couple has two children, both of whom are boys. What is the chance that the parents' next child will be a boy?
50%
To feed the population from 2000-2020, how much should the world cereal production increase by?
50%
What is the wavelength maximum or color of reaction product?
500 nm or red
How many probes could a complete whole-genome oligonucleotide array contain and how many genes would this represent?
500,000 probes, 30,000 genes
How long is the EcoRI recognition sequence?
6 bp
When using DNA fingerprinting to determine the paternity of a child, how many of the child's 13 str loci should match the father's?
6 or 7 -probably because half of your DNA is from dad, and half from mom, so half should match (at each loci, you have an allele from mom, and an allele from dad)
How many codons does the complete genetic code consist of?
64
How many sigma factors can E. coli produce?
7
How many clones are required for a 99% chance of discovering a particular sequence in a human genomic library with an average insert size of 20 kb?
700,000 clones
Plants are being "engineered" to be more resistant to insects or viruses, to be herbicide tolerant
75% of soybeans and cotton in the US are herbicide resistant
In chickens, rose comb (R) is dominant to single comb (r). A homozygous rose-combed rooster is mated with a single-combed hen. All of the chicks in the F1 generation were kept together as a group for several years. They were allowed to mate only within their own group. What is the expected phenotype of the F2 chicks?
75% rose comb and 25% single comb
A female guinea pig homozygous dominant for black fur color is mated with a male homozygous for white fur color. In a litter of eight offspring, there would probably be ___.
8 black guinea pigs
Suppose 10 000 units of energy are available at the level of the grasses. What is the total number of energy units lost by the time the third energy level is reached?
9990 units
What plant cell DNA-delivery method is used with a wide range of plants and tissues and is also easy and inexpensive?
Microprojectile bombardment
In homologous genetic recombination, RecA protein is involved in: A) Strand invasion to form Holliday intermediates. B) introduction of negative supercoils into the recombination products. C) nicking the two duplex DNA molecules to initiate the reaction. D) pairing a DNA strand from one duplex DNA molecule with sequences in another duplex, regardless of complementarity. E) resolution of the Holliday intermediate.
A
Which of the cells depicted in the line graph in Figure 8-8 are most likely cancerous?
A
Which of the following statements about E. coli RNA polymerase is false? A) Core enzyme selectively binds promoter regions, but cannot initiate synthesis without a sigma factor. B) RNA polymerase holoenzyme has several subunits. C) RNA produced by this enzyme will be completely complementary to the DNA template. D) The enzyme adds nucleotides to the 3' end of the growing RNA chain. E) The enzyme cannot synthesize RNA in the absence of DNA.
A
Which structure shown in Figure 11-3 would attract a free cytosine nucleotide?
A
Provide 5 domain for deptomycin biosythesis and the function of each
A domain: activate specific amino acid. T domain: bind activated amino acid and the intermediate. C domain: condensation of intermediates. E domain: epimerize d amino acid. TE domain: product cyclrelease
What is a leaky promoter?
A promoter that turns on without being told to
What is shotgun sequencing?
A rapid sequencing method that involves cutting DNA into random fragments then determining the base sequence in each fragment
Cancer is infectious, can be transmitted from person to person
ANSWER: disagree -cancer is not infectious and cannot be transmitted from person to person - one type of cancer that is transmissible is wiping out the Tasmanian devil - if you get cancer cells from someone else your immune system will kill it off, if you have a defective immune system it may be transmissible
Most cancers are caused by viruses
ANSWER: disagree there are some cancers that are caused by viruses, some viruses directly cause cancer, but most cancer doesn't have anything to do with virus
All cancer cells grow faster than normal cells
ANSWER: disagree there are some cancers that are fast growing and some that are slow growing, very diverse, prostate cancer is slow growing
Retrovirus explained and their way of replacing their DNA with the therapeutic DNA
A retrovirus is any virus belonging to the viral family Retroviridae. All The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of their hosts is in the form of DNA. When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes into the cell. This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be considered part of the genetic material of the host cell. Retrovirus genomes commonly contain these three open reading frames that encode for proteins that can be found in the mature virus. Group-specific antigen (gag) codes for core and structural proteins of the virus, polymerase (pol) codes for reverse transcriptase, protease and integrase, and envelope (env) codes for the retroviral coat proteins (see figure 1). The process of producing a DNA copy from an RNA molecule is termed reverse transcription. It is carried out by one of the enzymes carried in the virus, called reverse transcriptase. After this DNA copy is produced and is free in the nucleus of the host cell, it must be incorporated into the genome of the host cell. That is, it must be inserted into the large DNA molecules in the cell (the chromosomes). This process is done by another enzyme carried in the virus called integrase (see figure 2). Now that the genetic material of the virus is incorporated and has become part of the genetic material of the host cell, we can say that the host cell is now modified to contain a new gene. If this host cell divides later, its descendants will all contain the new genes. Sometimes the genes of the retrovirus do not express their information immediately. Retroviral vectors are created by removal op the retroviral gag, pol, and env genes. These are replaced by the therapeutic gene. In order to produce vector particles a packaging cell is essential. Packaging cell lines provide all the viral proteins required for capsid production and the virion maturation of the vector. These packaging cell lines have been made so that they contain the gag, pol and env genes. Early packaging cell lines contained replication competent retroviral genomes and a single recombination event between this genome and the retroviral DNA vector could result in the production of a wild type virus. Following insertion of the desired gene into in the retroviral DNA vector, and maintainance of the proper packaging cell line, it is now a simple matter to prepare retroviral vectors (see figure 3). One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert the genetic material of the virus in any arbitrary position in the genome of the host. If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted (insertional mutagenesis). If the gene happens to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur. This problem has recently begun to be addressed by utilizing zinc finger nucleases or by including certain sequences such as the beta-globin locus control region to direct the site of integration to specific chromosomal sites.
DNA fingerprinting
A test to identify and evaluate the genetic information-called DNA in a person's cells
5. Which of the following statements about tRNA molecules is false? A) A, C, G, and U are the only bases present in the molecule. B) Although composed of a single strand of RNA, each molecule contains several short, double-helical regions. C) Any given tRNA will accept only one specific amino acid. D) The amino acid attachment is always to an A nucleotide at the 3' end of the molecule. E) There is at least one tRNA for each of the 20 amino acids.
A) A, C, G, and U are the only bases present in the molecule.
9. Formation of the ribosomal initiation complex for bacterial protein synthesis does not require: A) EF-Tu. B) formylmethionyl tRNAfMet. C) GTP. D) initiation factor 2 (IF-2). E) mRNA.
A) EF-Tu.
3. Which of the following is not one of the five steps of protein synthesis? A) Termination and ribosome degradation B) Elongation of the peptide chain C) Initiation of peptide synthesis D) Activation of the amino acids E) Protein folding and posttranslational processing
A) Termination and ribosome degradation
18. The first step in two-dimensional gel electrophoresis generates a series of protein bands by isoelectric focusing. In a second step, a strip of this gel is turned 90 degrees, placed on another gel containing SDS, and electric current is again applied. In this second step: A) proteins with similar isoelectric points become further separated according to their molecular weights. B) the individual bands become stained so that the isoelectric focus pattern can be visualized. C) the individual bands become visualized by interacting with protein-specific antibodies in the second gel. D) the individual bands undergo a second, more intense isoelectric focusing. E) the proteins in the bands separate more completely because the second electric current is in the opposite polarity to the first current.
A) proteins with similar isoelectric points become further separated according to their molecular weights.
The fusion cyclin D1/CDK4 enzyme was constructed in cell line for what use/application? And purified for what use?
A): For cell-based assay where the activity of the fusion enzyme is based for Growth of cell lines. (B): For structural determination.
A virus isolated from monkeys contains a circular double strand of DNA. The virus, called Simian Virus 40, interests scientists because it causes cancer in laboratory animals. Using a restriction enzyme, the strand is separated into six unequal segments, as shown in Figure 13-2. A scientist hypothesizes that the segment of DNA causing cancer can contain no fewer than 600 base pairs. Using Figure 13-2, decide which segments of the virus have the highest chance of containing the segment of interest. Identify in DESCENDING order, from the HIGHEST change to the LOWEST.
A, B, C
What protocols have been devised for the transformation of corn and rice?
A. tumefaciens carrying Ti plasmid vectors
In crown gall tumors, what happens after the initial attachment step?
A. tumefaciens produces network of cellulose fibrils that bind the bacterium tightly to plant cell surface
What must be on either end of any genetic material that is inserted in the cleaved DNA in fig 13-5
AATT
What must be on either end of any genetic material that is inserted into the cleaved DNA in Figure 13-5?
AATT
How is inhibition sensing used to discover what enzyme
AHL lactonase enzyme binds to the substrate AHL, it hydrolyzes the substrate and then the substrate cannot bind to lux protein and its for the AHL lactonase, result is no activation of lux promoter and no expression of gfp. Enzyme detected- AHL lactonase
Which of the following disorders would be the easiest to treat by gene therapy and which would be the hardest? 1. Muscular dystrophy 2. Cystic fibrosis 3. Hemophilia
ANSWER: Easiest 3, Hardest 1 easiest to target the circulatory system that is why hemophilia is the answer -harder to target muscles
The role of the Dam methylase is to: A) add a methyl group to uracil, converting it to thymine. B) modify the template strand for recognition by repair systems. C) remove a methyl group from thymine. D) remove a mismatched nucleotide from the template strand. E) replace a mismatched nucleotide with the correct one.
B
If you have a genetic test to see if you are a carrier of cystic fibrosis and the test comes back negative does this mean your children cannot get the disease?
ANSWER: maybe, maybe not -Difficulty in genetic screening can only detect the known alleles, and we don't know all the alleles that cause the disorder with cystic fibrosis - we know one allele and that accounts for 80% of the people who have it, the other 20% of people who get it don't know where they got it from -Getting back a negative test doesn't mean you're not gonna get it, but a lesser chance, but that u may have a rare allele that can cause it that they didn't test for -If it comes back positive - then you will get it, you have the common allele that causes it
Making transgenic humans is considered unethical because:
ANSWER: we cannot control where the recombinant DNA will go when it integrates into the chromosomes -You can't control where DNA goes when you add it in, so you create the possibility of mutations in genes that are important - not unethical to make genetic modifications to certain cells, BUT if you make bad mutations in transgenic organisms it can be passed on to the next generation WHICH IS unethical
Using the simplified and descriptive name, provide the catalytic domain and one protein domain. each domain exists in what form?
AT: acyl transferase. KS- keto synthase. KR: keto reductase. DH: dehydratase. ER: enoate reductase. b. Acyl carrier protein c. dimer
What 2 enzymes express in a different tissue are involved in fat muscle loss associated in cancer
ATGL (adipose triglyceride lipase), HSL (hormone-sensitive lipase). If you can't remember specifics at least remember two lipases
What 2 enzymes contribute to cancer-associated chachexia?
Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL)
What is de novo sequence interpretation?
After getting MS-MS spectrum, calc aa masses Do a blast against DB sequence
Using alcohol dehydrogenases, how does all enzymes now shining in new clothes
Alcohol dehydrogenases normally used for the stereoselective reduction of ketones but can be used as racemases, the old enzymes are shining in new clothes.
Provide two enzymes involved in oxidation and reduction of polycyclic aromatic hydrocarbon
Aldo-keto reductase, expoxide hydrolase
Who discovered penicillin? Won the nobel prize and what year?
Alexander Fleming in 1945
What is allosteric multi subunit model by MOM
Allomeric Enzyme exists in two confirmations, T and L
List 5 post-translational modifications of enzyme, each with a different amino acid. And if you can what is the effect of each application
Amino terminal: acetylation for gene expression Carboxy terminal: amide bonding for pro-drug (delivery) Glycosylation: asparagine, for stability Phosphorylation: threonine, for metabolism signaling Disulfide linkage: cysteine, for stability
Provide 2 common methods in determining that tylosin reductase contained 2 non-identical subunits
Amino-terminal sequencing and peptide mapping
Enzymes called _____________ couple each amino acid to its appropriate tRNA molecule to create an _____________ molecule.
Aminoacyl-tRNA synthetases; Aminoacyl-tRNA
What is 1 I.U.?
Amount of enzyme required to convert one 1 micromol of substrate to the product under typically optimum activity
What plaque is an important feature of AD
Amyloid plaque
Assuming IEP 6.5 for PBP2a and the stability of the enzyme is 5-8, what pH of a buffer could be used for either chromatography?
Anion-exchange above IEP and cation-exchange below IEP (optimum is 1 unit above/below)
What 3 step procedure was used for PBP2a purification?
Anion-exchange, cation-exchange, dye-ligand affinity
Provide 4 chromotographic steps for cyclin D1/CDK4 purification. If the isolelectric point of the enzyme is 6.75 and the enzyme is stable between pH 6-7.5, then the enzyme can be purified by what exchange at what pH? And also by what other exchange and pH?
Anion-exchange, hyrdroxylapatite, HP cation-exchange and chromatofocusing. Anion-exchange is pH=7.75; cation-exchange is pH=6.75
What kind of substrate concentration would lead to what activity
Anything equal and above 10 km would lead to maximum activity
1. The difference between thymine and uracil is: A) one methylene group on the pyrimidine ring. B) one methyl group on the pyrimidine ring. C) one hydroxyl group on the ribose ring. D) one amine group on the pyrimidine ring. E) one methyl group on the purine ring.
B
If the segments in fig 13-6 are mixed with several restriction enzymes, which will not be cleaved
B
Processing of a primary mRNA transcript in a eukaryotic cell does not normally involve: A) attachment of a long poly(A) sequence at the 3' end. B) conversion of normal bases to modified bases, such as inosine and pseudouridine. C) excision of intervening sequences (introns). D) joining of exons. E) methylation of one or more guanine nucleotides at the 5' end.
B
The excision (splicing) of many group I introns requires, in addition to the primary transcript RNA: A) a cytosine nucleoside or nucleotide and a protein enzyme. B) a guanine nucleoside or nucleotide (only). C) a protein enzyme only. D) a small nuclear RNA and a protein enzyme. E) ATP, NAD, and a protein enzyme
B
Which segment in Figure 13-6 is not a palidrome?
B
Why does it work/What's the enzyme's function?
Convert substrate to normal product, which is needed for cell. Substrate is removed. A deficiency in this enzyme causes the substrate to accumulate, which is toxic.
What is the difference between a high copy, and low copy number plasmids?
Copy number refers to plasmid's ability to replicate itself
What three enzymes for Sc- are evolutionarily homologous? They share the same ancestor gene
Cyclase, expandase, hydroxlase
Provide a new enzyme target of B-lactam antibiotic other than PBP2a/PBP2x.
Cysteine LD-transpeptidases
1. The Ames test is used to: A) detect bacterial viruses. B) determine the rate of DNA replication. C) examine the potency of antibiotics. D) measure the mutagenic effects of various chemical compounds. E) quantify the damaging effects of UV light on DNA molecules.
D
10. When a DNA molecule is described as replicating bidirectionally, that means that it has two: A) chains. B) independently replicating segment. C) origins. D) replication forks. E) termination points
D
6. Which of the following is not required for initiation of DNA replication in E. coli? A) DnaB (helicase) B) DnaG (primase) C) Dam methylase D) DNA ligase E) DnaA (a AAA+ ATPase)
D
8. In contrast to bacteria, eukaryotic chromosomes need multiple DNA replication origins because: A) eukaryotic chromosomes cannot usually replicate bidirectionally. B) eukaryotic genomes are not usually circular, like the bacterial chromosome is. C) the processivity of the eukaryotic DNA polymerase is much less than the bacterial enzyme. D) their replication rate is much slower, and it would take too long with only a single origin per chromosome. E) they have a variety of DNA polymerases for different purposes, and need a corresponding variety of replication origins.
D
In homologous recombination in E. coli, the protein that moves along a double-stranded DNA, unwinding the strands ahead of it and degrading them, is:
D
The ABC excinuclease is essential in: A) base-excision repair. B) methyl-directed repair. C) mismatch repair. D) nucleotide-excision repair. E) SOS repair.
D
When bacterial DNA replication introduces a mismatch in a double-stranded DNA, the methyl-directed repair system: A) cannot distinguish the template strand from the newly replicated strand. B) changes both the template strand and the newly replicated strand. C) corrects the DNA strand that is methylated. D) corrects the mismatch by changing the newly replicated strand. E) corrects the mismatch by changing the template strand.
D
Which of the following is not needed in pyrosequencing of DNA? A) dNTPs B) Sulfurylase C) Luciferase D) ddNTPs E) Apyrase
D
Which structure shown in Figure 11-3 does not contain a nitrogenous base?
D
7. Which of the following statements about aminoacyl-tRNA synthetases is false? A) Some of the enzymes have an editing/proofreading capability. B) The enzyme attaches an amino acid to the 3' end of a tRNA. C) The enzyme splits ATP to AMP + PPi. D) The enzyme will use any tRNA species but is highly specific for a given amino acid. E) There is a different synthetase for every amino acid.
D) The enzyme will use any tRNA species but is highly specific for a given amino acid.
4. Which one of the following statements about ribosomes is true? A) The large subunit contains rRNA molecules; the small subunit does not. B) The RNA in ribosomes plays a structural, not catalytic, role. C) There are about 25 ribosomes in an E. coli cell. D) There are two major ribosomal subunits, each with multiple proteins. E) Ribosomes are relatively small, with molecular weights less than 10,000.
D) There are two major ribosomal subunits, each with multiple proteins.
What 2 other enzymes may be involved in lipid metabolism in an opposite direction
DGAT1&2
The design diel-alderase was created from a scaffold of what enzymeb. What is the major beneficial property of this design
DIPFPase i. Absolute stereoselectivity.
DNA upstream of the replication fork is unwound by a class of enzymes called what?
DNA helicases
What is the procedure that depends on the formation of stable base pairs between the probe and target sequence?
DNA hybridization
What are the four popular methods of identification used for target sequences?
DNA hybridization with labeled probe and radiographic screening for probe labe immunological screening for protein product assaying for protein activity genetic complementation
Why would host cells that carry a plasmid cloned DNA construct produce white colonies as opposed to blue colonies?
DNA inserted into a restriction endonuclease site within the multiple cloning site disrupts the correct sequence of DNA codons of the lacZ gene and prevents production of functional lacZ protein so no B-galactosidase is produced.
What is the primary objective of a metagenomic project?
DNA library of all microorganisms of location
How does alkaline phosphatase prevent production of the original plasmid DNA?
DNA ligase cannot join ends of dephosphorylated linear plasmid DNA
Since the hydrogen bonds of the four bases that pair are not strong enough to keep two DNA molecules together, what resolves the problem in molecular cloning and where does it come from?
DNA ligase, bacteriophage T4
What protein is responsible for binding deoxyribonucleotides and fitting the correct nucleotide into place, forming the phosphodiester linkage?
DNA polymerase
What are two factors important for regulating eukaryotic transcription?
DNA-protein interactions, protein-protein associations
What enzyme degrades double stranded DNA by hydrolyzing internal phosphodiester linkages?
DNase I
Name 3 thiol compounds or reducing
DTT, Glutathione, beta-ME
Name three factors or chemicals that do differential reactivation/stimulation for the enzymes (like expandase/hydroxylase)? Enzyme calculation.
DTT, ascorbate, ATP
What 3 cofactors and metal ion for expandase
DTT, ascorbate, ATP, and ferrous ion
What is the function of the reducing agent?
DTT: reducing agent. It takes S-S bond to S-H.
What six type of enzyme reaction are involved in producing final tdp sugar with very structure differences
Deoxygenation, ketoreduction, epimerization/isomerization, dehydration, transamination, methylation
How an inhibitor of resistant PBP2x may be designed to have antimicrobial activity against most both penicillin-resistant and -sensitive bacteria?
Design a molecule with the features of the branched-stem peptides and penicillin.
Analysis of cloned genes - DNA sequencing
Dideoxynucleotide sequencing - has an -H not an -OH at the 3' end so it terminates replication - no where to form phosphodiester bond to the next nucleotide - YOU NEED .... -DNA polymerase -DNA template -primer -dATP, dGTP, dCTP, dTTP -small amount of a didNTP that is labeled 1. primer is annealed to DNA template 2. Then reaction mixture with DNA poly, all the bases, and the ddNTP (tagged with fluorescent dyes, each base has a diff color that this is attached to AKA ddATP - pink and ddGTP is green etc) 3. Eventually there will be a ddNTP inserted at every location so that each strand differs in length by 1 nucleotide because during primer extension it sometimes puts in ddNTP instead of dNTP 4. the products of this are added to a single lane on a capillary gel electrophoresis, and the bands are read by a detector system 5. the sequence is found by the extension of the primer, and is read from new strand not template strand
Two major strategies for enzyme engineering glycosyltransferases
Directed evolution and sequence structure based engineering
Provide two major strategy in enzyme engineering of anomeric kinases.
Directed evolution-based random mutagenesis, structural based rational design.
The _____________ recognize Ori C sequence to initiate DNA replication in E. coli
DnaA
What is the tagging enzyme in LongSAGE and how many bp is it?
MmeI, 17 to 21
DNA from an organism is cut by a restriction enzyme and so is the vector DNA, both cut by the same enzyme, and then they can base pair with their complementary tails, and then gaps are sealed with DNA ligase THIS MAKES RECOMBINANT DNA
Done in a test tube at low temperature, this is necessary for hydrogen bonds to stabilize and stick together. Ligase seals the phosphodiester bonds and now they're bonded from two different sources
Misaligned sequence can be generated by what type of recombination?
Double cross over→ successful recombination Orthologous sequences
What 2 reasons for conformational change?
Drug and post-translational modification- phosphorylation
1. Which of the following is a feature of site-specific recombination? A) A specific recombinase enzyme is required. 3 B) The energy of the phosphodiester bond is preserved in covalent enzyme-DNA linkage. C) Recombination sites have non-palindromic sequences. D) Insertions or deletions can result from site-specific recombination. E) All above.
E
2. The phosphodiester bonds that link adjacent nucleotides in both RNA and DNA: A) always link A with T and G with C. B) are susceptible to alkaline hydrolysis. C) are uncharged at neutral pH. D) form between the planar rings of adjacent bases. E) join the 3' hydroxyl of one nucleotide to the 5' phosphate of the next
E
20. In what location do you expect to find single-stranded siRNA? A) Attached to the major groove of DNA by hydrogen bonds B) Bound to an operon Repressor site C) Bound to RNA polymerase D) Bound to the small ribosomal subunit E) Bound to mRNA
E
3. Chargaff's rules state that in typical DNA: A) A = G. B) A = C. C) A = U. D) A + T = G + C. E) A + G = T + C.
E
4. In the Watson-Crick model of DNA structure: A) both strands run in the same direction, 3' → 5'; they are parallel. B) phosphate groups project toward the middle of the helix, where they are protected from interaction with water. C) T can form three hydrogen bonds with either G or C in the opposite strand. D) the distance between the sugar backbone of the two strands is just large enough to accommodate either two purines or two pyrimidines. E) the distance between two adjacent bases in one strand is about 3.4 Å
E
5. In living cells, nucleotides and their derivatives can serve as: A) carriers of metabolic energy. B) enzyme cofactors. C) intracellular signals. D) precursors for nucleic acid synthesis. E) All of the above.
E
7. At replication forks in E. coli: A) DNA helicases make endonucleolytic cuts in DNA. B) DNA primers are degraded by exonucleases. C) DNA topoisomerases make endonucleolytic cuts in DNA. D) RNA primers are removed by primase. E) RNA primers are synthesized by primase.
E
A branched ("lariat") structure is formed during: A) attachment of a 5' cap to mRNA. B) attachment of poly(A) tails to mRNA. C) processing of preribosomal RNA. D) splicing of all classes of introns. E) splicing of group II introns.
E
An alternative repair system by error-prone translesion DNA synthesis can result in a high mutation rate, because: A) alternative modified nucleotides can be incorporated more readily. B) interference from the RecA and SSB proteins hinders the normal replication accuracy. C) replication proceeds much faster than normal, resulting in many more mistakes. D) the DNA polymerases involved cannot facilitate base-pairing as well as DNA polymerase III. E) the DNA polymerases involved lack exonuclease proofreading activities.
E
Splicing of introns in nuclear mRNA primary transcripts requires: A) a guanine nucleoside or nucleotide. B) endoribonucleases. C) polynucleotide phosphorylase. D) RNA polymerase II. E) small nuclear ribonucleoproteins (snRNPs).
E
Which of the following statements is false? In vitro, the strand-exchange reaction: A) can include formation of a Holliday intermediate. B) is accompanied by ATP hydrolysis. C) may involve transient formation of a three- or four-stranded DNA complex. D) needs RecA protein. E) requires DNA polymerase.
E
6. Which of the following statements about the tRNA that normally accepts phenylalanine is false? (mRNA codons for phenylalanine are UUU and UUC.) A) It interacts specifically with the Phe synthetase. B) It will accept only the amino acid phenylalanine. C) It's about 80-nucleotides long. D) Phenylalanine can be specifically attached to an —OH group at the 3' end. E) The tRNA must contain the sequence UUU.
E) The tRNA must contain the sequence UUU.
17. By adding SDS (sodium dodecyl sulfate) during the electrophoresis of proteins, it is possible to: A) determine a protein's isoelectric point. B) determine an enzyme's specific activity. C) determine the amino acid composition of the protein. D) preserve a protein's native structure and biological activity. E) separate proteins exclusively on the basis of molecular weight.
E) separate proteins exclusively on the basis of molecular weight.
What are the tac promoters recognized by?
E. coli RNA polymerase holoenzyme
Which strong regulatable promoters are the most widely used?
E. coli lac and trp tryptophan operons
deacylase is involved in generating what therapeutic agent
ECB antifungal agent
What specific interaction was observed
ES 1:1 ratio (B-lactam and enzyme)
What is another way to use mass spec in proteomics? What does it do? How does ESI work?
ESI- electrospray ionization Produces ions -Peptides/proteins to be anaylzed are in aqueous form -Makes use of functional groups in peptides acquire different charge at different pH -Usually acidic pH -Limited range more than TOF
What was the expansion of GenBank due to?
EST library, cDNAs, mapping of genome
What is the tagging enzyme in SuperSAGE and how many bp is it?
EcoP151, 26
What was the first type II restriction endonuclease?
EcoRI
Name the 4 5' phosphate extension restriction endonucleases that are common?
EcoRI, BamHI, Sau3AI, NotI
A cyclin needed for inhibitor design may be based on the interaction of staurosporine with what 4 amino acids of CDK4 at the atp binding site?
Glu94, Val96, Asp99 and Glu144
Describe briefly what is epigenetics?
Encompasses all mechanisms for the inheritance of biological traits that do not involve alterations of the coding sequence of DNA
What is optimally occurring enzymatic reaction for the 3 assays/what is the enzymatic function with B-lactam
Enzymatic acylation
What is the two step enzymatic de-racemization by stereo inversion
Enzymatic oxidation and enzymatic reduction
using the biocatalytical route in the manufacture of the diol precursor of TBIN and the key intermediate for Lipitor, what is the strategy for cofactor regeneration
Enzyme coupled using KRED and GDH as the enzymes
Repair pathways: Ap endonuclease UvrAF, C, D protein mutS-mutL complex Translesion DNA polymerase DNA photolyase
Enzymes BER NER MMR translesion synthesis Direction repair
Name 2 enzymes and one protein that are essential for autophagy
Enzymes are AMPK (increase autophagy) and mTorc1 (blocks autophagy). The protein is TFEB (transcription factor).
What 3 modern metabolism-related scientific disciplines are important for biotechnology
Epigenetics, circadian cycle, autophagy
What is epistasis and how might it contribute to directed enzyme evolution.
Epistasis is interactions between mutations; industrial biotransformation
Dimerizaton of what enzyme is a therapeutic strategy
Epoxide hydrolase
What did GenBank do?
Established in 1982 for the collection, management, storage, and distribution of sequence data. The servers were linked to NSFnet, until 1995 when NSFnet was replaced by internet
What 3 stabilized macrocin o-methyltransferase (M O-M)?
Ethanol, PMSF, SAM
The hyp that genetic code consisted of groups of 3 nulceotides rather than groups of 2 nuclotides supports
Every amino acid has at least 1 corresponding codon
Which plasmids carry their own information from one cell to another?
F plasmids
7. Many DNA polymerases have a proofreading 5' to 3' exonuclease.
F; 3 to 5
4. The ratio A+T/G+C is constant for all natural DNAs.
F; A+G=T+C
3. In the Watson-Crick model for the DNA double helix, there are two equally sized grooves that run up the sides of the helix
F; major and minor grooves are different in size
E. coli DNA polymerase III can initiate replication without a primer
F; needs a primer
5. In the Watson-Crick model of DNA structure: both strands run in the same direction, 5' to 3'; they are parallel.
F; opposite direction, anti parallel
What is the strategy used in which a stream of cells in single file passes a laser beam that detects the excitation of a fluorochrome that is either attached or inside the cell and sorts the fluorescent and nonfluorescent cells into separate collection vessels?
FACS
What happens in SIGEX after the catabolic operon is activated by the substrate and transcription continues through the gfp gene, producing gfp?
FACS 2nd time
Law and Regulation of GMOs
FDA/EPA/USDA - GM crops must be shown (by the manufacturer) to be "substantially equivalent" to nonGM versions of the crop. Toxicity and allergenicity of the crop are assessed based on the nature of the protein product of the genes that have been inserted. If they are deemed safe US law does not require labeling
For cofactor for NADPH recylin, provide 8 couple enzyme
FDH, GDH, G6PDH, PDH, ADH, NAD oxidase, NADPH oxidase, NAD hydrogenase
Santa Gertrudis cattle have the combined traits of good beef production and hot weather tolerance as a result of selection and INBREEDING.
False, hydridization
Many flowering plants such as roses, African violets, and orchids have been produced by the process of TEST CROSSING.
False, selective breeding
in 1990, Young survivor of ADA deficiency: treated at age 4 with transformed T-cells -in 2000, 20 babies are Successfully treated For SCIDX but 3 of them get lymphoma
First done in 1990, and she got a correction, she got 4 or 5 treatments, and she got 25% of her immune system back, it is enough that she will survive -in 2000, 20 babies treated for SCID X they did 2 bone marrow replacements on these kids, and they had 95% success with this, most kids got 50% of their immune system back, then 3/20 of he kids got cancer, and 2 were treated and 1 died, and in these kids this virus put DNA where regulation of cells was important thus causing cancer -in 2002 human gene therapy stopped to get around this problem
What's the enzyme that is deficient in Gaucher Disease?
Glucocerebrosidase
Improve chemoenzymatic synthesis with a key intermediate for hypertensive drug candidate involved what two enzyme
Glutamate oxidase and L-lysine aminotransferase
From B-lactam induced precipitation of PBP2a, what specific enzyme kinetic behavior was observed for the enzyme precipitation
First-order kinetics
Provide three major aspects of enzyme optimization process to produce industrial biocatalysts in the quickest time
Fitness function, diversity generation, search algorithm
What was the first FDA approved genetically modified food?
Flavr Savr tomato 1994
What is FACS and how does it work?
Fluorescence Activated Cell Sorting Labelled cells are sent under pressure through a small nozzle and pass through an electric field. A cell generates a negative charge if fluoresces and a positive charge if it does not Genomic scale for DNA that might have promoter or goi for specific function Can treat until has it and has positive charge
Provide 3 PBP2a assays
Fluorescence quenching, radioactive labeling, chromogenic assay.
Provide 2 common methods in determining the mw of enzyme and one common method in determining the subunit size of the enzyme
For molecular weight use gel filtration and ESI-MS (electrospray ionization mass spectrometry). For determining the subunit size of the enzyme use SDS-PAGE
Which of the following sequences is a palindrome and could be a restriction enzyme recognition site? >GAATTC> <CTTAAG<
GAATTC
What is an ideal in vivo marker for monitoring transgenic plants?
GFP
What are the 3 plant enzymes for downstream module and subsequent hydroxylation
GGPP synthase, taxadiene synthase and for hydroxylation is taxadiene 5-alpha hydroxylase
What is the process of recombinant DNA technology?
GOI cleaved and ligated into cloning vector, cloning vector transferred into host cell, transgenic host cells selected
What gene encodes a stable enzyme that is not normally present in plants and catalyzes cleavage of a range or B-D-glucuronides?
GUS
What are some examples of reporter genes?
GUS, GFP, LUC
What reporter gene products can be detected in situ in intact plant tissues?
GUS, firefly and bacterial luciferases, GFP
What four enzymes are involved in converting galactose into udp glucose
Galactose mutarotase, galactokinase, galactose-1-phosphate uridylytransferase, udp-galactose 4- epimerase
Excluding hydrophobic interaction and affinity provide 4 major chromatography types of enzyme purification and the separation basis of each
Gel Filtration, based on molecular weight Ion-Exchange, based on charge Hydroxylapatite Interaction, based on calcium phosphate Chromatofocusing, based on ion-exchange/isoelectric focusing
What was established in 1982 that revolutionized biology?
GenBank
The erythromycin PKS has been the most extensively studied for the development of combinatorial biosynthesis method and generation of polyketide derivative by what four type of combinatorial biosynthesis strategy
Gene activation, substitutional addition of domains, domain swaps, and glycosylation or methylation pathways.
___ is an application of the Human Genome Project that involves the insertion of normal genes into cells with defective genes in an attempt to correct genetic disorders.
Gene therapy
In 1974, Stanley Cohen and Herbert Boyer inserted a gene from an African clawed from into a bacterium. The bacterium produced the protein coded for by the inserted frog gene. The insertaion of a small fragment of frog DNA into the DNA of another species can most accurately be called
Genetic engineering
How many nrsp subunit, module, and catalytic domains are involved in deptomycin biosynthesis
Module: 13. Catalytic domains: 43. Subunits:3.
The development of what method for screening enzyme activity is critical for glycosyl transferase engineering, leading to what three potential improved enzyme properties
HT assays, enhanced catalytic efficiency, increased substrate promiscuity, novel activities
Who discovered TCA cycle and got a noble prize what year
Hans Kreb and 1953
Cloning and sequencing of genes is providing unexpected information on how genes function and evolve
Human Genome Project: Human - "finished" 2003 Drosophila -- done 1999 Caenorhabditis (worm) -- done 1997 Arabidopsis (plant) -- completed 2000 Yeast -- done 1996 E. coli -- done 1995 Mouse -- 2004 *Sequenced more than just humans - these different genomes are cross informative - more info about human genome by looking at other organisms than just humans alone -everything has been sequenced at this point - all in genbank
In Most Eukaryotes much of the Genome is repeated DNA sequences
Humans: 3 billion base pairs of DNA -50% repeated noncoding sequences: *most are transposable elements -45% single copy, noncoding sequences -5% codes for genes - this is the total of introns and exons (1.5% exons) ***L1 and Alu are the major transposable elements in the human genome
Provide 5 enzymes in preparing HMG-CoA reductase inhibitor
Hydrolase, G. candidum reductase, acinetobacter Reductases I,II,III, cytochrome P-450, and DERA
Provide 3 PBP2x assay to evaluate the potency of designed molecule.
Hydrolysis activity assay, radioactive labeling, fluorescence polarization assay
Suggesting what metabolic enzyme is evolved from what metabolic enzyme
Hydroxylase comes from the expandase
What is the purpose of microarray in transcriptomics?
ID genes whose expression changes in response to particular biological condition
10. In bacteria the elongation stage of protein synthesis does not involve: A) aminoacyl-tRNAs. B) EF-Tu. C) GTP. D) IF-2. E) peptidyl transferase.
IF-2
Which of the structures in Figure 11-1 are composed of RNA?
II and IV
What is the genotype of generation 1 in Figure 10-5?
Ii
A _______is a small ring of DNA found in a bacterial cell
Plasmid
___ is used to develop pure breeds.
Inbreeding
misaligned pairing using orthologous versus paralogous sequences...the recombination result?...how will it lead to substitution, insertion and deletion? How do you achieve them?
Insertion: more than 1 nucleotide Replacement (substitution): 1 nucleotide Deletion: delete nucleotide Consequences of mispairing at recombination cause mutation
what is combinatorial biosythesis? What are the two type of enzymes commonly used?
Involves the genetic engineering of biosynthetic clusters in order to create diverse libraries of compounds derived from natural products. i. PKSs and NRPSs
Cancer cause mutation in gene has been implicated with what 3 enzymes in TCA
Isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase (add water molecule)
Enzymes that have different functions but from same ancestral gene?
Isomerase and Hydrolase. Muconolactone isomerase and beta-ketoadipate enol-lactone hydrolase mediate sequential metabolic steps in the beta-ketoadipate pathway.
How is recombinant bacteriophage lambda maintained?
growth in E. coli and lytic cycles
Which of the following statements correctly describes the role of telomerase in eukaryotic cells
It synthesizes new DNA a the 3' end of the chromosome
What is the function of the sulfhydryl reagent?
It takes S-H to S-S.
Describe the three terms metagenomics, metagenome, and metagenomic techniques.
Metagenomics- the development of culture-independent methods to probe the total genetic content of an environment such as soil Metagenome- refers to the collective genomes of the organisms present in the environment Metagenomic techniques- comprise a varying collection of methods for identifying and cataloging the genetic diversity.
What are the two enzymes converting alpha KG to 3m glutamate
Methyl transferase, aminotransferase
Epigenetic drug works on solid tumors by inhibiting what enzyme? Involved in what modification of DNA? Leads to expression of what genes?
Methyltransferases. Methylation. (Has silenced) tumor-suppressor genes.
Two calculated kinetic constants for tungsten
Kcat and kcat/km Kcat- turnover number Kcat/Km- catalytic activity
What kinectic constant was used in comparing the two enzymes for all peptide
Kcat/km
To determine the potency of B-lactam what 2 kinetic constants are used in comparison with mic for mrsa
Ki and Km
The substrate specificity of complex and fusion cyclin D1/CDK4 was determined by using what two kinetic constants?
Km and vmax
What are 2 basic kinetics from M-M Eqn
Km in micromole and Vmax in nanomol product/min milligram of enzyme
Name 3 everyday biotechnology examples.
Lactic acid producing bacteria, insecticide on crops, nitrogen fixing bacteria in soil, antibiotics made from bacteria
Starting from sc-cyclase, what are the three evolution strategies to get to ca-hydroxylase?
Lateral transfer, vertical retroevolution and vertical modification.
a ___________________ show the relative location of genes on a chromosome
Linkage Map
Stepwise deracemisation or dynamic kinetic resolution employing what two enzyme system in catalyzing acetylation s mandalate to s acetate product with high yield and steroselectivity. b. What is the function of each:
Lipase and mandelate racemase lipase- resolution. Madelate racemase- racemization.
Using the particular substrate, DM-CH-DC, provide two enzymes that catalyze conversion to two opposite stereoisomers
Lipase from Candida Antarctica; Porcine Liver Esterase
Provide three enzyme related to racemistion strategy
Lipase or stereo specific enzyme, racemase using two stereo complementary catalyst, single non- stereoselective alcohol dehydrogenase
Who discovered the two models?
Lock and Key model was discovered by Fischer in 1902. Induced Fit by Koshland and people in 1998.
What protocols have been developed to produce longer tags?
LongSAGE and SuperSAGE
Why does the deficiency of the enzyme lead to Parkinson's Disease?
Look at step 2: Oligomers convert into insoluble alpha-syn amyloid fibrils. This leads to Parkinson's Disease
What is an example of mass spec used in proteomics? What's something you have to know in order to use mass spec? What does Trypsin cut between?
MALDI-TOF The genome sequence Argenine and Lysine
How should the top row of Figure 10-7 be read?
MMXX, MMXx, MmXX, MmXx
Deptomycin approved in US approved for treatment against gram positive pathogen is efficacious against what 4
MRSA, MRSE, VRE, PRSP
Combinatorial biosynthesis have been applied in generating what four types of novel natural products
Macrolides, cyclic lipopeptides, carotenoids, alkaloids
Enoate reductase involved in synthesis of carotenoid such as zeaxanthin, used for preventing what degeneration.
Macular degeneration.
Genome sequencing based on mapped "contigs" (overlapping genomic DNA clones)
Map-based Cloning: 1. large DNA clones are mapped 2. individual contigs are restriction mapped subcloned and subclones are sequenced (subclones derived from one clones, and then that subclones can be cut up by restriction enzymes and mapped to determine its sequence) -first an entire chromosome is cut into short over lapping sequences most likely by restriction enzymes, -overlapping clones are mapped with in situ hybridization -subclones derived from one of many cloned fragments -each subclone can be characterized by restriction mapping, and this way each subclone will be cut at restriction sites, fragments cloned, and nucleotide sequence determined
Metabolomics. Name technology:
Mass spectrometry, NMR: Isotopomer Analysis, HPLC Know the two enzymes on slide 6 in red.
What does MALDI stand for? How does MALDI work? What is produced by MALDI?
Matrix assisted laser desorption -Sample is mixed with chemical matrix -Spotted onto small slide to evaporate -Forms crystal lattice containing the peptide -Placed into the source with a laser -Chemical matrix absorbs the laser light, and transfers energy to the peptides which are ejected into gas phase -Both positive and negative ions -Add 1 to peptide mass because accepted a proton. [M+H]
In addition to the 5 common parameters for catalytic optimization from practical enzymology we have had before, provide 4 other factors for agent and the reason for each using optimization activity of cyclin D1/CDK4.
Me2SO: E conformation DTT: -SH EGTA: Heavy metals Triton X-100: E/S solubility
What does eukaryotic mRNA get capped with?
guanine nucleotide at 5' end
Which of the following factors significantly contributes to the ability of eukaryotic cells to complete DNA replcation during the short window of time offered by S phase
Mulitiple origins of replciation are activated per chromosome
What regions of oleosins are hydrophilic?
N and C terminal regions
What kind of extension is found at each end of ditags after amplified and treated with N1aIII to release adaptor sequences?
N1aIII extension
What corepressor is involved in this differentiation
NCoR
Discover and design of pharmaceutical biocatalyst most effectly based on combine use of what two major approaches
Natural biodiversity and directed evolution
Provide additional misfolded peptides and misfolded enzymes, which leading to what metabolic disorders?
Neuroligin alpha/beta hydrolase causes autism, insulin causes diabetic nephropathy CEL-mody causes protein misfolding disease
Provide three enzymes used in making Lipitor
Nitilase, halohydrin dehalogenase, KRED
What enzyme is used in producing Nicotinamide (Acrylamide)
Nitrile hydratase
What is the substrate for chromogenic assay for PBP2a?
Nitrocefin
Which of the following growth media would you expect to result in synthesis of high levels of mRNA for the enzymes of the E. coli lac operon?
No glucose High lactose
By no inhibition of ETNB indicate the enzyme has what
No requirement for metal ion
which enzymes is directly responsible for sliding DNA along the surface of an assembled nuclosome to transferring a nuclosome form one DNa helix to another
Nucleosome- remodeling complexes
Name 2 metals chelators that effect Ca- expandase/hydroxylase:
O-phenanthroline and EDTA
The DNA fragments synthesized in lagging strand is called
Okazaki fragment
What is the common name for Enoate Reductase
Old yellow because it contains either FMN or FAD cofactor
What genes cause cancer and why?
Oncogenic (cancer-causing ) Viruses carry oncogenes -DNA tumor viruses: Papilloma, Epstein Bar, Hepatitis B: Viral genes activate DNA replication in host -RNA tumor viruses - retroviruses: Infection by virus causes mis-expression of host genes -in humans RNA tumor viruses aren't a primary cause of cancer, because they don't directly cause cancer, but they may lead to mutations that do so cancer is a secondary affect of the virus -organisms other than human do have retroviruses that are the direct cause of cancer when the virus enters the cell and expresses its genes, it causes mis-expression of host genes
A(n) ___ shows the relative location of genes on a chromosome.
linkage map
Which mass spec do you use for peptide fragmentation?
Tandem mass spec
What is the kinetic mechanism of cyclin D1/CDK4? How was it determined in two major steps?
Ordered bi-bi (single displacement mechanism) Peptide substrate with variable atp, staurosporine with variable atp, adp with variable atp
Provide a nucleotide sequence observation suggesting that S rosiosporus appear to express three NRPS subunit from a single very large transcript
Overlap of stop and start codons in NRPS genes
One type of DNA microarray is constructed by spotting _____________ sequences from the mRNAs of a single cell or all or specific sets of the coding sequences of an organism onto a glass slide or nylon membrane. How many probes can be arrayed in a 1 cm area?
PCR amplified cDNA, 10,000
a technique that may be employed in the hUman Genome Projects is
PCR, automated gene sequencers, gel electrophoresis- All of these
What are the two strategies of directed evolution used to improve the enzyme; The two strategies include what four properties
PROSAR and Gene Shuffling; Activity, thermal robustness, solvent stability, and stereo-selectivity
Accumulation of what metabolite in Cephalosporin C biosynthesis suggests what metabolic enzyme is a major rate limiting step?
Penicillin
What beta-lactam serves as a weak substrate and competitive inhibitor of what enzyme?
Penicillin (weak substrate) and a competitive inhibitor with hydroxylase.
Provide 4 metabolic pathways involved in cancer. Provide 3 enzymes as potential target for cancer drug discovery and for what metabolic pathway...what's missing?
Pentose phosphate pathway, glycolysis, one-carbon metabolism and TCA cycle. Enzymes: MTHFD1L, SHMT2, MTHFD2. Glycine metabolism
How do we determine disulfide linkage
Peptide mapping under oxidize and reducing conditions
10. Which one of the following statements about the elongation phase of protein synthesis is true? A) At least five high-energy phosphoryl groups are expended for each peptide bond formed. B) During elongation, incoming aminoacylated tRNAs are first bound in the P site. C) Elongation factor EF-Tu facilitates translocation. D) Peptidyl transferase catalyzes the attack of the carboxyl group of the incoming amino acid on an ester linkage in the nascent polypeptide. E) Peptidyl transferase is a ribozyme.
Peptidyl transferase is a ribozyme
A Ribosome contains three binding sites for tRNA molecules: the_______ or P-site, holds the tRNA molecule that is linked to the growing end of the polypeptide chain, and the _____-site, holds the incoming tRNA molecule that is charged with an amino acid and a E-site.
Peptidyl-site; Aminoacyl (A)
Kinetic mechanism "ping pong" mechanism is also named as what? And "ordered bi-bi" mechanism is also named as what?
Ping pong→ double displacement Ordered bi-bi→ single displacement
Vectors
Plasmids -origin of DNA replication -antibiotic resistance gene -unique restriction sites *Size of the recombinant fragment is limited to ~10 kb Vectors are bacterial plasmids- they're modified for cloning. The basic features of all of them is that they can replicate (origin of replication), antibiotic resistant genes (so you can introduce this without DNA cells recognizing it) and it must have places around the plasmid where restriction enzymes only cut once, and only once. You want it to be open up so it can accept an insert, and the plasmids have been modified where the sites only cut once. -the plasmid vector is removed from host cell and cut by a restriction enzyme, DNA to be cloned is cut with the same restriction enzyme, and then these 2 pieces are ligated, and introduced into bacterial host cells by transformation
Name 1 method each in determining primary, secondary, tertiary and quartenary structure
Primary structure: amino acid sequencing Secondary structure: circular dichroism Tertiary structure: fluorescence Quaternary Structure: x-ray
Provide example for primary, secondary, tertiary & quaternary structure:
Primary: amino acid sequence, Secondary: alpha helix or beta sheets, Tertiary: structure containing disulfide bonds, Quaternary: structure of multi-subunit complex enzyme.
Provide two dynamic resolution enzyme and what is the chemical reaction to convert non substrate isomer to the racemic mixture
Protease, lipase. Chemical condition is mild base.
Explain overexpression of protein in terms of half lives.
Proteins have half-lifes. The half lives may vary when you express it in a cell vs in its organism You can add an amino acid to its N-terminus in order to increase the half life
Name the 3' hydroxyl extension restriction endonuclease that is common?
PstI
A useful device for predicting the possible offspring of crosses between different genotypes is the ___.
Punnett square
Name the 3 blunt ended restriction endonucleases that are common?
PvuII, HpaI, HaeIII
Which plasmids encode for antibiotic resistance?
R plasmids
What three enzymes are involved in reduction of haloketones and stereo specific epoxide formation
R selective ADH, S selective ADH, halohydrin dehalogenase
During the process of transcription, DNA serves as the template for making ___, which leaves the nucleus and travels to the ribosomes.
RNA
Primases synthesize a short stretch of ______ to prime further synthesis
RNA
Translation is to protein as transcription is to ___.
RNA
What does a strong promoter have a high affinity for?
RNA polymerase
What happens in the transcription of eukaryotic genes after binding of the TATA sequence?
RNA polymerase II binds to transcription complex
How is transcription initiated?
RNA polymerase binds to promoter
Which one of the following components is not required for DNA synthesis
RNTPs ribo Triphos
What enzyme degrades the RNA strand of a DNA-RNA hybrid molecule?
RNase H
What hydrolyzes the RNA of the streptavidin-bound RNA-DNA hybrids and what else does it do?
RNase H, releases full length cDNA into solution
In pea plants, inflated pods (R) are dominant to constricted pods (r). Which of the following crosses is a test cross between inflated pods and constricted pods?
RR x rr
In pea plants, inflated pods are dominant to constricted pods. Which of the following crosses is a test cross between inflated pods and constricted pods
RR x rr
Which of the 3 assays can determine activation and inhibition
Radioactive labeling
What enzyme is a heat stable DNA polymerase from Thermus aquaticus?
Taq DNA polymerase
___________are used to cleave DNA into fragments
Restriction Enzymes
___ are used to cleave DNA into fragments.
Restriction enzymes
Cyanobacteria aldehyde decarbonylase is similar in three dimensial structure active site in amino acid residue to what enzyme in e coli
Ribonucleotide reductase of e coli
B-form DNA in vivo is a ________-handed helix, _____ Å in diameter, with a rise of ____ Å per base pair
Right; 20; 3.4
Provide one extreme example of epigenetic inheritance with a mechanism.
Robust self-propagating changes in the folding of certain proteins known as prions
NEM?
S-H group
Provide alternative host and screening application for each
S. lividans- terragine/norcardamine natural product families P. putida- Naphthalene Dioxygenase
What method uses recombinant DNA techniques to clone randomly linked short sequences of cDNA prepared from extracted cellular mRNA that can be efficiently sequenced to identify expressed genes?
SAGE
What online resource is available to match tags to likely genes, determine the frequency of a tag among various SAGE libraries, and provides other pertinent information?
SAGE Genie
What is an example of a specialized gene expression system?
SIGEX
What is an example of a aid used to detect metagenomic clones?
SIGEX (Substrate Induced Gene Expression)
What anti-aging gene was identified as tumor suppressor in mice
SIRT6
Provide two enzymes involved in detoxification of corresponding catechol
SULTs and COMT
Criticisms of GMOs
Safety -- Food Allergens Consumer choice - Labeling Bio Pollution Patents and control of food supply US decided that these crops must be substantially equivalent - have to show toxicity of protein product of the gene you put in (protein that dna codes for) -test to see if these proteins bring with them issues of toxicity or Allergenicity - that you can directly test -Bio pollution - biological polltuion - can these plants hybridize with the genes of native plants and then this can spread **For Example - with corn and cotton, there are no wild plants for the hybrid to cross with so there are no genes for it to hybridize with > worried that it can get away into wild plants -issue of patents and food supply - big companies that are producing seeds farmers are using > they have control over food supply
What anchoring enzymes have been used to identify transcribed genes that do not have a convenient N1aIII site but can recognize and cleave at specific 4-bp sequences?
Sau3AI
Provide one scientific rationale and one medical rationale of why cyclin D1.CDK4 was a major cancer drug target for cell cycle regulation.
Scientific: Cyclin D1/CDK4 plays a major role in the intiation of the cell cycle (G0 to G1) and in the G1 to S transition. Medical: Deregulation of the cyclin D1/CDK4-pRb pathway leads to uncontrolled proliferation and cancer
During DNA replication the parental DNA strands serve as templates for the synthesis of new progeny DNA stands, this is called
Semi-conservation
What does SAGE stand for?
Serial Analysis of Gene Expression
How does the SAGE method work for transcriptomics? How many bp does SAGE use? How many bp does LongSAGE use? How many bp does SuperSAGE use?
Serial Analysis of Gene Expression -Have a cut site -Each tag is 14 bases in length -Called a ditag -When cutting tag, have overhang -Separation of clones by size -Clone into plasmid -Sequence from the tag 10-14bp 17-21 bp 26
PMSF contain what essential amino acid
Serine
Name 4 types of proteolytic enzymes and inhibitors for each type
Serine proteases (trypsin): PMSF Thiol proteases (papain): N-ethylmaleimide (NEM) Acid (carboxyl) proteases (pepsin): Pepstatin A Metalloproteases (collagenases): EDTA
Which one of the following is true about the genetic code? A) All codons recognized by a given tRNA encode different amino acids. B) It is absolutely identical in all living things. C) Several different codons may encode the same amino acid. D) The base in the middle position of the tRNA anticodon sometimes permits "wobble" base pairing with two or three different codons. E) The first position of the tRNA anticodon is always adenosine.
Several different codons may encode the same amino acid.
Application of proSAR for in vitro gene recombination lead to activity or thermostability improvement of what three enzymes
a. Halohydrin dehalogenase, proteinase K, cytochrome P450s
L shuffling improve what property, with what kinetic constant of what enzyme
Specific activity, kcat, beta glucosidase
PCR Uses DNA Polymerase to copy DNA in the test tube repeated rounds of replication
Specificity of PCR depends on choosing the right primers and the annealing Temp used
Who won a nobel prize for the discovery of prions and element of disease transmission and what year?
Stanley Prusiner in 1997
What is the binding site or basis for staurosporine and ADP?
Staurosporine: ATP binding site, since staurosporine is a competitive inhibitor of ATP ADP: E-ATP-pRb (ES), since ADP is an un-competitive inhibitor of ATP
How sc- expandase and hydroxylase differ structurally and catalytically from ca-expandase and hydroxylase?
Strucutrally, sc- expandase and hydroxylase are two separate enzymes, whereas ca- expandase/hydroxylase is one single enzyme. Catalytically, ca-expandase/hydroxylase does both functions (bi-functional enzyme) and sc- does two different things- the expandase does mostly expandase and the hydroxylase does mostly hydroxylase function.
What is SIGEX? What does it include?
Substrate Induced Gene Expression Allows us to figure out which cells express the protein, and which ones don't -Identifies catabolic genes that are expressed when their promoters are activated in the presence of a particular substrate -Uses fluoerscence-activated cell sorting -pUC-based plasmid -Green fluorescent protein (gfp) -Lac promoter (Plac)
What is the strategy for co-factor regeneration in manufactor of key alcohol intermediate
Substrate coupled- KRED as enzyme and isopropanol as the substrate
In applying the engineered microbial platform for direct biofuel production from Brown macroalgae, provide substrate, 2 DNA sources, 4 enzymes, 3 pathways needed, what cell, what optimization, what final product?
Substrate: alginate 2 DNA sources: fosmid library and auxillary genes 4 enzymes needed: alginate lyase, periplasmic lyase, oligoalginate lyase, DEHU reductase. Or simply say 3 lyases and 1 reductase 3 pathways needed: ED pathway, TCA cycle and glycolysis Cell: E. coli Optimization: Fermentation Final product: Ethanol
2. The DNA of virtually every cell is underwound (i.e., negatively supercoiled) relative to Bform DNA
T
Why is it necessary to include a right border sequence of the T-DNA region in Ti plasmid-based vectors?
T DNA integration into plant cell DNA
What happens after a Ti plasmid carrying cell of A. tumefaciens attaches to a host plant cell and the vir genes are induced?
T DNA transferred linearly and integrated as single stranded DNA into plant chromosomal DNA
Crown gall formation is the consequence of what bacterial plasmid DNA that gets integrated and expressed into the plant cell genome?
T-DNA
What enzyme catalyzes the transfer of the terminal gamma phosphate from a nucleoside 5' triphosphate to a 5' hydroxyl group of a polynucleotide?
T4 polynucleotide kinase
What does the bacteriophage T7 gene 10 promoter require for transcription?
T7 RNA polymerase
What is used to produce RNA copies, cRNA, from the second cDNA strand as a template in the presence of biotin-labeled ribonucleotides?
T7 RNA polymerase
How is the bacteriophage T7 gene 10 promoter utilized?
T7 RNA polymerase gene is inserted in E. coli chromosome on bacteriophage lambda lysogen under control of E. coli lac promoter and after cells are transformed by a plasmid with a cloned gene under control of T7 promoter, IPTG is added to the medium and T7 RNA polymerase is induced and synthesized, T7 RNA polymerase transcribes the cloned gene
What does a eukaryotic promoter consist of?
TATA, CCAAT, GC repeats
What are the two sequences of the DNA binding sites for the promoter region called in bacteria?
TATAAT Pribnow box, TTGACA
List 4 general denaturants
TCA, acetone, urea and granidium chloride
provide 4 naturally occurring and usually nucleotide diphosphate containing sugar. Provide one enzyme involved in producing all 4 ndp sugar
TDP, CDP, UDP, GDP- write sugar after; Mutase
which of the following is ture readign the dirveion of new DNA synthesis during relication
THe leading strand DNA polymerase synthesizes DNA in the same direction that the replication fork is moving
What are the 4 cofactors of alpha-ketoacid dehydrogenase
TPP, coA, NAD, FAD
The most abundant sequences in the human genome are
TRANSPOSABLE ELEMENTS - more transposable elements and repeats occur in more complex organisms
Which of the following are features of the wobble hypothesis? A) A naturally occurring tRNA exists in yeast that can read both arginine and lysine codons. B) A tRNA can recognize only one codon. C) Some tRNAs can recognize codons that specify two different amino acids if both are nonpolar. D) The "wobble" occurs only in the first base of the anticodon. E) The "wobble" occurs only in the third base of the anticodon.
The "wobble" occurs only in the first base of the anticodon.
Provide four major tools for combinatorial synthesis and two examples of each type
a. Heterologous hosts- e coli, streptomyces coelicolor b. Genome mining tools- conserved PKS & NRPS regions c. Synthetic Biology Tools- synthons and ligation by survival d. Protein Engineering Tools- Structure Bases design and Directed evolution
Using STRs to follow human history
The Y chromosome does not undergo recombination, so related males inherit identical STR alleles - haplotypes -Y chromosome is directly inherited from father to son - less genetic variability in Y chromosome than autosomal chromosomes (not sex) -even 2 unrelated males may have the same Y profile if they share a distant ancestor
White colonies carry a recombinant DNA plasmid Blue ones do not
The cells making beta G turn blue, and if they don't, they're white. The white colonies contain a plasmid with an insertion ability. The blue ones just have the plasmids by themselves. -Original plasmid has lac-z gene to make x-gal and form blue colonies > multiple cloning site is cut > DNA cut with the same restriction enzyme is added and ligased to plasmid > DNA frag inserted anywhere in multiple cloning site, the lac Z gene is disrupted and cannot make beta G > recombinant plasmid cannot metabolize x-gal and will form white colonies
What enzyme from fatty acid biosynthesis by competing with what enzyme regulate global histone acetylation
The enzyme is acetyle-coA carboxylase. It competes with HATs
Output of a DNA sequencing reaction using fluorescently labeled bases
The fluorescent tags on the detector and signal system: as you get to longer pieces the peaks go down and the widths become wider and then you can't distinguish the peaks anymore ***ALL OF THIS TECHNOLOGY CAME OUT OF HUMAN GENOME PROJECT
Name one critical parameter in analyzing activity and provide 2 common methods in analyzing in normal enzyme
The parameter is specific activity. The two common methods are SDS-PAGE and N-terminal sequencing
What is a fusion protein?
The product of two or more coding sequences from different genes that have been in-frame cloned together. The expression of this hybrid construct generates a single polypeptide
Name the enzyme that provides the cofactor for 11betaHSD-1 in vivo?
a. Hexose 6 phosphate dehydrogenase
Name 2 protein or protein complex...2 enzymes and 2 cofactor essential for circadian cycle metabolism:
The two protein complexes are clock/bmal1 and cry/per. The enzymes are SIRT1 and AMPK with LKB. The cofactors are AMP and NAD.
Whats interesting? Why do bacteria have them all?
These are sequences that cut DNA so youd expect it to cut its own DNA which would be lethal, but it makes restriction enzymes and a corresponding methlyase at the same time, which methylates the other strand which blocks cutting. This doesn't happen in humans, so these enzymes are for the bacteria's immune system. Foreign DNA is moved around and if the cell has the restiction enzyme it can cut the foreign DNA up and protect itself from being genetically modified. In the labs- we get rid of this function so DNA can be cloned
When is the microarray treated with streptavidin that is bound to the fluorescent protein phycoerythrin?
after hybridization
Why is a high selectivity- greater than 100- for 11betaHSD1 vs 11betaHSD2 critical for 11betaHSD1 inhibitor optimization in the drug recovery
a. High selectivity over 100- then cortisol will stay away from MR and there is no hypertension.
Southern Blot of DNA from 4 Species: Human, mouse, hamster, and cow -Cut with EcoRI or HindIII or PstI -Probed with a fragment of the human cystic fibrosis gene
This is called a zoo block - they use animal DNA and probed it with the human cystic fibrosis gene to see if it would hybridize - aka is their DNA complementary to our DNA sequence for cystic fibrosis gene? -The cow showed some hybridization to the human gene, yet not in the same places as the human -The mouse and hamster don't have any hybridization - showing a bigger evolution between us and hamsters/mouse than us and cows -When you have a piece of DNA from 1 species and hybridize it with another species the sequences aren't the same, so hybridization isn't the same
what are the five most important enzyme catalyzed reactions for application of organic synthesis.
a. Hyrolysis/esterification/amidation of ester/amide/amine/alcohol resolution b. Reduction of ketone to alcohol c. Conversion of keto acid to amino acid d. Hydroxylation e. C-C bond forming reaction
Combinatorial biosynthesis of what two pathways has generated indolocarbazole derivative with improve property for treating what three type of diseases
a. Rebeccamycin and staurosporine b. Cancer, neurodegenerative disorders and diabetes
Combinatorial biosynthesis was used to synthesize novel carotenoids with power antioxidant properties by co-expression in E coli of what six enzymes?
Three carotenoid desaturases, a carotenoid hydratase, cyclase, hydroxylase
What two enzymes are involved in producing initial TDP sugar
Thymidylyl transferase and 4,6- dehydratase
What plant cell DNA-delivery method is an excellent and highly effective system that is limited to a few kinds of plants?
Ti plasmid mediated gene transfer
Where is the opine catabolism gene located?
Ti plasmid not part of T DNA region
Why do large DNA fragments need to be cloned?
To ensure all genes encoding proteins in a pathway are captured
Human insulin (humulin) was the first human protein to be produced in bacteria using recombinant DNA
To make recombinant human insulin... -mature insulin in made of 2 polypeptide chains: A (21 amino acids) and B (60 amino acids) -synthetic genes to make A and B were made by oligonucleotide synthesis (63 nucleotides for A and 90 for B) -These oligonucleotide sequences are inserted into separate vectors at the tail end of a cloned E. coli lacZ gene -The recombinant plasmids were transformed into E. coli host cells, where the B-gal/insulin fusion protein was synthesized and accumulated in cells -B-gal/insulin fusion proteins were extracted from host cells and purified -insulin chains were released from B-gal by treating with cyanogen bromide -insulin subunits were purified and mixed to produce a functional insulin molecule
What is functional genomics? What does it measure? What is the aim to find? What are methods? What is needed before doing the experiment?
Transcriptomics Transcription patterns. Measure qualitative- which genes are expressed Measures quantitative- change in transcription levels Aim of gene expression studies is to discover genes that are up and down regulated under certain conditions DNA microarrays Serial Analysis of Gene Expression (SAGE) A norm to compare it to
_______are produced when DNA from another species is inserted into the genome of an organism which then begins to produce the protein encoded on the recombinant DNA
Transgenic Organism
In 1974, Stanley Cohen and Herbert Boyer inserted a gene from an African clawed from into a bacterium. The bacterium produced the protein coded for by the inserted frog gene. The bacterium containing functional frog DNA would be classified as a ______
Transgenic organism
___ are produced when DNA from another species is inserted into the genome of an organism, which then begins to produce the protein encoded on the recombinant DNA.
Transgenic organisms
Why would relaxed samples migrate faster in gel
Treatment w/ ethidium relaxed the negatively supercoiled cccDNA and positively supercoiled the relaxed cccDNA
What 2 different detergents used for recombinant enzyme or peptide/lipid substrate
Triton X-100 and Tween80
1.The linking number (Lk) of a closed-circular DNA molecule can be changed only by breaking one or both strands.
True
You have benefited from SELECTIVE BREEDING by having more agricultural produce than would have been possible otherwise.
True
What are the 2 amino acids that contribute to highest level of absorbance at 280 nm or 2800 A
Tryptophan and tyrosine
What is an isomeric enzyme?
Two different enzymes that perform the same function Use transcriptomics to differentiate
Beta beta carotene provided a reduced lipid storage capacticity of a liposide, therefore decreasing what two metabolic disorders
Type 2 diabetes and obesity
What kind of restriction endonuclease is N1aIII?
Type II
What kind of restriction endonuclease is BsmFI?
Type IIs
What stop codons end the elongation process?
UAA, UAG, UGA
Prenatal Testing: Amniocentesis ASO or FISH can be used for genetic diseases
Use amniotic fluid and obtain the fluid and the fetal cells from it - culture this on a pea tree dish and analyze it using recombinant DNA methods for looking at genetic and biochemical analysis -you can do it before insemination if you couple it with in vitro fertilization
If the isoelectric point of an enzyme is 5 and the enzyme is stable in pH 5-8, then the enzyme can be purified by what exchange chromatography? Using a buffer and what pH
Using isoelectric focusing precipitation. Use a pH higher than 5 you can use (like 6 or 7).
A gene gun and a virus may both be classified as______ because they are mechanisms by which foreign DNA may be transferred into a host cell
Vectors
What is catabolite repression?
When a bacteria chooses to eat the most energy efficient substance first
What is the physiological function of 11 beta HSD-1 and what is the cofactor
a. Reductase b. NADPH
11. It is possible to convert the Cys that is a part of Cys-tRNACys to Ala by a catalytic reduction. If the resulting Ala-tRNACys were added to a mixture of (1) ribosomes, (2) all the other tRNAs and amino acids, (3) all of the cofactors and enzymes needed to make protein in vitro, and (4) mRNA for hemoglobin, where in the newly synthesized hemoglobin would the Ala from Ala-tRNACys be incorporated? A) Nowhere; this is the equivalent of a nonsense mutation. B) Wherever Ala normally occurs C) Wherever Cys normally occurs D) Wherever either Ala or Cys normally occurs E) Wherever the dipeptide Ala-Cys normally occurs
Wherever Cys normally occurs
What's another way to determine the abundance of proteins?
With tagging dyes
What is the genotype in the bottom left-hand quadrant in Figure 10-6?
Ww
List 4 simple methods for determining a change in protein or enzyme conformation
X-ray crystallography methods Fluorescence methods Thermal activation methods: expandase and hydroxylase are two example enzymes Chemical modification methods: expandase and hydroxylase are two example enzymes
Proteomics is protein-protein interaction. Name 2 common methods:
Yeast 2-hybrid and APMS
Tissue culture allow us to grow entire plants from fragments of stems, roots or leaves
You can genetically modify plants in tissue culture > make genetic modification of cells and take these cells to make a plant -make pieces of plant by manipulating culture conditions > you make roots and put them in soil and they'll grow **Easiest to make genetic modification is in tissue culture
Why is keeping the gene for your protein of interest repressed until the cell numbers are higher important?
You can get more proteins expressed if there are more cells, and so that the protein of interest won't be lost or destroyed because it's foreign and/or be used yet until there are higher numbers
What is the first principle of directed evolution
You get what you screen for
Enoate reductase has been used in producing what type of commercially important compounds, producing pharmaceutical intermediate for drug candidate of what three diseases
a Carotenoids b. Non-insulin dependent diabetes, osteoporosis, Alzheimer
What are the important features of plasmid cloning vectors?
a choice of unique (single) restriction endonuclease recognition sites which the insert DNA can be clones and one or more selectable genetic markers for identifying recipient cells that carry cloning vector insert DNA construct
What do transformed genes have that makes them resistant to an antibiotic?
a gene as part of the vector that confers resistrance
What is the primary impediment to the release of a secretory protein?
a membrane
List three reasons as rationale why 11 beta HSD-1 is a prime target for metabolic syndrome and diseases?
a. Adipose specific overexpression of 11betaHSD1 can drive a complete metabolic syndrome phenotype b. Adipose specific overexpression of HSD1 can protect against the metabolic response to HF feeding c. Targeted deletion of 11betaHSD1 locus or small molecule inhibition of the enzyme protects against diet-induced obesity, glucose/insulin intolerance and hyperlidemia
In prokaryotes, what is responsible for the transcription of all types of RNA?
a single RNA polymerase
What is screening by protein activity used for?
a target gene that produces an enzyme not normally made by host cell
Gene Families Evolve by Gene Duplication followed by Sequence Drift
a-globin versus b-globin 50% sequence similarity d-globin versus b-globin 93% same (d is on beta chain) -The globin genes (myoglobin + hemoglobin (a/b)) - the relationships between them and the points at which the gene duplication occurs - based on sequence similarities -You get the timeline by figuring out when the gene gets an amino acid substitution (rate of mutation assuming its constant) and another way is to use the fossil record, knowing where different groups diverged from each other to see what alpha genes they do and don't have -We think these dates are accurate, divergences in gene families don't occur often but over time
What is a three step strategy of CLIPS-O or high throughput screening and for identifying what two kind of enzymes?
a. 1. Design the enzyme substrate spacer and reporter. 2. The enzyme from metagenomic clone convert the substrate to a stable intermediate. 3. Chemical oxidation convert the stable intermediate to the signal from the reporter b. lipase and epoxide hydrolase
Provide 5 major steps for construction and functional screening of metagenomic DNA library from soil
a. 1. Extract genomic DNA b. 2. Prepare DNA and vector for cloning c. 3. Ligation and transformation into host bacteria d. 4. Expression of genes from DNA insert e. 5. Screen for phenotypic change as a result of genes from the metagenomic DNA insert
Provide three major strategies for metabolic pathway and net work optimization
a. 1. Optimize the catalytic/thermodynamic properties of specific metabolic enzymes b. 2. After non-coding regions and manipulate non-metabolic gene functions to optimally adapt a heterologous pathway to its host environment. c. Inactivation of competing pathways and/or over-expression of precursor pathways
ECB deacylase can catalyze side chain removal from what two types of cyclic peptides
a. 6 amino acid cyclic peptide b. 10 amino acid cyclic peptide
provide the enzymatic differentiation between reduction between methyl and methyl ketone and trifluor ketone. Using adh and kred and indicate what selective reduction
a. ADH- methyl- s isomer b. Kred 1, 2- trifluoro- R isomer c. Kred 3- trifluoro- S isomer
For deptomycin biosynthesis by NRPS, what is the enzyme for fatty acid activation, what is the protein for fatty acid binding
a. AL- acyl CoA ligase b. ACD- acyl carrier protein
In addition to AD, gamma secretase has been implicated in what other human disease, and the other disease is associated with mutations in what three proteins of gamma secretase
a. Acne inversa- skin disorder b. Pen2, PS1, NCT
Which enzyme exhibit group stereo selectivity and group selectivity?
a. Adh vs kred 1,2,3 for group. b. Kred 1, 2 vs kred 3 for stereo selectivity
Provide three anti-bacterial natural products and two anti tumor products
a. Anti-bacterial- tylosin, erythromycin, vancomycin b. Anti-tumor- staurosporine, rebeccamycin
Provide three methods for diversity generation and for each describe how it is used for improving what two enzyme properties.
a. Arational methods. Use saturation mutagenesis of the whole gene. Goal to improve thermo stability and targeting active site of the enzyme for specificity and selectivity b. Semi-rational methods. Use phylogenic studies from related proteins to restrict search for beneficial stability/activity diversity into manageable libraries. c. Rational methods. Use evolutionary or computational analyses for site directed or site saturation mutagenesis to improve stability or specificity/selectivity of an enzyme
What kind, what number, of selectivity of inhibitor for 11betaHSD-1 and HSD-2
a. At least 100 fold- why- inhibitor does not inihibit HSD-2, if doesn't inhibit, will not cause hypertension
Sitagliptin-enzyme-technology-disease-mechanism
a. Biocatalyst: transaminase b. Technology: stucture base design c. Treatment: diabetes d. Mechanism: DPP4 inhibitor
Two enzymes are indistinguishable in what two biochemical and pharachological properties, therefore suggesting the transgenic APP mouse model should be useful in the development of base inhibitor for treatment of AD
a. Biochemical property is the catalytic efficiency in beta and beta' cleavage
Provide 5 criteria for metabolic syndrome and including the number and unit for each
a. Central obesity: Waist Circumference- men>40in women>35in b. Fasting blood glucose: greater than or equal to 110mg/dL c. Fasting triglycerides: greater than or equal to 150 mg/dL d. Fasting HDL Cholesterol: men- <40 mg/dL womaen <50 mg/dL e. Blood pressure: > or = syst
What is the two step bases for 11betaHSD1 reductase assay by LCM/MS
a. Chromatographic separation b. Mass differentiation
Each of three functional screen using a chromogenic substrate, a fluorogenic substrate, and a catabolic substrate, provide one substrate, one recombinant enzyme discovered and its phenotype
a. Chromogenic: substrate- X gal, b galactosidase, blue colony b. Fluorogenic: substrate- MU-DACB, chitinase, fluorescence c. Catabolics: substrate- catechol, catechol dioxygenase, yellow culture
What gene were combined by combinatorial biosynthesis in what heterologous host to produce what novel indolocabazole derivative
a. Combining genes from different microorganisms including those with staurosporine plus Rebeccamycin b. Heterologous host is streptomyces albus
What is the plot for competitive and uncompetitive- draw the plot
a. Competitive- parallel lines b. Uncompetive- y-intercept
Provide a selection based screen, for each of the 5 screen designs, provide resulting recombinant enzyme
a. Compound resistance: B lactam resistance- B lactamase, growth b. nitrogen or Carbon source- nitriles-nitrilase c. Host auxotrophy- amide linked leucine derivatives- amidase d. Engineered catabolic selection- glycerol- glycerol dehydrogenase e. Sigex- benzoate and naphthalene, naphthalene dioxygenase
How 11 beta HSD-1 play a pivotal role in the liver
a. Convert cortisone to cortisol b. Cortisol activates GR for increasing gluconeogenesis
What is the physiological function of 11 beta HSD-2 and what is the cofactor?
a. Dehydrogenase b. NAD
Provide three industrial applications and two enzymes for each.
a. Detergent and food processing- lipases/esterases and proteases b. Industrial processing- cellulase and amylases c. Biosynthesis of fine bulk chemicals- nitrilase and glycerol dehydratase
Drug discovery and development such two approaches can lead to six improved property of biocatalyst and catalyst
a. Enzymatic activities, binding efficacy of receptors, protein solubility, thermo stability, shelf life, regio and enantio specificity
Provide one enzyme and one protein involved in amyloid independent pathway to AD
a. Enzyme- PS1; protein- APP
Other than Tau, provide enzyme and one protein involved in amyloid dependent and amyloid independent pathway to AD
a. Enzyme- cdk5; protein ApoE4
Provide two enzymes and one protein and three compounds in lowering Abeta and provide the mechanism of each
a. Enzyme-Fbx2, mech- BACE 1 degradation b. Enzyme- NEP, mech- a beta peptide degradation c. Protein is prion, mech is BACE1 maturation and transport d. Compound 9- inhibition of atase 1 and 2 e. Ursolic acid- competitive binding to Abeta receptor f. Compound D737- inhibition of Abeta aggregation
Telaprevir, enzyme-creation technology-what disease-what mechanism
a. Enzyme: amine oxidase b. Creation technology: gene shuffling methods and PROSAR c. Treating what disease: hep C d. What mechanism: serine protease inhibitor
From the visceral fat, how 11beta HSD-1 contribute to glucose production
a. Fat contain a lot of elevated level of HSD-1, so HSD-1 in the fat convert cortisone to cortisol, high level of cortisol in the fat then get delivered to the liver and subsequently activate GR for glucose production
From the extreme thermophile, provide two enzymes for what biotech application and another two enzymes for what application
a. Food and detergent processing- lipases and proteases b. Industrial processing- xylanases, cellulases
Provide the 5 step principle for shuffling
a. Fragmentation, denaturation, hybridization to templates, overlaps elimination, ligation with a ligase
What two pathway are involved in posttranslational modification
a. Gluconeogenesis b. Oxidative PPP
Cancer cell make cope with oxidative stress thru glycoslylaion of what metabolic enzyme
a. Glycosylation of all GLC and AC b. Or glycosylation of specific sugar at serine site
V nitrogenase catalyzes what two reactions of synthesis in nature and has what two biotech applicationsi. What synthesis? What hydrocarbon formation f. Hydrocarbon formation by two enzymes was changed by what engineering
a. HB and FT syntheses b. Fertilizer and fuel production c. Function- carbon fixation d. Enzyme evolved toward nitrogen fixation after it had carbon fixation --photosynthesis ---i. C2H4 i. Reaction engineering
How is the selectivity of 11betaHSD1 against 11betaHSD2 defined?
a. HSD2-ki/HSD1-ki
From GalK engineering, each approach generated what enzyme mutant with what improved enzyme property
a. Increased substrate promiscuity- mutant: 1 amino substitution produced by directed evolution b. Structure based design produced two enzyme mutant. 1 single aa mutation and the other was a double enzyme mutation.
A selective inhibitor of 11 beta HSD-1 treat what four metabolic disorders?
a. Insulin resistance b. Obesity c. Cognition and dementia d. Cardiovascular abnormality
Engineering of plant that fix nitrogen and make their own fertilizer involved what enzyme complex
a. Iron-sulfur-molybdenum nitrogenase complex b. Cofactor: Mo-(Fe-S)-C
What precursor was optimized to increase production of what caratenoid
a. Isopentenyl diphosphate-IPP to increase production of lycopene
What is the equation for the inhibition constant ki?
a. Ki=EC50/(1+[S]/Km)
How dynamic resolution differ from kinetic resolution in the theoretical yield in producing one stereospecific isomerb. Provide two kinetic resolution enzymes, each of which involved in generating what pharmaceutical agent
a. Kinetic- 50% of desired enantiomer, dynamic- 100% i. Lipase-taxol and hydrolase- HCR inhibitor
Provide one kinetic observation and one enzyme observation suggesting oleoyl acp is the physiological substrate for acyl acp reductase
a. Kinetic: ratio of oleoyl acp/oleoyl-coA... catalytic efficiency is much higher for oleoyl acp b. Enzyme observation: acyl CoA synthetase-if deleted still get same hydrocarbon made, suggesting acyl coA is not the major physiological substrate and there for oleoyl acp is the major substrate
What are the three parameters demonstrating environmental friendliness or green chemistry of this biocatalytic route
a. Less solvent-6 b. Solvents used- environmental friendly- isopropanol, water toluene c. Biodegradable enzyme
Provide 5 enzymes, each for a different application, identify using functional metagenomic
a. Lipase/esterase b. Polysaccaharide hydrolysis: cellulase, amylase c. Oxygenase: polyphenol oxidase d. AHL-mediated quorum sensing activation and inhibition- AHL synthase, AHL lactonase e. Other: protease
For the Metrex Biosensor screen based on AHL- mediated Quorum sensing, first what is metrex? What are 3 DNA component of the plasmid reporter? From a metagenomic clone, describe how is activation sensed used to discover what enzyme?
a. Metrix is- metabolite regulated gene expression b. Lux R gene, lux promoter, GFP gene c.The enzyme makes AHL as a product, and then the product AHL binds to the LUXr, causing a conformational change, and then luxR activates the Lux promoter, leading to expression of GFP. Enzyme detected-AHL synthase
How did deracemisation of secondary alcohol compound can be carried out via concurrent oxidation reduction using isolated enzymes with cofactor and stereo preference. Answer include two main enzymes and two coupled enzymes and also the function of each enzymatic step
a. Nadph-specific R selective ADH, coupled enzyme: Nadph oxidase. Co-substrate: oxygen b. Nadph specific S selective ADH, couple enzyme: FDH. Cosubstrate is formate c. Function of first step- oxidation. Second step is reduction
For AD, exercise specifically rescues HFD induced deterioration of what enzyme activity therefore decreasing what accumulation
a. Neprilysin activity b. Decreasing Abeta
What is the kinetic mechanism of 11betaHSD-1
a. Order bi-bi- NADPH binds to the enzyme first and NADP released last
According to the proposed catalytic mechanism of enoate reductses for a symmetric reduction of alkane. What is the half reaction. c. What are the three amino acids interacting with the enzymes at the active site
a. Oxidative half reaction: FlavinH2 to Flavin for alkene reduction b. Reductive half reaction: Flavin to FlavinH2 for cofactor regeration i. Asn, His, Tyr
4. Overexpression of what two enzymes in nerve cells confer resistance to what peptide
a. PDK1 and LDHA b. A beta
Glucocorticoid stimulate what two enzymes and therefore increasing gluconeogenesis?
a. PEPCK, G6Pase
Gamma secretase is a multi protein complex of what five proteins and what are the function of each protein
a. PS1- catalytic core b. PS2- catalytic core c. Pen2- cofactor d. Nct- cofactor e. Aph1-cofactor
Overexpression of what protein complex lead to what Abeta42 over Abeta40 ratio
a. Pen2-nyc is overexpressed b. Leads to increased Abeta42:Abeta40 ration
How was each FRET peptide designed or FreT assay conducted for each enzyme using each peptide and measuring fluorescent change
a. Peptide design: peptide must contain beta and beta prime cleavage sites b. Assay conducted: BACE-cleavage of the peptide, releasing product containing fluorophore; therefore increasing fluorescence of the product.
Define phenomic, and provide one advantage of this technology
a. Phenomic- acellular in vitro technology, advantage- overcome regulation of transcription.
Provide two gate keeping enzymes for in vitro evolution and 5 tailoring enzymes together for producing novel products
a. Phytoene desaturase, lycopene cyclase- gate keeping b. monooxygenase, 2 oxygenases, desaturase, hydroxylase
provide two techniques or programs to create gene library by in vitro gene recommendation
a. ProSar and DOGS
In comparison to naturally produced peptides such as insulin, provide three unique features about deptomycin, one for its biosynthesis and two for its chemical structure
a. Produced by non ribosomal machinery b. Chemical structure: fatty acid side chain and 6 not natural amino acids
Provide three major applications for pathway for in vivo evolution and one example for each
a. Production of novel natural products-polyketide antibiotic b. Degradation of toxic organic compounds- pentachloropenol c. Industrial production of small molecules- lactate
Provide three enzymes that catalyze carbon carbon bond forming reaction. nobel prize. who and what year.
a. Pyruvate decarboxylase, DERA, hydroxynitrile lyase i. Nobel prize- 2010 and Negishi and suzuki.
Provide two strategies for cofactor regeneration or recycling, each with an enzyme and what cosubstrate involved.
a. S- coupled: ketoreductase, isopropanol as co-substrate b. E- coupled: ketoreductase, and GDH as co-substrate
Provide one scientific rationale and one medical rationale why the two secretases are important drug targets for Alzheimer disease
a. Scientific rationale- oligomerization- secretases allow for production of beta-amyloid peptide b. Medical rationale- age is the greatest risk factor for AD
Provide six modifiable risk factors for alzheimer disease in the US and provide one compound, one sport, and one food type that may help prevent or delay AD
a. Sport- table tennis b. Food type- green tea c. One compound- resveratrol d. Physical inactivity, depression, smoking, midlife hypertension, midlife obesity, low education and mental activity, diabetes
Provide three different types of environmental sample use for developing new biocatalyst
a. Strains isolation b. Microbial enrichment and DNA extraction c. Total DNA extraction
Provide three combinatorial biosynthesis strategy for deptomycin and af145 and one resulting efficacy observation. Molecular engineering combinatorial biosynthesis can successfully modify what kind of property of complex lipopeptide antibiotics
a. Subunit exchanges, module exchanges, domain exchange and fusion b. Improve efficacy- improve s aureus MIC\ Antibacterial
For erythromycin biosynthesis by a modular polyketide synthase, first how many subunits, how many modular and many total domains involved. b. What substrate and how many molecules each
a. Subunits: 3, modules: 6, domains 29; Propionyl-coA: 1 Methyl-malonyl-CoA: 6
What resin was used in purification in human BACE1 FC and murin BACE1 FC? what is the binding bases
a. The resin is protein A- agorose b. Binding bases is affinity between hu/muBACE1:FC and protein A
Gamma secretase catalyzes which three cleavage type from CTF99 to generate Abeta42 and Abeta40
a. Three cleavages: epsilon, zeta, gamma
Provide three enzymes involved in generating Crixivan for AIDS
a. Toluene dioxygenase, epoxide hydrolase, amidase i. Protease inhibitor
Opposite way, provide three enzymes, one critical partner, and one modified Abeta involved in increasing Abeta or AD and provide mechanism of each
a. Two enzymes- Atase 1 and 2- mech is acitivation of BACE1 b. Third enzyme- glutaminyl cyclase- producing pyroglutamate Abeta c. Partner- Tau- mech- axonal transport d. Modified Abeta- pyroglutamate Abeta; mech is rapid Abeta oligomer formation
What two enzymes are widespread in what bacteria for converting for converting what intermediate of fatty acid metabolism to what two hydrocarbon for potential fuel production
a. Two enzymes: acyl-acyl carrier protein reductase and aldehyde decarbonylase b. Comes from cyanobacteria c. For converting fatty acyl acp to fatty aldehyde to produce alkane and alkenes.
Two enzymes human BACE1: FC and murine BACE1:FC that catalyze beta side cleavage between what two amino acids from humban beta side mutant peptide and from wildtype peptide
a. WT: KM b. Mutant: LD
What is the Hanes-wolf plot?
a. Y axis is [S]/V b. X axis- [S]
Provide 6 types of reductase transformation catalyzed by old yellow enzyme family
a. alkene reduction, alkyne reduction, disproportionation, nitro ester reduction, nitro-group reduction, reduction of aromatics b. alkene reduction is most important.
for an inhibitor modes what are the sites for
a. competitive- active site b. uncompetitive- binding to ES c. noncompetitive- allosteric site or active-site overlapping
using a DI plot how do they relate
a. competitive- intercept on y axis b. uncompetive- parallel c. noncompetive- negative x axis
what is the three step principle of adrenaline test and also provide two enzymes from this test
a. design a suitable substrate with acetyl group. enzyme to convert substrate to diol. Thiol reacts with sodium peroxide using no color product. Adrenaline reacts with the remaining sodium peroxide producing adrenochrome with the wavelength maximum of 490nm. d. Enzymes- lipases and epoxide hydrolase
three step rule in developing an enzymatic process, the production of designing a pharmaceutical intermediate
a. design the ideal process, make it "green" by design, create the enzyme that enables this processs
pathway engineering of methymycin and pikromycin (macrolides) was based on what two strategies for glycodiversification in S Venezuela
a. first is: four mutated enzyme for sugar modification use b. two glycosyltransferases has broad substrate specificity
Provide 5 activity assays for 11betaHSD1 programming including enzyme function, kinetic purpose and methodology
a. hHSD1-dehydrogenase assay: IC50 by NADPH fluorescence b. hHSD1- reductase assay: IC50 by cell-based cortisol FRET c. hHSD1- reductase assay: Ki & MOA by LC/MS & HPLC d. hHSD2- dehydrogenase assay: Ki/selectivity by LC/MS & HPLC e. mHSD1- reductase assay: Ki & mouse model by HPLC & LC/MS
what two kinetic constants and what isomer parameters for the product
a. kcat and km b. diostereo specificity
briefly describe the three step method for Evosight
a. optimize the mutation load, apply l shuffling, and independent benefic mutations to optimize a particular enzyme or function system
what three biochemical properties were included in comparing DMOMT, MOMT, TR and how do we use these properties
a. physical, catalytic and kinetic b. ph optimum is more alkaline for the MOMT, DMOMT than the ph for tylosin reductase. DMOMT and MOMT require metal c. km is very low for DMOMT and MOMT-narrows substrate specificity, they are pathway specific
provide four potential benefits of neutral drift and adaptive evolution
a. polymorphisms, thermodynamic stability, mutational robustness, protein evolvability
directed evolution can be used on the enzyme level to improve what five enzymatic properties
a. substrate specificity, increase substrate promiscuity, create new catalytic functions, alter the pH range, increase thermal stability
Provide 6 scientific discipline needed for chemoenzymatic process
a. synthetic organic chemistry, biocatalysis, biochemistry, microbiology, genetic engineering and fermentation, and chemical and biochemical technologies.
one valuable in vitro glycodiversificaiton strategy involve a three enzyme procedure or two enzyme approach. Provide three enzyme and two enzyme respectively
a. thee enzymes: anomeric kinase, nucleotidyl transferase, glycosyl transferase b. two enzymes: two different glycosyl transferases
What genes are used for positive selection (spectinomycin and streptomycin resistance)?
aadA (aminoglycoside 3'-adenylyltransferase
Wind, humidity, and rocks are all ___ in a terrestrial ecosystem.
abiotic factors
What does the number of times each tag is sequenced represent?
abundance in initial sample
What plant phenolic compounds do bacterium respond to in wounded plants?
acetosyringone and hydroxysyringone
A cell moves particles from a region of lesser concentration to a region of greater concentration by ___.
active transport
What is peptide bond formation exclusively catalyzed by?
activity associated with large rRNA
What does BsmFI digestion release?
adaptor tag molecules from beads into solution to be recovered
What is ligated to the ends of cDNA molecules to facilitate cloning of the cDNAs into a vector?
adaptors with extensions for restriction endonuclease recognition sequences
How can condensed regions of chromatin be relaxed?
addition of an acetyl group to amino acids in the packaging of proteins or methyl groups to nucleotide sequences where proteins bind
What can both tac and trc promoters be induced by?
addition of lactose or IPTG to growth medium
What is the purpose of the gene tmr?
adds isopentenyl side chain to 5' AMP for synthesis of cytokinin
What does eukaryotic mRNA get polymerized with?
adenine nucleotide at 3' end
What is the consequence of a strong promoter that has a high affinity for RNA polymerase?
adjacent downstream region is frequently transcribed
substrate selection for new enzyme is based on what three criteria
affinity, reaction rate, detection method
Are most genes located with the T-DNA region activated before or after the T-DNA is inserted into the plant genome?
after
When does the tRNA become charged?
after binding of amino acid to its tRNA
What does the promoter tagging vector approach rely on?
agrobacterium mediated Ti plasmid transformation system
When setting out to transform a new plant species what is necessary to determine?
agrobacterium strain and Ti plasmid best suited for that plant
What is the purpose of the 5' cap and 3' tail?
aid in binding of ribosomes to mRNA to begin translation and increase stability
What enzyme prevents self ligation?
alkaline phosphatase
What enzyme removes the 5' phosphate groups of DNA molecules?
alkaline phosphatase
The Human Genome Project may make use of which of the following to diagnose genetic disorders before birth?
all of the above
The causes of cancer may include which of the following?
all of the above
What would be the result of the test cross in Figure 13-4 if the unknown were homozygous long ears?
all of the offspring would have long ears
What would be the result of the test cross in fig 13-4 of the unknown were homozygous long ears
all of the offspring would have long ears
A technique that may be employed in the Human Genome Project is ___.
all of these
Which of the following are applications of genetic engineering?
all of these
Genes located on homologous chromosomes may have alternate forms that control different forms of a trait.
alleles
What does SAGE principally detect for?
all transcripts in a sample
In comparison to conventional breeding, what advantages does genetic engineering present?
allows direct transfer of one or a few genes between close or distant related organisms, crop improvement achieved in short time period
How are two different mRNAs produced?
alternative splicing of exons
Structure III in Figure 11-1 represents a(n) ___.
amino acid
Provide one enzyme involved in each of the two steps in producing the sterospecific isomer used as what modulator in treating diabetes
amino acid oxidase and amino transferase. GLP1 modulator.
What property distinguises a protein that remains in the cytoplasm from one that is secreted?
amino acid sequence at its N terminus
What group is free in the first amino acid of a protein?
amino group, N terminus
What can be expressed to lower plant ethylene levels?
aminocyclopropane-1-carboxylate (ACC)
What type of plastid contains starch grains?
amyloplasts
What are some specific plastids?
amyloplasts, chloroplasts, elaioplasts, chromoplasts
The gamete that contains genes contributed only by the mother is ___.
an egg
What is IPTG?
an inducer of the lac operon which aids in the transcription and translation of the lacZ gene in the plasmid and prevents binding of the lacI repressor gene
The chromosomes shown in Figure 8-6 are in which state of mitosis?
anaphase
The phase of mitosis in which the sister chromatids separate from each other is
anaphase
The Neanderthal Genome has been sequenced
ancestors evolve into neanderthals 350,000-500,000 years ago - and they died out 30,000 years ago and modern homo sapiens take over -All non-African modern humans have 2-4% Neanderthal DNA sequences
In the nomenclature developed for SAGE, what is N1aIII called?
anchoring enzyme
Heparan sulfate congugate have what three potential pharmaceutical applications
anti viral, anti cancer, or anti coagulant.
The _______ in a tRNA molecule is designed to base-pair with a complementary sequence of three nucleotides, the ___________ in an mRNA molecule.
anti-codon; codon
What kind of selectable marker is usually used when introducing foreign DNA into the chloroplast genome?
antibiotic resistance gene flanked by specific chloroplast DNA sequences
What sequence plays a crucial role in the formation of the linear array of amino acids that constitute a protein?
anticodon sequence
How was the first genetically modified food enhanced?
antisense technology suppressed polygalacturonase gene expression to prevent fruit ripening
What plants have researchers reported the complete DNA sequences for?
arabidopsis thaliana, rice, poplar
How is ethylene produced?
as a consequence of agrobacterium infection of plants
How is transcription at the whole genome level assessed?
as a function of clinical conditions, mutations, natural or toxic agents, cells or tissues, cell division, organism development
What is the only way that a cloning vector can be maintained in A. tumefaciens?
as part of a cointegrate structure
What is the affinity of CAP for its binding site on DNA enhanced by?
association with cAMP
In which part of the cell does this process shown in Figure 11-1 take place?
at the ribosomes
Describe the synthesis of cytokinins.
attachment of isopentenyl moiety from isopentenyl disphosphate (IPP) to 5' AMP by isopentenyltransferase to isopentenyl adenosine monophosphate (IPA)
What is the initial step in the infection process of crown gall tumors?
attachmet of A. tumefacions at site of open wound usually at base of stem
What method can be used to study the resolution of fragments?
autoradiography or fluorography
manufactures food using energy from the sun or from chemical compounds
autotroph
What is an important component of a successful chloroplast transformation system?
availability of suitable selectable marker and reporter genes
How 11 beta HSD-2 play a protective role in kidney
b. Cortisone has low affinity for MR, so MR remains functional-normal hypertension. If 11beta HSD-2 is inhibited, low activity in the kidney, then the cortisol will remain and compete with the native ligand for MR. When cortisol binds to MR it messes up the conformation of MR and leads to secretion of sodium to the blood, causing hypertension
to derive x intercept and y intercept in competitive inhibition
b. y int- 1/v=1/V
Where does the 5'-3' phosphate and deoxyribose components lie?
backbone of molecule
Where are many reporter genes from?
bacteria
What is T DNA transfer similar to?
bacterial conjugation
What are some derivatives of pCB301 that facilitate its use?
bar gene used with plant promoter and transcription termination region, encoding phosphothricin acetyltransferase inserted into MCS so transformants express this gene to be easily selected
What makes up a nucleotide?
base, sugar pentose ring, phosphate
What are chimeric oligonucleotides designed to have?
bases that don't pair with the endogenous plant DNA sequence so that after microprojectile bombardment, DNA repair enzymes recognize the mismatches between targeted gene and large molar excess of chimeric oligonucleotide
Lyases
catalyze cleavage of C-C, C-O, C-N, and other bonds by other means than by hydrolysis or oxidation. Example is pyruvate decarboxylase
Why is it possible for changed chromosomal DNA in the plant genome to be readily detected phenotypically if the mutation created is dominant or codominantly expressed?
because plants are diploid and only one copy of the genes is changed in this procedure
Where specifically is the EcoRI recognition sequence cut at?
between guanine and adenine on each strand, specifically between oxygen of 3' carbon of the sugar of one nucleotide and phosphate at 5' carbon of adjacent sugar
How can achievement of cloning vectors transferring and integrating the T-DNA region into recipient plant cells be done?
binary vector system or cointegrate vector system
What is the first step in the initiation of transcription of eukaryotic structural genes?
binding of TFIID to TATA sequence
What is transcription from the lac promoter also regulated by?
binding of catabolite activator protein (CAP) or cAMP repressor protein (CRP) to DNA upstream of promoter region
What does a natural transformation process usually entail?
binding of double stranded DNA to cell wall, entry of DNA into periplasm where it is protected from nucleases, transmission of one strand into cytoplasm while other is degraded, and if linear DNA then integration into host chromosome
How is translation initiated in prokaryotes?
binding of small ribosomal subunit to mRNA by base pairing at Shine-Delgarno sequence
In addition to gene expression studes, what else can DNA microarrays be used to determine?
binding sites for DNA binding proteins, sites where transcription of genes starts and stops
What is the term used for the extensive use of computers for storage and analysis of molecular data?
bioinformatics
group formed by several populations
biological community
To explain and show how the amount of living materials at each trophic level of a food chain changes, you could use a pyramid of ___.
biomass
Ecosystems, biotic factors, and abiotic factors make up the ___.
biosphere
In a pond ecosystem, ducks, mosquitoes, pond plants, and frogs are ___ factors.
biotic factors
What does streptovidin specifically bind to?
biotin residues of hybridized cRNA
What color light is needed to fluoresce the cells that have gfp?
blue
Under what conditions are a ditag formed?
blunt end molecule, adaptor A and B tags mixed, T4 DNA ligase
How does peptide bond formation discharge the initiator tRNA?
bond between carboxyl or methionin and tRNA is cleaved to make carboxyl available for peptide formation and so tRNA gets ejected
Which of the following explains why a cell's size is limited?
both a and c
Hydrolases
catalyze hydrolysis of various (like C-O, C-N, C-C) bonds. Example is phospholipase A2
Ligases
catalyze joining of two molecules with concomitant hydrolysis of the pyrophosphate bond in ATP or a similar triphosphate. Example is acetyl-coA synthetase.
Oxidoreductases:
catalyze oxidoreduction reactions with NAD/NADP or FAD/FMN as cofactors. Example is dehydrogenase, 11beta-HSD1)
Transferases
catalyze transfer of a group. Example is serine hydroxymethyltransferase
What does DNA ligase do?
catalyzes formation of phosphodiester bonds at ends of DNA strands that are already held together by base pairing of two extensions and also joins blunt ends when both bound to the enzyme
What happens to effector molecules that block repression?
broken down by cellular activity
How are few cells transformed?
by recircularized plasmid DNA that escaped dephosphorylation by alkaline phosphatase, they acquired ligated and nonligated nonplasmid DNA, or by plasmid insert DNA construct
How is the "intron problem" overcome in eukaryotic genes?
by synthesizing double stranded DNA copies or cDNA of mRNA that lack introns and cloning cDNA into a vector to create a cDNA library
How are the second complementary DNA strands generated?
by treating the heteroduplex of RNA-DNA with RNase H
In SAGE, why is each N1aIII cut site that is closest to the 3' end of a cDNA retained and all unbound fragments washed away?
cDNA are bound to beads
What happens to two samples of mRNA once extracted and cDNA is synthesized?
cDNA labeled with different fluorophores, mixed, hybridized to same microarray, laser determines intensities of color for each probe cell
cDNA = "complementary DNA" made from mRNA
cDNA synthesis - uses Reverse Transcriptase to make a DNA copy of mRNA 1) mix mRNAs with oligo-dT primers (single stranded) and these bind to the poly A tail. oligo primer is (TTTTT) 2) reverse transcriptase extends the oligo-dT primer and makes a complementary DNA copy of the mRNA 4) The RNA part of the hybrid (because now you have one strand of DNA and 1 RNA) is partially digest by RNAse H, the 3 ends of remaining RNA serves as primers for DNA poly 1, to make the second strand of DNA 5) and the opposing strand of DNA is made with DNA poly 1 and ligase to make the final double stranded cDNA molecule
What is the pL promoter controlled by?
cI repressor protein of bacteriophage lambda
How can competence be induced in E. coli?
calcium chloride or heat shock
The uncontrolled division of cells that results in a malignant growth is known as
cancer
What is the purpose of a partial digest?
capture all possible fragment lengths containing GOI
15. Prosthetic groups in the class of proteins known as glycoproteins are composed of: A) carbohydrates. B) flavin nucleotides. C) lipids. D) metals . E) phosphates.
carbohydrates
What source can opines be used as and what gene must the A. tumefaciens carry??
carbon and nitrogen, Ti plasmid borne gene
What parts of amino acids are linked by a peptide bond to form a protein?
carboxyl and amino groups
How is a specific amino acid enzymatically linked to a specific tRNA?
carboxyl end of amino acid to 3' end of tRNA
Wolves would be considered ___.
carnivores
What does SIGEX identify?
catabolic genes that are expressed when promoter actived in presence of substrate
Isomerases
catalyze changes within one molecule. Example is isopenicillin N epimerase.
for the diagnosis of a genetic disorder, many cells are required, but only a few need to be taken from the individual. These cells are grown in _____________so that enough DNA can be obtained to run the necessary tests
cell culture
The Human Genome Project may make use of which of the following to diagnose genetic disorders before birth?
cell cultures, PCR, automated gene sequencers---All of the above
The sequence of growth and division of a cell makes up the
cell cycle
Which series is arranged in order from largest to smallest in size?
cell, nucleus, chromosome, DNA, nucleotide
How does a metagenomic library of soil, mud, or intestine get constructed?
centrifuge, extract DNA, clone to vector, transform e coli with mlibrary, screen for novel genes or operons
Unlike plant cells, animal cells contain ___.
centrioles
A chromatid is attached to a spindle fiber by the ___.
centromere
The structures that hold together sister chromatids are
centromeres
What are some chloroplast genes transcribed by?
chloroplast encoded RNA polymerase plus a nucleus encoded RNA polymerase sigma factor or by just a nucleus encoded RNA polymerase
In order to modify chloroplast functioning or to produce foreign proteins, where must foreign DNA be inserted?
chloroplast genome because plant chromosomal DNA is much too large
What is DNA called with its associated packaging proteins?
chromatin
What type of plastid contains pigments?
chromoplasts
The dark-staining structures that carry the genetic material are the
chromosomes
What feature is included between the oleosin and the target protein so that the recombinant protein can be recovered by cleavage of the fusion protein?
cleavable linker
What do bacteria normally use restriction endonucleases for?
cleavage of foreign DNA such as bacterial viruses
What is the purpose of treatment with RNase I?
cleaves single stranded RNA so that incomplete cDNAs and nonpaired segments are degraded so that only RNA-DNA biotinylated capped segments remain
What is the biological activity of the ribozyme dependent on?
cleaving the target mRNA
What are two possible sources of probes for screening a genomic library?
cloned DNA from closely related organism (heterologous probe) and probe produced from chemical synthesis
What must be done once the library is created?
clones with target sequence must be identified
What does SIGEX rely on?
cloning of regulatory elements found upstream of genes they control
Recombinant DNA are currently used to produce
clothing dye, cheese, and laundry products
Recombinant DNA are currently used to produce ___.
clothing dye, cheese, and laundry products
What gene is used for negative selection (5-fluorocytosine sensitivity)?
codA (cytosine deaminase)
Each set of three nitrogen bases representing an amino acid is referred to as a(n)
codon
What happens to the nonfluorescent cells?
collected and grown in presence of substrate, screened by FACS again to identify substrate inducible reg elements
relationship between organisms in which one organism benefits and the other is neither harmed nor benefited
commensalism
What type of production was less efficient?
commodity production on large scale by natural microbes
An ecologist who studies how several species in an area interact is interested in the biologist organization called a(n) ___.
community
What is the response to a biological condition determined by?
comparing fluorescence intensity for each gene or probe, averaging replicates under different conditions and calculating ratio
How can up and downregulated mRNAs be identified?
comparing frequencies of tags in different samples
What are not intrinsic properties of E. coli?
competence and transformation
What is a cell that is capable of taking up DNA said to be?
competent
How do you clone and select targeted DNA from prokaryotes?
complete DNA of organism is cut with restriction endonuclease and each fragment inserted into a vector then the specific clone that carries target DNA sequence must be identified, isolated, and characterized
What is the name of the linear form or lambda DNA that is composed of several contiguous lengths of 50-kb units?
concatemers
What gets purified and cloned into a plasmid vector?
concatemers with 20 ditags
What do the columns represent in a gene expression profile?
conditions or samples
What influences both the integration and expression of foreign genes to plants?
configuration of vector
What is usually required before an optimal level of expression is found?
consideration of the distinctive features of a cloned sequence
In an oligonucleotide microarray, what is the purpose of the T7 primer?
contains the sequence for bacteriophage T7 RNA polymerase promoter
What is the purpose of the defective Ti plasmid?
contains vir genes but no T DNA region so that T DNA cannot be transferred but vir genes helps mobilize the binary cloning vector T DNA to be inserted into plant DNA
What does the ability to regulate a promoter enable the cell to do?
control transcription
How can you overcome the drawback of plasmid instability?
control transcription so that cloned gene is expressed only at a specific stage in host cell growth cycle and only for a specified duration
What must happen to lactose before it can act as an inducer?
convert to allolactose by B-galactosidase which is synthesized when system is repressed
What does treatment of plasmid DNA with calcium chloride do?
create transient openings in the cell wall that enables DNA to enter cytoplasm
Why is a high level of continuous expression of a cloned gene often detrimental to the host cell?
creates energy drain which impairs essential host cell functions and plasmid carrying a continuously expressed gene may be lost after several divisions
What important features of computer programs makes it possible so that all target sequences can bind to their complementary probe sequences under the same conditions?
cross-hybridize, no secondary structure or foldback, similar annealing temp
The exchange of genetic material between homologous chromosomes
crossing over
Using Figure 10-3, which process would result in the formation of chromosome C from chromosome A and B?
crossing over
When an area of a chromatid is exchanged with the matching area on a chromatid of its homologous chromosome, ___ occurs.
crossing over
As a result of agrobacterium tumefaciens genetically transforming plant cells as a normal part of its life cycle, what unfortunate problem occurs?
crown gall tumors
What interferes with the normal growth of an infected plant?
crown gall tumors
Which plasmids have no apparent functional coding genes?
cryptic plasmids
Adenine is to thymine as guanine is to
cytosine
ECB deacylase has the highest activity with what substrate
daptomycin
In microarray, what color is usually assigned for decreasing negative log ratios?
dark to bright green
In microarray, what color is usually assigned for increasing positive log ratios?
dark to bright red
Nitrogen is released to the abiotic parts of the biosphere from the processes of death and ___.
decay by bacteria
breaks down dead organisms
decomposer
Before plants can reuse many organic materials, the materials must be broken down by ___.
decomposers
Water, carbon, and nitrogen are released back into the atmosphere during ___.
decomposition
In the binary cloning vector approach, what acts as the helper plasmid?
defective Ti plasmid
Which plasmids carry specific sets of genes for the utilization of unusual metabolites?
degradative plasmids
A DNA nucleotide may be made up of a phosphate group, along with ___.
deoxyribose sugar and thymine
Explain a common 5'-end-labeling procedure?
dephosphorylation of the 5' ends of a linear DNA molecule with alkaline phosphatase and addition of gamma phosphate from ATP to 5' OH end by T4 polynucleotide kinase then labeled fragments are separated by column chromatography before gel electrophoresis
What determines which reporter gene will be used?
desired outcome of experiment
What method involves a dehydrated embryo that can uptake foreign DNA when rehydrated with DNA solution?
dessication
What is the goal of identifying genes that are coexpressed under different conditions or over a different period of time?
determining which gene products function in a given pathway
Which of the following would be an example of gene therapy technology?
development of a nasal spray that contains copies of the normal gene that is defective in persons with cystic fibrosis
which of the following would be an example of gene therapy technology
development of a nasal spray that contains copies of the normal gene that is defective in persons with cystic fibrosis
What kinds of plants does crown gall disease affect?
dicots (grapes, peaches, roses)
movement of particles from an area of higher concentration to one of lower concentration
diffusion
A cross involving two different traits
dihybrid
What does high temperatures do for second strand cDNA synthesis?
diminish intrastrand folding and increased efficiency of full length strand
Cells containing two alleles for each trait are described as ___.
diploid
What is the role of sigma factors?
direct RNA polymerase to promoter
What does the cointegrate vector recombine with?
disarmed Ti plasmid lacking tumor producing genes and lacking right border of T DNA within A. tumefaciens
What are one of the aims of gene expression study?
discover genes that are up and down regulated under specific conditions
How often does the sequence information in GenBank increase?
double every 18 months
What compound binds to a particular repressor protein and changes its conformation, thus preventing it from binding to its operator region?
effector
What is maximized by using the steps of a bioengineered biotechnology process?
efficiency
How is chloroplast DNA maintained in most angiosperm plants?
egg cells
If the sides of a cell double in length, its volume increased by ___ times.
eight
What type of plastid contains oil?
elaioplasts
After the target gene has been cloned into the multiple cloning site in the minivector, how is the final construct introduced into A. tumefaciens?
electroporation
What method involves foreign DNA being taken up by protoplasts in the presence of a high voltage electric field?
electroporation
What method is an effective way to transform E. coli with plasmids containing inserts longer than 100 kb?
electroporation
what derivative was produced from the combinatorial biosynthesis of three metabolic pathways using three glycosyltransferases; new indolocarbazoles were produced by pathway engineer of what two pathways in what heterologous host
elloramycin, urdamycin, landomycin; host is S albus; two pathways: rebeccamycin and Staurosporine
How does the mRNA get removed once separated from the other RNA?
eluted from column by treatment with a buffer that breaks A:T hydrogen bonds
How can hybridization of cRNA be detected?
emissions from phycoerythrin
How does the Ti plasmid act as a helper plasmid?
enables T DNA from binary cloning vector to be inserted into plant DNA
What are the two major functions of genetic material?
encodes info for protein production, reproduced with high accuracy
What happens after the second DNA strand synthesis is terminated?
end of cDNA are blund ended with T4 DNA polymerase to remove 3' extensions and fills in from 3' recessed ends
Which of the following is NOT a form of passive transport?
endocytosis
What is the name of the nucleases that cut nucleic acid molecules internally?
endonucleases
Where do protein modifications in eukaryotes occur?
endoplasmic reticulum and golgi apparatus
What is one way around the problem of keeping purification costs to a minimum?
engineer plants to secrete foreign proteins through the roots through a process called rhizosecretion
provide three enzymes involved in chemoenzymatic synthesis of vancomycin analogl; from such a chemoenzymatic synthesis from which one derivative display what property against s aureus and e faecium
engineered anomeric kinase, engineered nucleotidylyltransferase, wild type glycosyltransferase; improve antibiotic activity.
Glo Fish and Glo Pigs!
engineered to express the gene for green fluorescent protein cloned from jelly fish
What are the regions on the DNA that bind to enhancer binding proteins which interact with transcription factors and RNA polymerase to maximize the level of transcription from a particular promoter?
enhancer elements
Why is it necessary to put prokaryotic in origin, neomycin phosphotransferase gene under the control of eukaryotic plant transcriptional regulation signals with both promoter and termination polyadenylation sequence?
ensure efficient expression in transformed plant cells
What are the two possibilities after bacteriophage lambda infects E. coli?
enter a lytic cycle that leads to lysis of the hose cell and release of 100 phage particles or the bacteriophage lambda DNA can be inegrated into the E. coli chromosome as a prophage and maintained as a benign guest or lysogen through cell division
What kinds of probes might oligonucleotide microarrays consist of?
entire genome, single chromosome, select genomic regions, select coding regions
Restriction maps
establishes the number of, order of, and distances between restriction sites along a cloned segment of DNA > thus providing information about the length of the cloned gene and the location of restriction sites within the clone.
What significantly decreases the transfer of genes to plant genomes in agrobacterium-mediated plant transformation?
ethylene
What are the less condensed regions of chromosomes called?
euchromatin
Which has larger ribosomal subunits, prokaryotes or eukaryotes?
eukaryotes
Which type of proteins require modifications such as glycosylation, acetylation, sulfation, and phosphorylation?
eukaryotic
What is unique about the A. tumefaciens strain?
evolved to genetically engineer plant cells to be biological factories for production of carbon compounds
What can cause plants to develop tumorous growths such as crown galls?
excess auxin and cytokinins
release of wastes of cell products from inside to outside a cell
exocytosis
What is the name of the nucleases that degrade from the ends of nucleic acids?
exonucleases
What is adjacent to the bar gene but opposite in orientation?
expression cassette including 35S promoter, DNA sequence to target protein for expression, translational enhancer element, portion of MCS, and transcription termination sequence.
How can inhibition of mRNA expression be achieved?
expression of an additional copy of a gene using cosuppression, addition of an antisense version of a gene, or use of ribozymes
What is the primary objective of gene cloning for biotechnological applications?
expression of cloned gene in selected host organism
What is the name for the plasmids constructed to control transcription?
expression vectors
Typically for most gene expression profiling experiments that utilize microarrays, what is the protocol?
extract mRNA, purify, synthesize cDNA with reverse transcriptase from mRNA template
How do you prepare a metagenomic library for organisms that cannot be grown outside of their natural environment?
extract total genomic DNA from sample and express in a host bacterium
Who discover what as a treatment for rheumatoid arthritis and what year was he awarded?
f. EC Kendall, cortisone, 1950
What is the purpose of the polylinker (MCS) in Ti plasmid-based vectors?
facilitate insertion of cloned gene into region between T DNA border sequences
What cell process is responsible for the effect shown in Figure 8-5?
facilitated diffusion
What is the name for a visualized representation of a clustered microarray?
gene expression profile
What is the term for the growth or fermentation of the target microorganism in a large bioreactor with the consequent production of a desired compound which can be, for example, an antiobiotic, an amino acid, or a protein?
fermentation and transformation
How does a metagenomic library of marine or freshwater get constructed?
filtered, extract DNA, clone to vector, transform e coli with mlibrary, shotgun sequence
What is good about the derivatives of the minivector pCB301?
flexible, easy to use, contain variety of unique restriction enzyme sites in MCS
What does FACS stand for?
fluorescence activated cell sorting
Why is GFP an ideal in vivo marker for monitoring transgenic plants?
fluoresces green when excited with UV or blue light and does not require substrates or cofactors
How can GUS activity in plant extracts be quatitativly and sensitively assayed?
fluorometric analysis involving hydrolysis of substrate 4-methylumbelliferyl B-D-glucuronide for form fluorescent product
What can bring DNA regions close to one another in eukaryotes?
folding, looping, bending
simple model for showing how matter and energy move through an ecosystem
food chain
Tropic level and food chain are parts of a ___.
food web
network of interconnected food chains
food web
What have researchers used the constitutive 35S promoter for?
for the small subunit of RUBISCO
What is the cosmid vector that carries up to 40 kb of insert DNA and a cos site for in vitro bacteriophage lambda packaging?
fosmid
If the sides of a cell double in length, its surface area becomes ___ times as large.
four
In SIGEX, what happens to the DNA from a microbial community?
fragmented and cloned into p18GFP
What can happen to large plasmids greater than 10 kb during microprojectile bombardment?
fragmented and produce lower levels of foreign gene expression
What can make it difficult to find a specific target DNA sequence?
fragments too large to be cloned
A(n) ___ involves the addition or deletion of a single base in a DNA molecule.
frameshift mutation
What parameters are used to assess the success of DNA transformation?
frequency and efficiency
How can genes of interest be located for cloning through their ability to restore wild type phenotype to a mutant organism?
functional complementation
What type of genomics is concerned with the patterns of transcription either qualitatively to determine which genes are expressed or quantitatively to measure changes in the levels of transcription of genes?
functional genomics
What are two ways a specific foreign protein can be introduced into the chloroplast or mitochondrion?
fusion gene inserted into nuclear chromosomal DNA and after synthesis the recombinant protein can be transported into target organelle, or gene for foreign protein inserted directly into chloroplast or mito DNA
The ___ produced by each parent are shown along the sides of a Punnett square.
gametes
The process used to separate DNA segments of different lengths is
gel electrophoresis
The process used to separate DNA segments of different lengths is ___.
gel electrophoresis
The Human Genome Project has involved sequencing and mapping the human genome. The most important benefit of this information has been the diagnosis of genetic disorders. Once a genetic disorder is diagnosed, ___ can be used as a possible treatment.
gene therapy
The human Genome Project has involved sequencing and mapping the human genome. The most important benefit of this information as been the diagnosis of genetic disorders. Once a genetic disorder is diagnosed, ______ can be used as a possible treatment
gene therapy
_______is an application of the Human Genome Project that involves the insertion of normal genes into cells with defective genes in an attempt to correct genetic disorders
gene therapy
What is contained in the right arm of bacteriophages?
genes for DNA replication and cell lysis
What is contained in the middle fragment of bacteriophages?
genes for integration and excision processes
What genes are expressed continuously in bacteria?
genes that encode proteins for basic cellular function
What was required for three different proteins: GFP, xylanase, and alkaline phosphatase, to be efficiently exuded by the roots of transgenic tobacco plants?
genetic construct must include DNA fragment encoding a signal peptide placed upstreat at 5' end of gene whose protein was targeted for secretion
In 1974, Stanley Cohen and Herbert Boyer inserted a gene from an African clawed frog into a bacterium. The bacterium produced the protein coded for by the inserted frog gene. This insertion of a small fragment of frog DNA into the DNA of another species can most accurately be called ___.
genetic engineering
When DNA from a bacteriophage is cut with BamHI, three segments are created. What is contained in the left arm?
genetic information for heads and tails
Crossing over results in a ___.
genetic recombination
Agrobacterium tumefaciens is a phytopathogen that does what as a normal part of its life cycle?
genetically transforms plant cells
The intergenic parts of the human genome consist of which types of DNA sequences
genome-wide repeats, microsatellite DNA and regulatory sequences
What is the process called by which subdivision of genomic DNA into clonable elements and inserting them into host cells must be done?
genomic library
What is the term for the area of research that generates, analyzes, and manages the mountains of information about genome sequences and all the genes that are transcribed in various cell types and tissues?
genomics
What is the term for which the study of all features of genomes and individual genes at the DNA level, including mutations, polymorphisms, and phylogenetic relationships that are based on sequence differences?
genomics
Explain the naming protocol for restriction enzymes?
genus first letter capitalized, species two letters lowercase, strain designation or serotype, roman numerals for order of characterization of different restriction endonuclease of same organism
What gene does the vector of the SIGEX system utilize?
gfp
What gene is considered the reporter gene?
gfp
what intermediate or amino acid is coupling carbon assimilation with nitrogen metabolism
glutamate
What is the term for containing specific sugars attached to the hydroxyl group of either serine or threonin or the amide group of asparagine?
glycosylation
Where does processing, glycosylation, and posttranslation modifications take place?
golgi stack
Is Agrobacterium tumefaciens a gram-negative or gram-positive soil bacterium?
gram-negative
What does a bound activator protein do for the movement of RNA polymerase along DNA?
grease the wheels and enhance the rate of transcription of the operon
What happens to the gfp negative cells in SIGEX?
grown in presence of a low MW substrate
What are some causes of misleading interpretations?
growth conditions, RNA extraction, cDNA synthesis, labeling efficiencies, hybridization efficiences, variation of concentrations of probes, unequal distribution of targets
What other products soon followed the production of insulin?
growth hormone, viral antigen, therapeutic proteins, interferon
What plasmids are represented by 10 to 100 copies per host cell?
high copy number plasmids
7. advantage of the machine learning-guided logorithm is that it can explore the high gradient direction, increasing the odds of reaching higher fitness on Mount Improbable.
high gradient direction, higher fitness
When inducer is present and there is no repressor bound to the operator, what can lead to a high level of transcription of the genes downstream of the lac promoter?
high intracellular concentration of cAMP
What is the source of particle propulsion in microprojectile bombardment?
high pressure helium
What is second strand cDNA synthesis carried out with?
high temps with DNA polymerase, RNase H, and DNA ligase
How can the uptake of free DNA be induced?
high voltage electric field
Assembly of nucleosomes requires
histone "chaperones"
in add'l to histon composition and chromatin-remodeling, what is the 3rd way that the chromation structure can be regulated?
histone modification
What type of protein is EcoRI and what does it bind to?
homodimeric, DNA with a specific palindromic sequence
What do the cointegrate cloning vector and the disarmed helper Ti plasmid both carry?
homologous DNA sequences providing a shared site for in vivo homologous recombinations which lie inside T DNA region
What is the normal mode of DNA integration into the chloroplast genome?
homologous recombination
What type of protein are multiples of the same polypeptide chain required for an active protein molecule?
homomeric
The alleles present for a trait are the same
homozygous
A white mouse whose parents are both white produces only brown offspring when mated with a brown mouse. The white mouse is most probably ___.
homozygous recessive
The entire collection of genes within human cells is referred to as the
human genome
What was the first commercial product produced using recombinant DNA technology?
human insulin
Where are enhancer sequences located?
hundreds and thousands of base pairs from +1 base pair
Santa Gertrudis cattle have the combined traits of good beef production and hot weather tolerance as a result of selection and ____________
hybridization
What do DNA microarrays rely on?
hybridization and signal detection
Water moves into a cell placed in a(n) ___ solution.
hypertonic
Water moves out of a cell if the cell is placed in a(n) ___ solution.
hypertonic
the concentration of dissolved substances outside the cell is higher than the concentration inside the cell
hypertonic solution
What are the six parameters showing the benefit of the biocatalytical process over the chemical process
i. Crude substrate load, substrate/catalyst ratio, conversion, stereochemical purity, solvent use, ambient temperature and pressure
What genes does the T-DNA region include?
iaaM, iaaH , tmr
What is the purpose of growing the gfp-negative cells in the presence of a low MW substrate?
identify clones that carry segments required for activation of genes by interaction with target substrate
What is a major purpose of a microarray experiment?
identify genes whose expression changes in response to particular biological condition
Under what context is it best to remove the reporter gene?
if reporter gene taints a commercial product
How can refining and carefully controlling conditions help transformation of corn and rice by A. tumefaciens carrying Ti plasmid vectors?
immerse immature corn embryo in A tumefaciens cell suspension, incubate at room temp, transfer to medium with selective antibiotic allowing only transformed plant cells to grow, maintain in dark, transfer to different growth media containing plant hormones, incubate in light to permit regeneration of whole transgenic plant
What is an alternative method used to screen a library when a DNA probe is not available?
immunological assay
What are the three major reasons for developing transgenic plants?
improve agriculture, horticulture, ornamental value, bioreactors for proteins and metabolites, studying actions of plant genes during development
__________ is used to develop pure breeds
inbreeding
What modification of tissue culture conditions has been found to increase transformation frequencies of grape, ride, corn, and soybean?
inclusion of antioxidants during transformation
Why are the cells grown in the presence of IPTG?
induce expression of gfp from lac promoter
What does the high temperature do for secondary structure in mRNA?
increase likelihood that complete molecules will be synthesized so disrupts the intrastrand base pairs
What good would an ethylene lowering gene do?
increase transformation frequencies
As matter and energy move from grasses to coyotes, the amount of available energy ___.
increases or decreases but population size remains the same
What is the drawback to a shuttle vector when adding a segment of DNA containing the second origin of replication?
increases size of vector and reduces amount of DNA that can be inserted, not efficiently propagated in the host cell, can be unstable and lost
Which of the following rxns w/ covalently closed circular DNA does NOT result in a new lk
incubation w/ ethidium
The statement: "In meiosis, the way in which a chromosome pair separates does not affect the way other pairs separate," is another way of expressing Mendel's law of ___.
independent assortment
How can you identify the recombinant bacteriophage that carries the target gene in the bacteriophage lambda library?
individual plaques are lifted onto matrix and screened with DNA probes or antibodies
What must a secreted protein pass through in gram-negative bacteria?
inner membrane, periplasmic space, and outer membrane
What are some of the genetically determined traits that can be introduced into plants by a single gene or possibly a small cluster of genes?
insecticidal activity, protection against viral infection, resistance to herbicides, protection against pathogenic fungi and bacteria, delay of senescence, tolerance to stresses, altered flower color, improved nutrition, increased shelf life, self incompatibility
What is restriction endonuclease cleavage in molecular cloning important for?
inserting target DNA into a cloning vector
Where does the A-T and G-C base pairs lie?
inside of molecule
The processivity of a DNA polymerase depends on all of the following
interaction b/w the thumb domain of DNA polymerase and the DNA 2. associateion of the DNA polymerase w/ a sliding clamp 3. interaction b/w minor goorve of DNA and the palm domain of the DNA polymerase
What are the noncoding regions of eukaryotes called?
introns or intervening sequences
12. The chirality of an amino acid results from the fact that its α carbon: A) has no net charge. B) is a carboxylic acid. C) is bonded to four different chemical groups. D) is in the L absolute configuration in naturally occurring proteins. E) is symmetric.
is bonded to four different chemical groups
What is the first enzyme of dioxylate cycle
isocitrate lyase
What was replica plating originally intended for?
isolate mutant bacterial colonies that require a supplement for growth
What happens after concatemers are formed from ditags?
isolated and cloned into e coli plasmid, sequenced, recorded
What is recombinant DNA technology?
isolation and transfer of genes from one organism to another
After the prototype, what are additional restriction endonucleases that attack the same sequence as the prototype?
isoschizomers
What kind of pairs of restriction endoculeases can determine the methylation status of genomic DNA?
isoschizomers
the concentration of dissolved substances in the solution is the same as the concentration of dissolved substances inside the cell
isotonic solution
In conventional breeding, what are some disadvantages?
limited to exchanges between same or closely related species, no guarantee of obtaining desired gene combination from millions of crosses, undesirable genes transferred along with desirable, long time to achieve desired results
Under conditions of nutritional or environmental stress, what happens to the bacteriophage lambda DNA?
it is excised and enters a lytic cycle
What happens if X-Gal is present in medium?
it is hydrolyzed by hybrid B-galatosidase to blue which means pUC19 is unmodified
What happens if introduced DNA is a plasmid?
it is maintained in cytoplasm after second strand is synthesized
Why is 5' exonuclease activity abolished in DNA probing?
it would degrade some of the newly synthesized DNA
DNA binding proteins primarily recongize a specific seqof double stranded DNA through
itneractions w/ the exposed chemical groups in the major groove of the DNA
Recombinant DNA
joining of DNA from different organisms in the test tube •isolation of individual genes (Gene Cloning) •study of gene function and regulation of expression •cloned genes can be sequenced and their base sequences can be modified •transgenic organisms - insertion of cloned genes into animals, plants or bacteria "GENETIC ENGINEERING"
How can the resolution of fragments for restriction endonuclease mapping be enhanced?
labeling pieces of DNA at 5' ends with a radioactive compound or fluorescent dye and determine lengths after electrophoretic separation with autoradiography or fluorography
Which promoter has been used extensively for expressing cloned genes?
lac operon of E. coli
From which promoter does the -10 region of the tac and trc promoters come from?
lac promoter
In SIGEX, What is the gfp gene under the control of?
lac promoter in pUC based plasmid p18GFP
What are both tac and trc promoters repressed by?
lac repressor
What encodes for the enzyme B-galactosidase?
lacZ gene of lac operon
Why can't the cloning vectors effect the transfer and integration of the T-DNA region into recipient plant cells?
lack vir genes
An Okazaki fragment is a segment of DNA that is an intermediate in the synthesis of the leading strand
lagging strand
Why are special computational tools required for obtaining, storing, and analyzing the results from gene expression profiles?
large amounts of data obtained
What makes up the initiation complex?
large ribosomal subunit combined with fMet-tRNA-mRNA-small subunit complex
When is transformation more efficient in biolistics?
linear DNA used
By the end of prophase, each of the following has occurred except ___.
lining up of chromosomes in the cell
What is a problem that arises when using binary vectors?
large size makes difficult and inconvenient to manipulate in vitro and larger plasmids have fewer unique restriction sites for cloning
What method involves a UV laser making holes in the protoplasts to introduce foreign DNA?
laser microbeam
What problem can occur with repression of the trp promoter?
leaky leading to continuous low level of transcription even when supposedly turned off
How can transcription occur even when there are more repressor molecules than copies of promoters?
leaky promoter
Why can't the trp promoter-operator be used to express genes that might be toxic or deleterious to the growth of E. coli?
leaky promoter
Which border in the integration of T DNA is not involved?
left
Where is the selectable marker usually placed?
left border
Evolutionary rln b/w complexity of organisms and gene density
less complex organisms have increased gene density
What DNA sequences do many transcription factors bind to in eukaryotes and what are these sites called?
less than 10 bp, idiosyncratic
What happens after PCR amplification and digestion of ditags?
ligated to form concatemers 500bp long?
What were many of the early plant tansformation experiments conducted with?
limited host range strains for agrobacterium
What could happen if you leave a gene which encodes for opine synthesis in a transgenic plant?
lower final plant yield by diverting plant resources into opine production
What can MS determine?
m/z peptides of one or more proteins actual sequence of one or more peptides
In eukaryotes, how does mature mRNA emerge in contrast to in prokaryotes?
mRNA leaves the nucleus and is bound by ribosomes in cytoplasm and after splicing gets capped and polymerized
What does a cDNA library often represent?
mRNA sequence from a single specific tissue
mRNA leaves the nuclus and enter the cytoplasm. These data supported th yp that
mRNA serves as the template for protein synthesis
What are the factors required of translation?
mRNA, charged tRNA, ribosomes, proteins
How are the beads separated from the solution?
magnet
What is the disadvantage of cosmids?
maintained at higher copy numbers which leads to deletions or rearrangements of insert DNA
What is the aim of shotgun sequencing?
make DNA contigs from many genomes and identify novel and homologous sequences
An application of using DNA technology to help environmental scientists would be
make transgenic bacteria that can be used to clean up oil spills more quickly than do the natural bacteria
An application of using DNA technology to help environmental scientists would be ___.
make transgenic bacteria that can be used to clean up oil spills more quickly than do the natural bacteria
What is the purpose of the polyA tail of mRNA?
means for separating mRNA from rRNA and tRNA
The type of cell division that produces gametes
meiosis
Why were mutant strains not successful?
metabolic functions impaired
What is the term used for the study of collective genomes of uncultured organisms from environmental and clinical samples?
metagenomics
At the end of translation, what amino acid is commonly cleaved?
methionin at N terminus
what is the new cycle for nature combinatorial synthesis in Haloarchaea
methyl- aspartate cycle
What prevents restriction endonucleases from cutting at the sites?
methylation of cytosine resdues in host DNA
Energy flows from ___.
mice to cats
Among the following, the term that includes the other is ___.
mitosis
The process by which nuclear material is divided equally between two new cells is
mitosis
The numbers in Figure 10-1 represent the chromosome number found in each of the dog cells shown. The processes that are occurring at A and B are ___.
mitosis and fertilization
What does the recipient strain, A. tumefaciens, carry in the binary cloning vector approach?
modified or disarmed Ti plasmid containing complete set of vir genes but lacks portions or all of T DNA region so T DNA cannot be transferred
How does glycosylation affect modification of plant cells?
modify plant so that it does not add problematic carbohydrate residues, glycan side chains limit use of final protein
DNA polymerase ensures that the correct nudlotide is incorporated into the growing strand of DNA by
monitoring the ability of the incoming nucleotide to form either an AT or GC bp
DNA polymerase ensures that the correct nucleotide is incorportated into the growing strand of DNA by
monitoring the ability of the incoming nucleotide to form either and AT of GC bp
What types of plants are not readily transformed by A. tumefaciens?
monocots (rice, wheat, corn)
What do negative log ratios represent?
more Cy3 than Cy5 and lower expression of gene in test than reference
What do positive log ratios represent?
more Cy5 than Cy3 and greater expression of gene in test than reference
How many unique tags are collected from a single experiment?
more than 10,000
How many SAGE tags have been assembled from humans?
more than 30 million
What kind of cloning site does pUC19 have that pBR322 does not?
multiple
What are the important features of pUC19?
multiple cloning site B-galactosidase gene, lacZ regulatable promoter lacZ lac1 that produces repressor gene for lacZ expression
What is the cause of the eukaryotic replication process to include enzymatically ligating segments of newly synthesized DNA together with phosphodiester bonds?
multiple origins of replication
In systems that use the lac promoter, what is often used to decrease transcriptional leakiness under noninduced conditions such as transcription of a cloned gene in the absence of inducer?
mutant form of lacI gene (lacIq), it produces much higher levels of lac repressor
When parts of chromosomes are broken off and lost during mitosis or meiosis, the result is a(n)
mutation
Even after treated with alkaline phosphatase, the two phosphodiester bonds are still sufficient to hold the two molecules together despite the nicks, but they will not replicate following introduction into a host cell because they do not have what?
no origin of replication
What are the requirements of the transformed cells?
no restriction endonucleases, should be recombinant negative so as not to exchange GOI with native DNA, should not produce the endonuclease encoded by endA1 gene so they have increased transformation efficiency
What has been used to isolate plant promoters from several plant species?
promoter tagging vectors
X rays, ultraviolet light, and radioactive substances that can change the chemical nature of DNA are classified as ___.
mutegens
Both the alga and the fungus are benefited from their relationship in a lichen. This relationship is one of ___.
mutualism
Some birds are known as honey guides because they may be followed by humans to wild beehives. When the humans take honey from the hives, the birds are able to feast on the honey and bees, too. This type of relationship can BEST be described as ___.
mutualism
relationship between organisms in which both organisms benefit
mutualism
How is the calculated ratio of the response of gene expression expressed?
n fold change
What kinds of plasmids have less specific origins of replication and can replicate in a number of bacterial species?
narrow and broad host range plasmids
starting from environmental sample, provide four most important technology strategy for discovering an optimizing enzyme with industrial biocatalyst processes
natural diversity, new high throughput screening assays, molecular directed evolution technology, process development
What will be the result of the mutation in Figure 11-4?
nearly every amino acid in the protein will be changed
How is the trp promoter regulated?
negatively (turned off) by trp repressor protein complexed with tryptophan which binds to trp operator and prevents transcription of trp operon
What is the regulation of transcription by a repressor protein called?
negatively controlled system
What are isoschizomers that cleave at different positions within the same recognition site called?
neoschizomers
How do oligo(dT) beads separate the mRNA from other RNA?
oligodT beads are packed into a column and the polyA tail base pairs to the oligodT chains while the tRNA and rRNA pass through the column
In SAGE, what does polyadenylated mRNA get captured by?
oligodT sequence labeled with biotin attached to streptavidin coated magnetic bead
What does an alternative microarray system utilize?
oligonucleotides as probes representing thousands of genes
What influences the folding of the protein into a particular 3D shape and therefore its function?
side chain properties
The hershey and case exp using S and P labeled T2 bacteriophage supported the hyp that DNA is the genetic material b/c
new T2 progeny contained P-labeled DNA
Cougars are predators that often eat weakened or diseased animals. This is a description of the ___ of cougars.
niche
What is a break in the phosphodiester bond in one strand of duplex DNA called?
nick
What does RNase H do?
nick mRNA to provide a free 3' OH group for initiation of DNA synthesis
The pairing of ___ in DNA is the key feature that allows DNA to be copied.
nitrogen bases
Where does the base of DNA attach to the deoxyribose?
nitrogen to the 1' carbon
Are vir genes present on a binary cloning vector?
no
Can pollen transmit the contents of the chloroplast genome to the zygote in angiosperms?
no
Do plasmids with inserts that do not induce expression of gfp produce gfp in the presence of IPTG?
no
Does insertion of a gene into a cloning vector ensure that it will be successfully expressed?
no
Why is DNA a 2'-deoxyribose?
no hydroxyl group on 2' carbon
What happens when concentration of the corepressor decreases?
on state of operon resumes because repressor is unable to bind to operator
How is chloroplast DNA inherited?
non mendelian fashion
What is produced when less than 38-kb of DNA is packed into a bacteriophage head?
noninfective bacteriophage particle
What types of cells are sensitive to an antibiotic?
nontransformed cells
What are libraries containing small DNA fragments, inserted into a plasmid, screened for?
novel genes
What genes are used for positive selection (kanamycin resistance)?
nptII (neomycin phosphotransferase)
What plays an essential role in determining whether an operon is transcribed?
nucleotide sequences on or around the RNA polymerase binding site
An RNA molecule is a polymer composed of subunits known as ___.
nucleotides
Thymine, adenine, guanine, and cytosine are classified as
nucleotides
What is DNA made up of?
nucleotides
What is the transformation efficiency?
number of transformed cells as a function of the amount of DNA originally added to cells
What does the design of the probe for a microarray generally depend on?
objective of experiment and degree of resolution required
What does dominant marker selection provide the means for?
obtaining only transformed cells in culture
What system has the potential to significantly lower the costs of purifying target proteins produced in plants?
oleosins
What proteins are found in the seeds of a wide range of plants?
oleosins (oil body proteins)
Mendel's law of segregation states that during meiosis, the factors that control each trait separate, and only ___ from each fair is/are passed to the offspring.
one factor
What does the transcription of an operon give rise to?
one large mRNA
What happens to each aliquot of cDNA sample in SAGE?
one ligated with adaptor A, other with adaptor B
What is the inherent limitation with the host cell for selection schemes that depend on transcription and translation of the cloned gene?
only 40% of hetero genes expressed
Why is the ability to join different DNA molecules not useful by itself?
only if it can be perpetuated in host cell
What does DNA microarray principally detect for?
only sequences that correspond to probes on array
What would happen if the selectable marker were adjacent to the right border?
only small portion of T DNA would yield plants containing selectable marker but not gene of interest
What are the bacterial structural genes of an operon under the transcriptional control of?
operator and promoter region
What are most chloroplast-borne genes organized into?
operons
What are the bacterial structural genes that encode proteins and are required for several steps in a single metabolic pathway which are contiguous in the chromosome?
operons
What are libraries containing larger DNA fragments, inserted into a fosmid, cosmid, or bacterial artificial chromosome vector screened for?
operons or metabolic pathways
What other genes besides auxin and cytokinin does T-DNA carry?
opine
What genes are unique and unusual condensation products of an amino acid and keto acid or amino acid and sugar?
opines
What level of organization is shown in Figure 8-7?
organ system
Which of the following structures is the most complex?
organ system
What sequence site on a plasmid is essential for the replication in a host cell?
origin of DNA replication
Any 50-kb DNA molecules must contain what two things in order to be perpetuated?
origin of replication and cos ends
What is the difference between a cosmid and a fosmid?
origin of replication of a fosmid is derived from the E. coli F factor or sex plasmid
If a cell is placed in salt water, water leaves the cell by ___.
osmosis
diffusion of water molecules through a selectively permeable membrane
osmosis
Name three sulfhydryl reagants:
p-HMB, DTNB, NEM
Name 3 sulfhydryl reagants
p-HMB. DTNB, NEM
What is the name of a commonly used binary vector?
pBIN19
Name the best studied and most often used general purpose plasmid cloning vector?
pBR322
What was the commonly used binary vector constructed into to make it smaller?
pCB301
What are the five parameters that are essential for activity optimization of an enzyme
pH and temperature Buffers and salts Enzyme concentration and reaction time Substrate saturation Cofactor and metal ion
What was developed as an alternative to pBR322?
pUC19
What is an operon generally under the control of?
promoter
Where are most significant cleavages in tandem mass spec? Where is the positive charge retained?
peptide backbone The y-ion on the C-terminus of original peptide ion Obtain doubly charge ions so y- and b- ions are produced b left y right
What links adjacent amino acids to form a protein?
peptide bond
How does initiator tRNA get discharged?
peptide bond formation
What is excreted by susceptible wounded plants?
phenolic compounds
What do the wound response compounds in plants resemble?
phenylpropanoid metabolism, major plant pathway for synthesis of plant secondary metabolites, lignins and flavonoids
The backbone of a DNA molecule is made of which two components?
phosphate molecules and deoxyribose sugars
What joins the nucleotide subunits of DNA?
phosphodiester bonds
bonds that link adjacent nucleotides in both RNA and DNA
photodiester
What light-directed process is a commonly used platform of microarray where the probes are synthesized directly on a solid surface?
photolithography
Carbon dioxide in the atmosphere enters the biotic parts of the biosphere through ___.
photosynthesis
What are the limitations of Ti plasmids as routine cloning vectors?
phytohormones in transformed cells prevents regeneration into mature plants, opine synthesis not useful, too large, doesn't replicate in e coli, transfer of T DNA from right border doesn't always end at left border and sometimes goes past the left but isn't tested for
What prevents transformed cells growing in culture from being regenerated into mature plants? What can you do to change this?
phytohormones, remove the genes from Ti plasmid derived cloning vector
What can microprojectile bombardment introduce foreign DNA into?
plant cell suspension, callus cultures, meristematic tissues, immature embryos, protocorms, coleoptiles, pollen in monocots and conifers
What can be used to facilitate recombinant plant protein purification that would be fused to the foreign protein?
plant oleosins
What components are included in a cointegrate vector system?
plant selectable marker gene, target gene, right border, e coli origin of DNA replication, bacterial selectable marker
What were YACs engineered to contain?
plant selectable markers and yeast selectable markers
What organisms became biofactories or bioreactors for producing new or altered gene products that could never have been created by mutagenesis and selection or by crossbreeding?
plants and animals
The structure most responsible for maintaining cell homeostasis is the ___.
plasma membrane
A(n) ___ is a small ring of DNA found in a bacterial cell.
plasmid
Multiple cloning site
plasmid vectors have this which allows them to have many restriction sites for many restriction enzymes- allows scientist to clone a range of different fragments generated from many common restriction enzymes
What is the term for self-replicating, double stranded, circular DNA molecules that are maintained in bacteria as independent extrachromosomal entities?
plasmids
What properties do cosmids combine?
plasmids and bacteriophage lambda vectors
Where can foreign DNA be targeted for expression besides as part of the chloropast genome?
plastid
What group of plant organelles do chloroplasts belong to?
plastids
A DNA segment is changed from-AATTAG- to -AAATAG-. This is a ___.
point mutation
Where does the oligo(dT) sequence bind to?
polyA tail of mRNA
What do most chloroplast-borne genes produce?
polycistronic mRNAs
What chemical is used to treat protoplasts to induce uptake of macromolecules and foreign DNA by changing the permeability of the plant cell membrane?
polyethylene glycol (PEG)
A small amount of DNA obtained from a mummy or from frozen remains of a human may be cloned. In order to clone small amounts of DNA, ___ needs to be used to generate larger quantities of the DNA.
polymerase chain reaction techniques
A small amount of DNA obtained from a mummy or from frozen remains of a human may be cloned. In order to clone small amounts of DNA, ____needs to be used to generate larger quantities of the DNA
polymerase chain reaction techniques
It enciricles one of the 2 DNA strands upstream of the fork and uses the energy provided by ATP hydrolysis to separate the strands, The unwinding of the DNA introduces ______ into the double strandedDNA upstream to the fork that need to be removed by topoisomerase
positive supercoils
What is it caled when a regulatory protein increases the rate of transcription instead of repressing it?
positively controlled system
What is the process by which a certain group such as phosphate, lipids, or carbohydrates are enzymatically added to certain amino acids of a protein to create new proteins that mediate specific cellular activities?
posttranslational modification
How to make cDNA? Use reverse transcriptase- turns RNA to DNA, and the source of this is retroviruses. The retroviruses are RNA viruses that replicate by turning their genome into DNA and inserting it into genomes, (HIV) To clone a mRNA for eurkaryote—isolate total RNA from the cell, add to it a short piece 20 bases long of just T's (oligo dt primer) and they will base pair with the poly A tail. That serves as a primer site for reverse transcriptase site, and it will start the replication . RNA and DNA together now, and RNA H degrades RNA and it only degrates RNA that is attached to DNA. You add this in to take away the RNA but not all, then add DNA poly 1 and it will fill in the spaces and will use the primer to chug along and cut out RNA and replace it with DNA. Add DNA ligase & seal, now you have a DNA piece that coresponds with a messanger. To get into a plasmid, you must manipulate more steps (don't need to know) Just know reverse transcriptase & enzyme Rnase H For HIV- it has RNAse into it (read)
powerpoint notes
The relationships between the cats and mice could best be described as ___.
predator-prey
How can the GUS activity in transformed plant tissues be localized?
presence of blue color formed after hydrolysis of uncolored substrate 5-bromo-4-chloro-3-indolyl B-D-glucuronic acid
Why are the cells grown in the presence of ampicillin?
prevent growth of untransformed cells
Why do the adaptors have different primer sequences?
prevent intrastrand base pairing (snap back) during PCR
How do lactose and IPTG enable transcription to occur?
prevent lac repressor from binding to lac operator
What is a dual function oligonucleotide called?
primer adaptor
What are ditags flanked by?
primer sequences
in vitro DNA synthesis requires DNA polymerase DNTPsand a what junction
primer/templcate
What reaction is responsible for linear amplification and biotinylation of cRNA?
production of RNA copies from the second cDNA strand
What is biotechnology concerned with production of?
products made by microorganisms
What are some concerns about selectable marker genes?
products toxic or allergenic, antibiotic resistance genes used as selectable markers may be transferred to pathogenic soil microorganisms, more difficult to transform transgenic plant with additional foreign genes since SM cant be used more than once
In which organisms does transcription and translation occur concurrently and why?
prokaryotes, no nucleus
What molecular biological features have been manipulated to modulate gene expression?
promoter, transcription terminator sequences, strength of ribosome binding site, number of copies of
The longest phase of the cell cycle is ___.
prophase
What does the presence of 5-methyl-dCTP do?
protect DNA from being cleaved by certain restriction endonucleases
Why is hemimethylation done at the end of making a cDNA library?
protects a cDNA from cleavage if it contains the same restriction endonuclease sites used for cloning
What happens if a plant engineered for rhizosecretion is grown hydroponically?
protein secreted directly into culture medium
When does the RNA polymerase holoenzyme form?
protein sigma factor combines with core proteins of RNA polymerase
A gene is a segment of DNA that controls the production of ___.
proteins
What is the term for the studies of the entire protein populations of various cell types and tissues and the numerous protein-protein interactions?
proteomics
What is the first restriction endonuclease that is discovered to bind to a particular recognition site designated as?
prototype
16. For the study of a protein in detail, an effort is usually made to first: A) conjugate the protein to a known molecule. B) determine its amino acid composition. C) determine its amino acid sequence. D) determine its molecular weight. E) purify the protein.
purify the protein
Peptide bond formation is catalyzed by a catalytic activity that is thought to be mediated by the ________ molecule in the large ribosomal subunit
rRNA
What kind of RNA makes up most of a metabolically active cell?
rRNA
Ribosomes are made of ___.
rRNA and protein
How can hybridization between the DNA probe and DNA sample be detected?
radiography
What determines ditag formation?
random
What sites does insertion of T-DNA into the plant DNA occur?
random
What does the effectiveness of deactivating a repressor protein and activating transcription depend on?
ratio of number of repressor protein molecules to number of copies of promoter sequences
What is transformation frequency?
ratio of transformed cells to total number of treated cells
How do ribozymes decrease the amount of protein encoded by a specific mRNA?
recognize and cleave one specific mRNA
How are Ti plasmid contraints overcome?
recombinant DNA technology
What pivotal development enabled biotechnology?
recombinant dna technology
What color is used to denote gene overexpression?
red
What color do wild-type plants normally fluoresce if they do not contain the gene for GFP?
reddish purple
In DNA probing, why is the 3' exonuclease retained?
reduces misincorporation of erroneous dNTPs during synthesis of new DNA strand
In microarray, how can you avoid variation due to inherent and sequence-specific differences in labeling efficiences between Cy3 and Cy5?
reference and test samples are reverse labeled and hybridized to another microarray
Why can't the minivector pCB301 be introduced into A. tumefaciens by conjugation? What is an alternative means?
regions of DNA required for conjugal transfer were deleted, electroporation
What must be done in order to minimize the deleterious effects on plant growth from the expression of foreign genes?
regulate expression of introduced genes and amount of foreign protein produced
What happens to the transcription of the remaining structural genes that are not expressed continuously in bacteria?
regulated
What is usually close to the promoter of the catabolic operon and therefore most likely a portion is present on the cloned segment?
regulatory elements, genes encoding proteins, and DNA elements that bind to substrate
What do the tac promoters interact with to provide a controllable switch for turning on or off specific transcription of adjacent cloned genes?
regulatory proteins
What does the second RNase H treatment do?
remove any RNA that escaped the previous treatment
Why are metagenomic libraries prepared in p18GFP and initially screened using FACS?
remove clones that emit gfp in absence of catabolic substrate
Why do the ditags get amplified by PCR and then treated with N1aIII?
remove primer sequences and generate sticky ends
Why does functional eukaryotic mRNA not have introns?
removed by splicing
What do the rows represent in a gene expression profile?
reordered genes
What component of RNA polymerase recognizes the two binding sites of the promoter for bacteria?
sigma factor
Since only one or at most a few of the chloroplasts actually incorporates foreign DNA, what is required so that the chloroplast population becomes enriched and dominated by the transformants?
repeated rounds of growth on selective antibiotics
What types of sequences are not included in genomic DNA microarrays?
repetitive sequences
The process by which DNA makes a copy of itself is called
replication
The process by which a DNA molecule is copied is called ___.
replication
What is the process of DNA synthesis called?
replication
What does quantification and other applications when determining level of expression of a gene require?
reporter genes that encode an activity to be assayed
What happens to the E. coli lac promoter in the absence of lactose in the growth medium?
repressed (turned off) by lac repressor protein preventing transcription of lac operon
What type of regulatory protein can bind to an operator region and prevent RNA polymerase from binding to the promoter and ultimately block transcription?
repressor
What happens when levels of effector molecule are reduced?
repressor binds to operator region and off state reestablished
What functions do unique restriction endonuclease sites have?
required for inserting DNA into a cloning vector and allow inserted DNA sequence to be recovered from vector
What are transgenic plants that express high level RAD54 genes readily selected on the bases of?
resistance to high levels of gamma irradiation
How are most transformed chloroplasts selected?
resistance to spectinomycin, streptomycin, kanamycin which inhibit protein synthesis on prokaryotic type ribosomes
Due to the fact that operons are not found in eukaryotes, what does each eukaryotic structural gene have its own set of?
response elements
In SAGE, what cuts the cDNA?
restriction endonuclease N1aIII
When FACS is done for the 2nd time, what happens to the cells that glow?
retained
What does the oligo(dT) sequence use to prime the synthesis of cDNA from mRNA?
reverse transcriptase
In SAGE, how is double-stranded cDNA synthesized from purified mRNA?
reverse transcriptase to synthesize first strand from oligodT primer and mRNA template, DNA polymerase to synthesize second
When rRNA combines with specific proteins, what is formed?
ribonucleoprotein complexes that make up ribosomal subunits
Where does biotin chemically attach to?
ribose sugars of cap at 5' and 3' ends of mRNA
What are small RNA molecules with the ability to act as sequence-specific endoribonucleases called?
ribozymes
Where is transfer of T-DNA initiated from?
right border
Which is more promising, collecting root or collecting leaf exudate?
root
Which part of the RNA polymerase holoenzyme directs the binding of the holoenzyme to promoter regions on the DNA?
sigma factor
feeds on dead organisms
scavenger
Why does Reverse transcriptase not always produce full length cDNA copies from every mRNA template?
secondary structures such as hairpin loops
What is required for acquiring nutrients, cell to cell communication, protection, and structures that reside on the outer surface of the cell membrane?
secretory proteins
What is required to be able to detect the foreign DNA that has been integrated into plant genomic DNA so that those cells that have been transformed can be identified?
selectable marker
What is used to detect the presence of T-DNA inserted into the plant chromosomal DNA?
selectable marker
When a foreign gene is introduced into plants, what is also usually introduced at the same time?
selectable marker gene
What components do Ti plasmid-based vectors contain?
selectable marker gene, origin of replication for e coli, right border sequence, polylinker or multiple cloning site to facilitate insertion of cloned gene, killer gene to prevent unwanted vector DNA past left border
Many flowering plants such as roses, Aftrican violets, and orchids have been produced by the process of __________________
selective breeding
You have benefited from ______________ by having more agricultural produce than would have been possible otherwise
selective breeding
Gel electrophoresis is a technique used to
separate DNA fragments by charge and length
Gel electrophoresis is a technique used to ___.
separate DNA fragments by charge and length
The law of independent assortment states that the inheritance of alleles for a different trait if the genes for the traits are on ___.
separate chromosomes
What is most of the specificity of transcription in plants controlled by?
sequence specific transcription factors and enhancer binding proteins
What does the integration of T DNA into the plant genome depend on?
sequences at right border of T DNA
What has been developed to exploit the strength of the T7 promoter?
series of plasmids called pET vectors
Where is synthesis of second strands often initiated?
several nucleotides from the 5' end of mRNA
In bacteria, what precedes each start codon?
shine delgarno sequence
What is often produced during the insertion of T-DNA into the plant chromosomal DNA?
short deletions of plant DNA at the junction between T DNA and plant DNA
How can metagenomic clones be characterized?
shotgun sequencing
What happens to a bacterial protein that is required by a cell?
signal system turns on gene
Explain the immunological assay.
similar to DNA hybridization, proteins are treated with a primary antibody then a secondary antibody that only binds to primary antibody then examined for color reaction from secondary antibody reaction
The two halves of a doubled chromosome structure are called
sister chromatids
What other modification can be done using RNA-DNA chimeric molecules?
site specific insertion or deletion of a single base
One major purpose of the protein called what is to tether replication components to the DNA, in particular to enhance the processivity of DNA polymerase
sliding clamp
What kind of plasmids are preferred for recombinant DNA experiments? What can you do to change Ti plasmids?
small, remove nonessential large segments
What must happen for molecular cloning to happen?
source DNA containing target sequence and cloning vector must be cut into reproducible fragments
What systems aid in detecting metagenomic clones that carry genes with certain functions?
specialized gene expression systems
In a dividing cell, the football-shaped structure consisting of thin fibers is the
spindle
What is disaccharide trehalose added to reverse transcriptase for?
stabilize the enzyme for DNA synthesis to proceed at a high temperature
What type of cleavages occur in type IIS restriction endonucleases?
staggered
What is produced by the symmetrical staggered cleave of DNA by EcoRI?
sticky ends
What does Biotin have a high affinity for?
streptavidin
What is the minimum requirement for an effective gene expression system?
strong and regulatable promoter sequence upstream from a cloned gene
What is used in SAGE to retain the magnetic beads with attached cDNAs during successive treatments and washings?
strong magnet
The historical method used to assign genes to particular human chromosome was to
study linkage data from human pedigrees
Parasitism, commensalism, and mutualism are examples of ___.
symbiosis
What is the purpose of the genes iaaM and iaaH?
synthesize auxin (indoleacetic acid)
What does E. coli DNA polymerase I do in the synthesis of mRNA to double stranded DNA?
synthesizes second DNA strand
What does reverse transcriptase do in the synthesis of mRNA to double stranded DNA?
synthesizes the first DNA strand
Applicants for the second job of the Help Wanted ad in Table 11-1, "Accuracy and Speed," could qualify if they were ___.
tRNA
The molecule ___ brings amino acids to the ribosomes for the assembly of proteins.
tRNA
How is translation initiated in eukaryotes?
tRNA binds to small ribosomal subunit then mRNA moves along until the anticodon UAC base pairs with the start codon of the mRNA
What promoters are estimated to be stronger and more effective than the trp and lac promoter?
tac and trc
What two promoters are commonly used hybrid constructs that are similar to each other, differing only by a single base pair?
tac and trc
What is the segment of cDNA produced by the BsmFI treatment called?
tag
What kind of database is searched to identify the corresponding gene of a concatemer?
tag to gene
In SAGE, what is BsmFI known as?
tagging enzyme
Northern blotts are also used to study splicing reactions - like in this case ^, the splicing is done differently in the heart and the brain because they have different amount of RNA
tells us about alternative splicing, size of mrna transcript, and estimate relative transcriptional activity of a gene
What special enzyme is used for the completion of the linear ends of each chromosome?
telomerase
What event can cause repressed or nonexpressed genes to become activated?
temperature increase or hormones
To determine if an individual with a dominant phenotype is homozygous or heterozygous, a ___ is used.
test cross
To determine if an individual with a dominant phenotype is homozygous or heterozygous, a ____________ is used
test cross
What happens when an effector molecule binds to an activator protein?
the Act-E complex doesn't bind to activating site and rate of transcription of operon is diminished
What has been used to stably change the genomic DNA of plant cells?
the RNA-DNA chimeric molecule
19. The term specific activity differs from the term activity in that specific activity: A) is measured only under optimal conditions. B) is the activity (enzyme units) in a milligram of protein. C) is the activity (enzyme units) of a specific protein. D) refers only to a purified protein. E) refers to proteins other than enzymes.
the activity in a mg of protein
Eukaryotic Genomes Vary Greatly in Size
the bigger the genome, the more repeated sequences there are
Genome
the complete set of DNA in a single cell of an organism
Which of the following would be least likely to happen as a result of a mutation in a person's skin cells?
the person's offspring have mutated skin
Water is lost to the abiotic parts of the biosphere from the biotic parts by the process of ___.
transpiration
Why would the absence of DNA primers in PCR cause the rxn to fail
the rxn lacks a primer: template junction and thus a free 3/'OH group when primers are absent
Examine the pieces of DNA represented in Fig 13-1. Why are the nucleotide sequences on both strands referred to a palindromes
the sequences are the same but run in opposite directions
Examine the pieces of DNA represented in Figure 13-1. Why are the nucleotide sequences on both strands referred to as palindromes?
the sequences show chromosome mutation
Watson and Crick were the first to suggest that DNA is ___.
the shape of a double helix
Proteomics Describe the 2D gels
the study and comparison of all the proteins that result from an organism's genome Run the sample in one dimension: Ran in pH, separated by charge Run the sample in the 2nd dimension: Take the tube just run and put in crevice. Separated based on molecular weight, and charge. Size of protein. Small proteins at bottom. Larger on top.
13. At the isoelectric pH of a tetrapeptide: A) only the amino and carboxyl termini contribute charge. B) the amino and carboxyl termini are not charged. C) the total net charge is zero. D) there are four ionic charges. E) two internal amino acids of the tetrapeptide cannot have ionizable R groups.
the total net charge is zero
Pollination can best be described as ___.
the transfer of the male pollen grain to the female organ
What are the names of the cytokinins?
transzeatin and transribosylzeatin
Each bacterial colony is a clone, carrying a specific cloned DNA sequence
the whole thing plated on the medium is a colony, and each are a clone of some specific DNA sequence
evosight improve what property of what enzyme?
thermostability enhancement of CALB
What must a secreted protein pass through in a gram-positive bacteria?
they are transported across a single cytoplasmic membrane
Why would cells with recircularized plasmids be able to grow with ampicillin in the medium and produce blue colonies?
they can form B-galactosidase
If cancer is present, what is the likely explanation for what happened to cells B and D?
they died off because the cancerous cells deprived them of nutrients
What strains of A. tumefaciens can induce crown gall tumors?
those possessing Ti plasmid
What are the only cells that will survive in the presence of IPTG and antibiotic?
those that carry a plasmid with a DNA insert
The cell produced when a male gametes fuses with a female gamete
zygote
Cells that work together to perform the same function are organized into
tissues
Why is mRNA usually extracted from 2 or more sources?
to compare expression profiles
What were cloning vectors developed for?
to overcome the issue of missing information for cellular maintanence
What could make it difficult to induce transcription?
too many repressor protein molecules
What is the term for an entire plant being regenerated from a single plant cell?
totipotent
What technique can be used to rid the transgenic plant of the selectable marker?
traditional breeding where the two genes are separated by chromosome segregation during a few rounds of mating
How can the RAD54 gene be deleted?
traditional genetic crosses with wild type plant, selecting for plants containing desired altered gene but not RAD54
Where do secretory proteins emerge from?
trans face of golgi
Messenger RNA is formed in the process of ___.
transcription
What is the production of RNA from DNA called?
transcription
How are the protein coding genes decoded?
transcription and translation
What happens if the TATA sequence is deleted or grossly altered?
transcription cannot occur
What happen in SIGEX if a catabolic operon is activated by the substrate?
transcription continues through gfp and gfp produced
What is the control of transcription in eukaryotes mediated by?
transcription factors
What happens to a bacterial protein that is not needed by the cell?
transcription of that gene is turned off
What is the other term for functional genomics?
transcriptomics
What are the products of the vir genes essential for?
transfer and integration of T DNA region into genome of plant cell
In the cointegrate vector system, after recombination, when the cloning vector becomes part of the disarmed Ti plasmid, what are the vir genes necessary for?
transfer of T DNA to host plant cells
What is recombinant DNA technology?
transfer of genetic information from one organism to another
What is the introduction of DNA into a bacterial host cell called?
transformation
What is the name of the process in which free DNA is introduced into a bacterial cell?
transformation
What is the process called of introducing purified DNA into a bacterial cell?
transformation
Cells that do not produce gfp in the presence of IPTG are considered what?
transformed
What is another name for the DNA construct?
transformed cells
How do you remove cells that produce green fluorescent protein in the presence of IPTG?
transformed cells are subject to FACS
Which of the following are applications of genetic engineering?
transgenic bacteria in agriculture, transgenic plants and animals, transgenic bacteria in industry- all of these
In 1974, Stanley Cohen and Herbert Boyer inserted a gene from an African clawed frog into a bacterium. The bacterium produced the protein coded for by the inserted frog gene. The bacterium containing functional frog DNA would be classified as a ___.
transgenic organism
The process illustrated in Figure 11-1 is called ___.
translation
The chromosome abnormality that occurs when part of one chromosome breaks off and is added to a different chromosome is ___.
translocation
How do you create a genomic library?
treat DNA with a four cutter restriction endonuclease to divide into clonable elements and insert them in host cells
How is the production of the original closed circular plasmid DNA reduced in the cloning vector pBR322?
treat with alkaline phosphatase to remove 5' phosphate groups from the linearized plasmid DNA
What happens after the adaptors are ligated to N1aIII digested cDNA in SAGE?
treated with BsmFI
How is the uptake of plasmid DNA usually achieved?
treating mid-log-phase cells with ice cold CaCl2 and exposing them for 2 min to high temps
step in the passage of energy and matter through an ecosystem
trophic level
From which promoter does the -35 region of the tac and trc promoters come from?
trp promoter
Describe the auxin pathway.
tryptophan to indole 3-acetamide by tryptophan monooxygenase AND indole-3-acetamide to indoleacetic acid by indole-3-acetamide hydrolase
What is T-DNA a part of?
tumor-inducing (Ti) plasmid carried by most strains of A. tumefaciens
How can you overcome the limitations of shuttle vectors?
two different recognition sites for SfiI with different variable sequences to prevent self ligation amongst SfiIx and SfiIy sites, then insert two sequences into E. coli and host cell plasmids so that E. coli plasmid has selectable marker gene and cloning site and host cell plasmid has origin of replication
Which secretion pathways are Sec independent?
type I and type III
What pathway are most secreted proteins passed through?
type II
What are the enzymes that cut DNA at specific base pair sequences?
type II restriction endonucleases or restriction enzymes
What genes are used as reporter genes?
uidA and gfp (beta glucuronidase and green fluorescence protein)
What does the killer gene prevent? What happens if this happens anyways?
unwanted vector DNA past left border from being incorporated into transgenic plants, transformed cells die
How much inserted DNA do plasmid-based vectors used for cloning DNA molecules generally carry?
up to 10 kb
Does RNA polymerase bind upstream or downstream of the coding sequence?
upstream
In SIGEX, where are the regulatory elements found?
upstream of catabolic genes
What is the term for the preparation of the microorganism and the raw materials required for the microorganism to grow and produce the desired product?
upstream processing
What are the 3 key stages of an industrial biotechnology process that uses microorganisms for producing a commercial product?
upstream processing, downstream processing, fermentation and transformation
How can you increase the likelihood of identifying additional novel genes?
use broad host range vectors and other host cells
What are type IIS restriction endonucleases?
used in multiplex polony sequencing where they cut DNA in both strands, a fixed number of nucleotides away from one end of the recognition site
What are some methods for normalization to calibrate data among replicate microarrays?
using fluorescent intensity of a gene not expressed under different conditions as reference point, include spiked control sequences that are different from target and bind only to control probe, adjusting total fluorescence intensity for each microarray to similar value
Michaelis-menten equation
v=vmax/1+Km/[S]
What enzyme is associated with vancomycin resistance in enterrococcus faceaelis.
vanG ligase
How can the extent of particle penetration into the target plant cells be controlled?
vary intensity of explosive burst, alter distance particles must travel, use different size particles
What is often not tested for in the transfer of T DNA?
vector DNA sequences past left border
What is the SfiIx-SfiIy procedure called?
vector backbone exchange
A gene gun and a virus may both be classified as ___ because they are mechanisms by which foreign DNA may be transferred into a host cell.
vectors
What is the advantage of fosmids?
very stable single copy vectors
What compounds can plants be engineered to synthesize?
viral antigenic determinants for vaccines
What plant cell DNA-delivery method is not an effective way to deliver DNA to plant cells?
viral vectors
As a cell grows, its ___ increases more than its ___.
volume, surface area
The ___ consists of evaporation, precipitation, transpiration, runoff, and respiration.
water cycle
proto-oncogenes encode transcription factors that stimulate expression of other genes, signal transduction molecules that stimulate cell division, and cell cycle regulators that move cells through the cycle
when a proto-oncogene is mutated or overly expressed and contributes to the development of cancer it is an ***ONCOGENE***
When is cAMP's level the highest?
when amount of glucose in medium is lowest
Mutations in the EGF (epidermal growth factor) and neu growth factor RECEPTORS result in stimulation of cell division even in the absence of the hormone
when it loses the functional growth factor receptors, it causes it to not need the hormone to bind to cause cell division, so it is always active -some mutations cause it to bind the hormone and dimerize it doubling the output of the cell division -mutation of the neu growth factor receptor is a base substitution -mutation in EGF receptor is a deletion
In prokaryotes, what initiates translation?
when new mRNA emerges, a ribosome binds to ribonucleotide sequence at 5' end of mRNA
What method involves silicon carbide fibers and protoplasts being vortexed and then the fibers punch holes in the plasma membrane which allows passage of DNA into the cell?
whisker mediated
Where are the opines synthesized?
within crown gall
Where do most common molecular-cloning protocols cleave?
within sites of 4 and 6 nucleotide pairs
What gene promotes integration of homologous introduced DNA into the plant genomic DNA?
yeast RAD54 gene
How can large segments of DNA be introduced into plants?
yeast artificial chromosomes (YAC)
Do cells that carry plasmids without inserts (self-ligating) produce gfp in the presence of IPTG?
yes
Do plasmids with inserts containing promoters that are constitutively active produce gfp in the presence of IPTG?
yes
Do plasmids with inserts that do not prevent transcription of gfp from the lac promoter produce gfp in the presence of IPTG?
yes
