BIO 325 Exam 3
Uses of expressing cloned genes in Research:
-Help explain its cellular function. -Aids in purification of large amounts of protein needed for biochemical or biophysical studies.
Uses of expressing cloned genes in Biotechnology:
-Introduced into bacteria to make pharmaceutical products such as insulin. -Introduced into plants & animals to make transgenic species with desirable traits.
PCR: 3 Steps
1. Denaturation 2. Annealing 3. Extension
Steps in Base Excision Repair
1. N-glycosylase recognizes an abnormal base and cleaves the bond between the base & sugar 2. If a purine is removed, an apurinic site is created. If a pyrimidine is removed, an apyrimidinic site is created. 3. The region is recognized by AP endonuclease, which makes a cut at the 5' side of the abnormal nucleotide. 4. DNA polymerase removes the abnormal region and fills it in with normal DNA 5. DNA ligase seals the repaired strand.
Steps in Nucleotide Excision Repair in E. coli:
1. UvrA/UvrB complex scans along DNA until encounter a thymine dimer 2. UvrA dimer is released 3. UvrC binds to UvrB 4. UvrC makes two cuts in the damaged DNA strand (one 8 nt 5' to the site, one 5 nt 3' from the site) 5. UvrD (a helicase) binds to the site and causes the damaged strand to be removed 6. UvrB and UvrC are then released 7. DNA Polymerase resynthesizes a complementary strand 8. DNA ligase makes the final connection
a small effector molecule that causes transcription to decrease can happen in what two ways?
1. corepressor (binds to a repressor & causes the protein to bind to DNA) 2. inhibitor (binds to activator protein & prevents it from binding to DNA)
Researches often measure the amount of ____ in a sample of DNA to determine the extent of oxidative stress
8-oxoG
Name the cause of this spontaneous mutation: Abnormal crossing over may cause deletions, duplications, translocations, and inversions
Aberrant recombination
Name the cause of this spontaneous mutation: Abnormal chromosomal segregation may cause aneuploidy or polyploidy
Aberrant segregation
A ___ protein can remove methyl or ethyl groups from guanine bases that have been mutagenized by alkylating agents.
Alkyltransferase
(BER/NER) is particularly important for the repair of oxidative DNA damage
BER
Name this DNA Repair System: An abnormal base or nucleotide is first recognized and removed from the DNA. A segment of DNA is excised, and then the complementary DNA strand is used as a template to synthesize normal DNA.
BER & NER (Base Excision Repair and Nucleotide Excision Repair)
the lacZ gene encodes the enzyme _____
Beta-galactosidase
Name 4 medical conditions that overaccumulation of ROS has been implicated.
Cardiovascular disease, Alzheimer disease, chronic fatigue syndrome, aging
(BER) Base excision repair involves _____ enzymes that recognize an abnormal base & cleave the bond beween it and the sugar backbone, creating an apurinic or apyrimidinic site.
DNA N-glycosylases
(Gel retardation assay/DNase I footprinting) can determine the regions of a DNA molecule that are bound by a protein
DNase I footprinting
Name the cause of this spontaneous mutation: Cytosine and 5-methylcytosine spontaneously deaminates to create uracil or thymine
Deamination
Name the cause of this spontaneous mutation: linkage between purines & deoxyribose spontaneously breaks. If not fixed, can lead to a mutation
Depurination
Name this DNA Repair System: An enzyme recognizes an incorrect alteration in DNA structure and directly converts it back to a correct structure
Direct repair
Name the cause of this spontaneous mutation: Mistake in DNA polymerase causing a point mutation
Errors in DNA replication
T/F ROS is always harmful
False (ROS are produced by the immune system as a means of killing pathogens and some ROS are used in cell signaling)
(Gel retardation assay/DNase I footprinting) can determine if a protein binds to a specific DNA fragment or RNA molecule because the binding of the protein slows down the movement of the DNA through a gel.
Gel retardation assay
Name the Technique of Gene Cloning that: Uses labeled DNA strandes from a cloned gene to identify similar or identical genes or RNA. Southern & Northern blot analysis methods are used. Used to localize genes within intact chromosomes and DNA fingerprinting.
Gene probes
Name the Technique of Gene Cloning that: Provides enough DNA to subject the gene to DNA sequencing . The sequence can reveal the gene's promoter, regulatory sequences, and coding sequence. Important in the identification of alleles that cause cancer and inherited human diseases.
Gene sequencing
Name this DNA Repair System: Occurs at double-strand breaks or when DNA damage causes a gap in synthesis during DNA replication. The strands of a normal chromatid are used to repair a damaged chromatid.
Homologous recombination repair
Name this DNA Repair System: Similar to an excision repair except the DNA defect is a base pair mismatch, not an abnormal nucleotide. The mismatch is recognized, and a segment of DNA is removed. The parental strand is used to synthesize a normal daughter strand of DNA.
Mismatch repair
The number of mutant genes divided by the total number of genes within a population
Mutation frequency
The likelihood that a gene will be altered by a new mutation. (expressed as the # of new mutations in a given gene per cell generation)
Mutation rate
In (NER/BER) several nucleotides in the damaged strand are removed from the DNA--and the intact strand is used as a template for re-synthesis of a normal compelementary strand.
NER
Name this DNA Repair System: Occurs at double-strand breaks. The broken ends are recognized by proteins that keep the ends together; the broken ends are eventually rejoined.
Nonhomologous end joining
(Southern/Northern/Western) blotting uses a labeled DNA probe to detect a specific RNA within a mixture of many different RNAs
Northern
____ refers to an imbalance between the production of ROS and an organism's ability to break them down
Oxidative stress
UV light is a Physical Mutagen that can cause what effect on DNA structure?
Promotes pyrimidine dimer formation, such as thymine dimers
______ ____, also called ___ _____, cut chromosomal DNA and vector DNA to produce sticky ends that will hydrogen bond with each other. _____ ____ is needed to make a covalent link in the DNA backbone.
Restriction enzymes; restriction endonucleases; DNA ligase
cDNA can be made from mRNA via _____ ____
Reverse Transcriptase
Name the Technique of Gene Cloning that: Can be manipulated to change its DNA sequence. Mutations within genes can help to identify gene sequences such as promoters & regulatory elements. This study can also help to explain its normal function and how its expression may affect the roles of other genes. Can reveal which amino acids are important for a protein's structure and function.
Site-directed mutagenesis
(Southern/Northern/Western) blotting uses a labeled DNA probe to detect the presence of a particular gene sequence within a mixture of many gene sequences. At low stringency, gene families may be detected.
Southern
Name the cause of this spontaneous mutation: Spontaneous shifts in base structure causing mutations if they occur immediately prior to DNA replication
Tautomeric shifts
Name the cause of this spontaneous mutation: The products of normal metabolic processes, such as reactive oxygen species (ROS) may be chemically reactive agents taht can alter teh structure of DNA
Toxic metabolic products
Name the cause of this spontaneous mutation: elements inserting themselves into the sequence of a gene
Transposable elements
Name the cause of this induced mutation: physical phenomena that can damage the DNA
UV light & X-rays
Uses of expressing cloned genes in Clinical Trials:
Used in clinical trials involving gene therapy
In Prokaryotes, the NER system requires four key proteins:
UvrA, UvrB, UvrC, and UvrD (& help of DNA polymerase & DNA ligase)
(Southern/Northern/Western) blotting uses antibodies to detect a specific protein within a mixture of many different proteins
Western
How do tautomers interconvert?
a chemical reaction involving the migration of a hydrogen atom and a switch of a single bond and an adjacent double bond.
the lac operon is regulated by a (repressor/activator)
activator
positive control occurs with (repressors/activators)
activators
The common, stable form of adenine and cytosine is the (amino/imino) form
amino
Deamination involves removal of an ____ from the ___ base, producing ___
amino group; cytosine; uracil
what does an effector molecule bind to?
an activator or repressor
Some TNRE disorders have the unusual fature of a progressively worsening severity in future generations, called _____ or ____ _____
anticipation; dynamic mutation
X-rays are Physical Mutagens that can cause what 4 effects on DNA structure?
base deletions, single-stranded nicks in DNA, cross-linking, chromosomal breaks
tautomers are ___ which exist in ___ and ___ or ___ and ___ forms.
bases; keto and enol or amino and imino
inducible operons typically encode (catabolic/anabolic) enzymes, whereas repressible operons often encode (catabolic/anabolic) enzymes
catabolic; anabolic
the lac operon can be regulated by (catabolite/anabolite) repression and the trp operon can be regulated by (catabolite/anabolite) repression
catabolite; anabolite
If ROS accumulate, they can damage _____ including ___, ___ and ___
cellular molecules including DNA, proteins and lipids
Name the cause of this induced mutation: substances that may cause changes in the structure of DNA
chemical agents
what controls the switch during the lytic cycle?
cro protein
Nitroous acid is a Chemical Mutagen that ______
deaminates bases
Most common type of chemical change that occurs naturally:
depurination
mutation of a regulatory element/operator site may ___ the ability of the gene to be properly regulated
disrupt
an inducer is a small effector molecule that causes transcription to _____
increase
mutation of a promoter sequence may ______ the rate of transcription
increase or decrease
3. Primer Extension: temp & function
incubate at slightly higher temp, allows DNA synthesis to occur
_____ mutations are caused by environmental agents
induced
lac operon and ara operon are (inducible/repressible) systems
inducible
trp operon is a (inducible/repressible) system
inducible
Allolactose binds to the repressor and prevents repressor from binding to operator, (inhibiting/inducing) transcription
inhibiting
lac repressor protein binds to lac operator and (inhibits/induces) transcription
inhibits
Reverse transcriptase PCR (is/is not) extraordinarily sensitive and (can/cannot) detect the expression of small amounts of RNA from a single cell.
is; can
The common, stable form of guanine and thymine is the (keto/enol) form
keto
the ara operon is similar to the (lac/trp) operon
lac
what controls the switch during the lysogenic cycle?
lambda repressor
2. Primer Annealing: temp & function
low temp allows primers to bind to template DNA
the expression of cII protein favors the (lytic/lysogenic) cycle. The expression of cro protein favors the (lytic/lysogenic cycle)
lysogenic; lytic
Reverse transcriptase PCR begins with ___ to study gene expression
mRNA
mutatino of the 5'-UTR/3'-UTR may alter the ability of the ___ to be translated and may alter ___ stability
mRNA; mRNA
In eukaryotes, ROS are _____ produced as (wanted/unwanted) by-products of energy production in mitochondria
naturally; unwanted
when tryptophan levels are high, tryptophan acts as a corepressor that binds to the trp repressor protein and binds to the operator site, turning (on/off) the operon.
off
(ROS) Reactive oxygen species (such as hydrogen peroxide, superoxide, and hydroxyl radical) are products of ____ metabolism in all ____ organisms.
oxygen; aerobic
Cloning vectors can be derived from ___ or ___
plasmids; viruses
feedback inhibition & covalent modification are forms of (transcriptiona/translational/posttranslational) regulation
posttranslational
mutation of the splice recognition sequence may alter the ability of ____ to be properly spliced
pre-mRNA
what do constitutive genes frequently code for?
proteins that are continuously needed for the survival of the bacterium
Depurination involves removal of a ___ from the DNA, creating an ___ site
purine; apurinic site
the genes that corepressors & inhibitors regulate are (inducible/repressible) genes
repressible
the trp operon is regulated by a (repressor/activator)
repressor
negative control occurs with (repressors/activators)
repressors
1. Denaturation: function & temp
separates DNA strands; high temp
_______ mutations are changes in DNA structure that result form abnormalities in biological processes
spontaneous
What enzymes do cells use to prevent buildup of ROS?
superoxide dimutase and catalase (antioxidants like Vitamin C)
intergenic suppressor
suppressor mutation in a different gene from the first mutation (find info about proteins that have similar or redundant functional roles)
deamination of 5-methylcytosine prouduces ____
thymine
Nucleotide Excision Repair (NER) can repair what 4 types of mutations?
thymine dimers, chemically modified bases, missing bases, certain types of crosslinks
What is the purpose of vector DNA
to act as a carrier of the DNA segment that is to be cloned
antisense RNA is form of (transcriptiona/translational/posttranslational) regulation
translational
constitutive genes are (unregulated/regulated) genes
unregulated
deamination of cytosine produces ____
uracil
A ___ is a small DNA molecule that can replicate independently of host cell chromosomal DNA and produce many identical copies of an inserted gene.
vector
intragenic supressor
when the second mutant site is within the same gene as the first (change in protein structure that compensates for an abnormality in protein structure caused by the first mutation)