Genetics
I=1
-"complete interference" -no double recombination
Genes on same chromosome..
-"synthetic" -form linkage groups -number of linkage groups = number of chromosomes -non additivity summed up map distances can b >.5
Allele Specific Oligonucleotides (ASOs)
-short single stranded DNA fragments (~20 bases) that can be used to identify single nucleotide polymorphisms (SNPs)
What enzymes were used traditionally to study regulatory elements?
Galactosidase and glucuronidase
Cells halt progress through the cell cycle if..
DNA replication, repair, or chromosome assembly are aberrant
what are the 3 steps of translation?
Initiation, Elongation, Termination
Key stages of Meiotic Prophase I
Meiosis I "reduction division" -homologous chromosomes pair up (synapsis) -form bivalents or tetrads containing 4 copies of homologous chromatids -crossing over of homologs (recombination) -chiasmata move to end of chromosomes
What method has led to helping dog breeders reducing genetic disorders?
PCR
Restriction map
establishes the number and order of restriction sites and the distance b/w restriction sites in a cloned DNA segment
Histones near inactive genes tend to be..
hypoacetylated
proteins
much more variable structure and charge
nucleotide
nucleoside + a phosphate group added
What has the FDA approved for treatment of myeloid lukemia and myelodysplastic syndrome?
decitabine, marketed as Vidaza
Degree of Penetrance
fraction of individuals w/ a certain phenotype ex.) 6 of 8 dogs w/ bb genotype were yellow dog at penetrance of yellow = 6/8 = 75%
"lost" allele
frequency = 0
"fixed" allele
frequency = 1
p
frequency of an allele
q
frequency of other allele
How can genomic libraries be made?
from sub genomic fractions such as single chromosome generated by methods such as flow sorting and pulsed field gel electrophoresis
Immunoglobulins
function in the immune system of vertebrates
Shine Dalgarno sequence
involved in binding in prokaryotes and precedes start codon and base-pairs with a region of 16S rRNA of 30S small subunit
Pericentric Inversion
involves centromere
Base Excision Repair (BER)
involves recognition of the erroneous base by DNA glycosylase and cutting of the DNA backbone by AP endonuclease
Use of bacteria..
is very worthwhile and has no significant downside ex.) Human insulin is now produced in bacteria
Why is yeast useful?
it can do things to proteins that bacteria cannot
how does bacterial DNA differ from eukaryotic DNA?
it is single and circular and synthesis originates at a single point
Why is the triphosphate form significant?
it is the precursor molecule during nucleic acid synthesis
Polyploids
organisms w/ 3 or more complete chromosome sets
Where does DNA replication begin?
origins of replication
2 classes of Homologs
orthologs & paralogs
Inherited dominant autosomal mutations will be expressed..
phenotypically in the first generation
What kind of bond links nucleotides?
phosphodiester bonds between the phosphate group at the C-5' position and the OH group on the C-3' position
Ethical, Legal, and Social Implications (ESLI)
program was established as an adjunct to the to the Human Genome Project to address these issues
Recombinant DNA technology
technology used to copy or clone DNA and allowed scientists to isolate and study specific DNA sequences
What processes make up the central dogma of molecular genetics?
Transcription & Translation
Oncogene
a mutated or abarrently expressed proto oncogene that contributes to development of cancer (gain-of-function alteration: dominant)
Silent Mutations
alter codon, but do not change the amino acid encoded ex.) CCT to CCC
Translation requires..
amino acids, messenger RNA (mRNA), ribosomes, transfer RNA (tRNA)
What does the degree of methylation in transposons promoter dictate?
amount of yellow pigment produced and varies b/w individuals, even in genetically identical mice
In vitro fertilization (IVF) in farm animals
can cause problems w/ imprinted genes during embryonic growth and development
what is the fx of RecB, RecC and RecD proteins?
can cleave DNA strands and help unwind the duplex
Epigenetic changes
can modify gene expression, potentially leading to tumor progression
A-DNA
slightly more compact than B-DNA
Making cDNA for cloning..
...
p+q
=1
what relieves supercoiling?
DNA gyrase
Physical Maps
map genes based on DNA sequences
what enzyme removes primers by looping the lagging strand?
DNA polymerase I
blastx
translated nucleotide vs protein database
which is the only enzyme that exhibits 5' to 3' exonuclease activity?
DNA polymerase I
Meiosis II
"equation division"
Monoploid
# of chromosomes in the basic set
Genotype Counts
# of individuals with each genotype ie. MN blood groups
what happens during RNAi?
(RNA interference) -short interfering RNAs (siRNAs) 21-25 nucleotides long are produced by 2 molecular machines -DICER recognizes double stranded RNA and cuts it into short double stranded pieces -RNA-induced silencing complex (RISC) binds pieces, makes them single stranded, then uses them to target and breakdown complementary mRNAs
TERRA
(telomeres repeat-containing RNA) - integral component of telomeres, acts as an inhibitor of telomerase
Structural Genomics
(transcriptomics) focuses on sequencing genomes and analyzing them to identify genes and other important sequences such as regulatory elements
Inversions
-ABCD -> ABDC -often forms loops in Meiosis
Cancer Statistics
-1 M cases diagnosed a year -500,000 deaths/year (300,000 are >65) -1 in 3 people are diagnosed with cancer in their lifetime
mono-allelic inheritance
-1 of the 2 parental alleles is non-functional BUT no changes seen in the DNA sequence -associated w/ methylation of DNA, usually at CYTOSINE residues
Copia elements in Drosophila
-10 to 100 copies -may move to different chromosome locations throughout the genome ex.) eye color and segment formation are due to copia insertions w/ in genes -the copia elements demonstrate regulatory effects at the point of insertion in the chromosome
What percentage of human cancers are associated w/ viruses?
-15%
Quaternary Structure
-4 chains: 2 beta chains & 2 alpha chains -heme group (Oxygen binds to Fe group)
Genetic disorders that dogs and humans share
-400 single-gene disorders -sex-chromosomes aneuploidies -multifactorial diseases ie. epilepsy -behavioral conditions ie. obsessive compulsive disorder
Major Surprises from the HGP
-<2% of the genome codes for proteins and that there are probably only 20,000 protein-coding genes -many genes for multiple proteins through alternative splicing - produces an incredible diversity of proteins beyond the number of human genes -human genome is 99.9% the same (regardless of racial and ethnic origins) with most genetic differences resulting from.. -Single Nucleotide Polymorphisms (SNPs) -Copy Number Variations (CNVs)
Genotyping (DNA) microarrays
-ASOs can be used to simultaneously examine SNPs in thousands of genes -each SNP is represented by 5 oligonucleotides that differ by 1 base or a deletion -many sets exist for mutations in genes including p53 and BRCA1
What happens to DNA during apoptosis?
-DNA becomes fragmented, internal structure are broken down and the cells dissolve into small spherical structures (apoptoic bodies) -apoptoic bodies are engulfed by white blood cells (phagocytes)
Oxidative damage
-DNA damage by the by-products of normal cellular products -reactive oxidants (often radiation induced) free radicals - >100 chemical modifications of DNA have been found ie. Superoxides, Hydroxyl radicals, Hydrogen Peroxide -loss of modification of bases leads to miss-pairing
What did Watson and Crick propose about DNA structure?
-DNA is a right handed double helix in which the 2 strands are antiparallel and the bases are stacked on one another -the two strands are connected by A-T and G-C base pairing & there are 10 base pairs per helix turn -one chain runs 5' to 3', the other runs 3' to 5'
Error rates in DNA sequencing are caused by..
-DNA polymerases are not 100% faithful (especially in vitro) -proofreading is absent or reduced relative to in vivo -sequencing from highly repetitive regions can be very hard to align w/ certainty
what did the Meselson-Stahl experiment demonstrate?
-DNA replication is semi-conservative in bacteria -each new DNA molecule consists of one old strand and one newly synthesized strand
Transposons
-DNA segments that can move w/ or w/ out replication -integrations into new genomic locations can act as naturally occurring mutagens -insertions, deletions, chromosomal breakage
molecular hybridization
-DNA strands (or DNA and RNA strands) can be denatured and renatured to each other -basis for many techniques for studying DNA -single stranded polynucleotides (DNA or RNA) will pair up in appropriate conditions -% identity needed to pair can be manipulated -length and GC content -buffer contradictions (salt concentration & pH) -temperature
A strong link b/w defective mismatch repair and cancer has been documented
-Hereditary nonpolyposis colon cancer -Leukemia -Lymphoma -Tumors of the ovary, prostate, and endometrium
2 types of bacterial Transposons
-Inversion of sequences (IS elements) -Tu elements
Ac-Ds System in Maize
-Maize has 2 transposable elements, Ac and Ds -Ds moves only if Ac is present, but Ac is capable of autonomous movement
Recombinant DNA
-a single DNA strand made from > source strand -recovered copies for producing copies of recombinant DNA molecule are referred to as clones and can be used for study
Duchenne Muscular Dystrophy
-severe -typically a result from a frameshift mutation in the dystrophin gene-premature stop codon -typically leads to nonfunctional truncated protein
triplet binding assay
-Nirenberg and Leder developed this technique to determine other specific codon assigments -ribosomes bind to a single codon of 3 nucleotides, and the complementary amino acid-charged tRNA will be able to bind -long RNAs with di, tri, and tetranucleotide repeats were used for in vitro translation to determine more codon assignments
DNA strand is doubled each cycle, and the new strands along w/ the old strand serve as templates in the next cycle. As a result..
-PCR is rapid (carried out in 1 - a few hours) -PCR is very sensitive, amplifying sequences from very small DNA samples
New technologies resulting in higher speeds, greater accuracies and reduced cost of sequencing of human genome research
-Personal Genome Project (PGP) -Human Microbiome Project ($115 million, 5 year project to complete the genome of 600-1000 microorganisms, bacteria, viruses, and yeast that live on and inside humans)
RNA vs. DNA
-RNA has ribose instead of deoxyribose -Uracil replaces Thymine -most RNA is single stranded, but some RNAs form double stranded regions as they fold into different secondary structures -some viruses have a double stranded RNA genome
how is RNA synthesis initiated? how is reaction expressed?
-RNA polymerase directs the RNA synthesis from a DNA template -no primer is reequired for initation & the enzyme uses ribonucleotides instead of deoxyribonucleotides n(NTP)*(DNA/RNA polymerase)*(NMP)(n+n)(PP1)
SNP
-Single Nucleotide Polymorphism -differences among genomes at particular (single) site -can occur w/ in or b/w genes -can lead to differences in restriction sites
Pyrimidine dimers
-UV radiation creates pyrimidine dimers that distort DNA conformation in such way that errors tend to be introduced during replication
Amino Acids all have..
-a carboxyl group -an amino group -an R group bound to a ventral Carbon atom
DNA vs. RNA codons
-a codon is always the sense strand (template DNA is anti sense) -only difference is that the RNA table uses U rather than T
Introducing transgenes into organisms
-a number of possible methods -vary according to organism type
What did analysis of adult hemoglobin reveal?
-a single amino acid substitution: valine for glutamic acid: at the sixth position of the beta chain -shows that a single gene provides the genetic information for a single polypeptide chain -a mutation causing a single amino acid substitution can affect phenotype
Adult Hemoglobin
-a tetramer made of 2 identical alpha chains and 2 identical beta chains in quaternary structure
Ubiquitin
-added in chains to other proteins -small 76 amino acid protein found in all cells of all eukaryotes -targets proteins for destruction by the 26S proteosome -short lived proteins such as cell cycle regulators or damaged or mutant proteins are particular targets
When does methylation in mammals take place? Where does it occur?
-after replication and during cell differentiation -addition of methyl group to cytosine bases located adjacent to guanine base forming a CpG dinucleotide
Ion torrent
-again a 1 base at a time principle, but detecting the 1 proton added each time -a cross b/w a PCR machine and a micro-pH meter -much cheaper
Gene Annotation
-aims to identify and label important structural features of genes (known or unknown) -regulatory elements in promotors and enhancers -exons and introns (splice sites) -translation start and stop sites -polyadenylation site
Insertion Sequences
-all IS elements contain two essential features -a gene encoding for the enzyme transposase makes staggered cuts into DNA, allowing the IS element to insert -IS element ends contain inverted terminal repeats (ITRs) which act as recognition sites for the binding of transposase
effects of Transposable elements on gene expression and chromosome structure..
-alter protein-encoding genes -alter or provide new regulatory elements -change splicing patterns -serve as DNA methylation sites -recombination b/w transposons can cause chromosomal rearrangements -retroviruses can also move genes in and out of genomes and alter host gene expression
What does alteration of nucleosome structure cause?
-altering the contacts b/w DNA and histones -altering the pathways of DNA around the nucleosome -altering the structure of the nucleosome core itself *all make the DNA more accessible to transcription factors
Primary Structure
-amino acid sequence -which amino acids, what order -often plays a major role in determining protein localization -also determines upper levels of structure
Synthetic Genome
-an artificially constructed genome to generate artificial cells or designer organisms -in 2008 scientists chemically synthesized the complete genome of the bacteria myoplasma genitalium, but could not transplant it into another bacterium
Temperature-sensitive Mutation
-an example of conditional mutations depending on environment -gene product functions at one temperature, but not another (temperature sensitive coat color variations in Siamese cats and Himalayan rabbits)
Carcinogenic
-any substance or event that damages DNA has potential to be carcinogenic if it causes mutations in proto-oncogenes or tumor suppressor genes -including chemicals, radiation, some viruses, and chronic infections
Cloning vector
-anything that will replicate DNA with a cell -different organisms require different vectors -different "origins of replication" -choice depends on needs
DNA microarrays (genotypic microarrays)
-are being used to detect mutations in genomic DNA and gene expression patterns in diseased tissue -most human genes are represented -DNA microarrays have been designed to scan for mutations in many disease-related diseases, including the p53 gene, which is mutated in a majority of human cancers & the BRCA1 gene, which when mutated predisposes women to breast cancer
Histone modifications in cancer cells
-are disrupted -genes that encode histone-modifying enzymes are often mutated or abarrently expressed in cancer cells
Phosphorylation and Dephosphorylation of proteins
-arguably the most important event in cell signaling -regulate enzyme activities and protein-protein interactions -protein kinases phosphorylate other proteins -protein phosphates dephosphorylate
Where is heterochromatin common?
-around telomeres and centromeres -tend to have fewer genes and lower recombination frequencies
Individuals suffering from sickle cell anemia suffer from...
-attacks when red blood cells aggregate in capillaries where oxygen tension is very low -various oxygen-deprived tissues suffer severe damage including: kidneys, muscles, joints, brain, gastrointestinal tract, and lungs -rapid loss of red blood cells, which are quickly destroyed, leads to their increased production -these mechanisms lead to abnormal bone size and dilation of the heart
PCR based methods using ASOs
-available to screen for many disorders, including sickle cell anemia and cystic fibrosis -can be used in early stage human embryos created for in vitro fertilization = preimplantation genetic diagnosis (PGD) -amplify gene by PCR -spot onto DNA binding membrane -hybridize to specific ASO probes under "high stringency" condition (determined by salt concentration and temperature) -tricky technique, controls are critical, DNA sequencing often used to confirm positive diagnosis
SMART Sequencing
-back to using different fluorochromes for each base (can use all nucleotides at once so much faster) -attached to pyrophosphate group, cleaved off rather than incorporated -flash of light of a specific wavelegth as each base is added -works in real-time - at the speed of the DNA polymerase
what is the function of DnaA?
-binds to the "origin of replication" and is responsible for the initial steps in unwinding helix
How is chromatin remodeling brought about? What is involved in chromatin remodeling?
-by remodeling complexes -repositioned nucleosomes make regions of the chromosome accessible to transcription regulatory proteins -transcription activators and RNA polymerase II
What is the significance of reversal of histone acetylation?
-modification is reversible and acetyl groups can be removed, changing the chromatin from an "open" to a "closed" configuration, silencing genes by making them unavailable
DNA based tests
-can be used to diagnose disease -usually mutations associated w/ single gene disorders ex.) Huntingtons Disease, Hemophilia, Sickle Cell Anemia, Cystic Fibrosis, Muscular Dystrophies -tests are also being developed for complex disorders like cancer
Gene expression microarrays
-can be used to study diseases -many forms of cancer show distinctive patterns of gene expression -these patterns can be detected by microarrays, and mRNA expressed only in cancer cells represent targets for drug development
Replication Slippage
-can cause small insertions or deletions -repeat sequences are hot-spots (Fragile X syndrome, Huntington's disease: hereditary disease caused by slippage mutation)
Migration
-can introduce new alleles into a local population -"gene flow"
PFGE
-can separate up to 2 mb -separation based on re-orientation time of DNA strands
Cancer cell methylation
-cancer cells are less methylated than normal cells and the promotors of some genes can be hyper methylated ie. the DNA repair genes MLH1 and BRCA1 are transcriptionally silenced in certain cancer cells
Nutritional (biochemical) Mutations
-cause loss in stability synthesize an amino acid or vitamin -biochemical mutations can have an effect on the well being and survival of the affected individual
Alkaptonuria
-caused by mutation that leads to metabolic block preventing breakdown of homogentisic acid -accumulates in cells and tissues and excreted in urine -oxidation products are black and detected in diapers, accumulates cartilaginous areas, causing ears and nose to darken -homogentisic acid accumulates in joints leading to benign arthritis -pedigree analysis of these diseases indicated that human diseases can have a genetic basis -afflicted individuals had parents who were first cousins (consanguine)
Polygenic
-caused by variations in several genes -most human genetic diseases are polygenic
What happens when tumor suppressor genes are mutated or inactivated?
-cells cannot respond normally to cell cycle checkpoints or are unable to undergo apoptosis if DNA damage is extensive -inactivation of both tumor-suppressor genes have been identified
Normal cells in G0 stage vs cancer cells in G0 stage
-cells that stop dividing enter G0 and become metabolically active -cancer cells are unable to enter G0 and cycle continuously -cells in G0 can often be stimulated to re-enter cell cycle by external growth signals via signal transduction pathways -cancer cells often have defects in these pathways leading to continuous stimulation to divide
Anaphase II
-centromeres divide
Anaphase
-centromeres divided -chromatids separate and daughter chromosomes move to opposite poles (due to shortening of spindle fibers)
Metaphase II
-centromeres line up at metaphase plate -attach to spindle fibers
Alternatives to Dideoxynucleotides
-chain terminates w/ addition of dideoxynucleotide -need a method to monitor base incorporated w/ stopping the reaction -Prosequencing (detection of pyrophosphate release NOT chain termination) -better, but have to flush w/ one nucleotide at a time and luciferase is expensive
Proteins
-chains of the 20 naturally occurring amino acids, covalently linked by polypeptide bonds -much more diverse than nucleic acids -variety in structure/shape = variety in function -diversity comes from chemistry of amino acid (R groups)
Mis-sense Mutations
-change a codon -result in an altered amino acid w/in a protein-coding portion of a gene ex.) GAC to GAG
Nonsense Mutations
-changes codon into a stop codon -result in premature termination of translation ex.) TAC to TAA
Chromatin is not uniform..
-changes through cell cycle AND varies across genome
p53 becomes more stable and transcriptionally active in response to..
-chemical damage to DNA -double stranded breaks in DNA by ionizing radiation -DNA-repair intermediates generated by UV light
"ordered" code
-chemically similar amino acids often share one or two middle bases in their codon -an ordered code potentially buffers effects of mutation on protein function
What effects does famine have on pregnant women?
-children w/ increased risk of obesity, diabetes, and coronary heart disease -as adults, these individuals had significantly increased risks for schizophrenia and other neuropsychiatric disorders -the F2 generation also had abnormal patterns of weight gain and growth
What have electron microscopic observations of chromatin revealed?
-chromatin fibers are composed of a linear array of spherical particles -chromatin is bound up in nucleosomes (the "beads") with positively charged proteins called histones or less positively charged non histones
What happens to chromatin during Interphase? Cell division?
-chromatin is dispersed through the nucleus during interphase -chromatin condenses and chromosomes become visible during cell division (length contraction of 10,000x for each chromatin fiber; human DNA/nucleus > 2m)
Telophase I
-chromosomes reach poles -cell divides -forms 2 products each w/ 1 homolog (2 cells w/ 1 n chromosome)
Telophase II
-chromosomes reach poles -nuclear membrane forms -cell division
Telophase
-chromosomes reach poles -cell divides
Prophase II
-chromosomes shorten
Prophase
-chromosomes thicken and shorten, attached at centromeres -nuclear membrane breaks down -spindle fibers form stretch between poles of cell
Prokaryotes Genome (usually)..
-circular genome -<5 Mb -500-5000 genes -"compact" genomes - gene density ~ 1kb
Self Fertilization
-selfed plants to create true breeding strains/lines -breed true for a particular trait
Excision Repair
-collective name for number of light independent repair mechanisms -cut and paste mechanisms w/ 3 steps: 1. Distortion in helix is recognized and cut out by a nuclease 2. DNA polymerase fills in the gap 3. DNA ligase repairs the backbone ex.) Nucleotide Excision Repair (NER) repairs bulky lesions and involves uvr genes
What did Feinberg and Vogelstein observe?
-colon cancer has much lower levels of methylation than normal cells derived from the same tissue -complex changes in DNA methylation patterns are associated w/ cancer
Comparative Genomics
-compares genomes of different organisms to answer questions about genetics and other aspects of biology -incorporates the study of gene and genomic evolution -explores the relationship b/w organisms and the environment -studies differences and similarities b/w organisms and how differences contribute to phenotype, life cycles, etc..
The most important applications of bioinformatics..
-comparing DNA sequences -identifying genes in genomic DNA sequences -finding gene-regulatory regions (promotors and enhancers) -identifying structural sequences ie. telomeres -predicting amino acid sequences -identifying protein structure and function -deducing evolutionary relationships b/w genes and organisms based on organism sequence
Significance of R group
-confers specific chemical properties -can be polar or non polar, hydrophobic or hydrophilic, charged/non charged
What major roles do imprinted genes play?
-control growth during embryonic and prenatal development
Retinoblastoma protein (pRB)
-controls G1/S cell cycle checkpoint -pRB protein activity varies throughout the cell cycle, depending on its phosphorylation state
What do RITS complexes do?
-convert euchromatic regions into heterochromatin, silencing genes w/in regions -reversible
Human Genome Project (HGP)
-coordinated international effort to sequence and identify all the genes in the human genome -began in 1990 under direction of James Watson -budget of $3 billion and a 15 year plan
Mismatch pair
-corrects errors that remain after proofreading -corrects DNA strand is recognized based on DNA methylation of the parental (original) strand -involves endonuclease, exonuclease, DNA polymerase, and DNA ligase
Mendel's monohybrid cross
-created hybrid offspring by crossing green & yellow -first filial generation (F1) were yellow -"yellow" = dominant -"green" = recessive -allowed F1 individuals to self fertilize, produced F2 generation
Bt crops
-cry: gene from Bacillus thuringiensis -makes a protein that crystallizes in guts of insect larvae and kills them -Bacillus thuringiensis is the major method of pest control for organic famers
To study DNA at a basic level we need these methods to..
-cut up DNA in a predictable way -separate DNA fragments -amplify DNA -identify specific DNA sequences -sequence DNA
How is cell cycle progress regulated?
-cyclin proteins and cyclin dependent protein kinases are synthesized and degraded in a precise pattern -incorrect timing or levels of cyclins disrupts the progression
How does age increase risk of cancer?
-daily, the human body suffers ~10,000 lesions due to the actions of oxygen free radicals -when DNA repair enzymes miss those damages, they become permanent mutations -the cumulative nature of mutations leading to cancer lead to cancer rates increasing dramatically with age
SOLID (supported oligonucleotide ligation and detection)
-developed by the company Applied Biosystems (ABI) produces 6 gigabases of sequence data per run -3rd generation sequences are available and will be widely used in the next few years, providing direct sequencing of DNA in a single strand
Prenatal Tests
-diagnosis of embryos that will develop disease -identification of carriers -prediction of future disease development -tests performed on samples from amniocentesis and chorionic villus sampling (CVS: fetal part of placenta)
What happens after implantation?
-differential genomic remethylation recalibrates which maternal and paternal alleles will be reactivated *imprinted alleles stay silent in all cells, while silenced genes by epigenetic methylation can be reactivated by external signals during or after differentiation
Short interspersed elements (SINES) and long interspersed elements (LINES)
-dispersed throughout the genome rather than tandemly repeated and constitute over 1/3 of the human genome -these "transposable elements" are segments of DNA generated via an RNA intermediate and are referred to as retrotransposons -probably evolved from retroviruses but now never leave the cell (endogenous parasites?)
Standard gel electrophoresis can..
-separate DNA up to 50 kb -can adjust agarose concentrations for different size
similarities b/w eukaryotic and prokaryotic DNA synthesis
-double stranded DNA unwound at replication origins -replication forks -bidirectional synthesis creates leading and lagging strands -eukaryotic polymerases require four deoxyribonucleoside triphosphate, a template, and a primer
How does RNA-induced silencing control gene expression? (2 ways)
-double stranded RNA is cleaved by DICER (double stranded RNase) and can take two pathways: 1. SiRNAs associate with an RNA-induced silencing complex to regulate gene expression in the cytoplasm of plants, animals, and fungi by repressing translation or triggering mRNA degradation 2. miRNAs associate w/ RNA-induced initiation of transcription (RITS) complex in the nucleus and alter chromatin structure to repress transcription in a phenomenon known as RNA induced gene silencing
How does RISC work?
-double stranded RNA is incorporated into a protein complex where one RNA strand is removed and degraded, forming a mature RNA-Induced Silencing Complex (RISC) containing the remaining single miRNA strand -RISCs act as post transcriptional repressors of gene expression by binding to and destroying target mRNA molecules with sequences complementary to RISC miRNA
Neanderthal Genome and Modern Humans
-draft of Neanderthal genome has provided 2/3 of the human genome -comparative genomic analysis will help identify genomic areas w/ humans have undergone rapid evolution since diverging from Neanderthals -99% identical, 78 new protein coding sequences since divergence -genomic studies suggest that interbreeding took place b/w Neanderthals and modern humans b/w 45 -80,000 years ago -gonome of non-African H. sapiens contains approximately 1-4% of sequences inherited form Neanderthals
Natural Selection
-driving force of adaptive evolution in wild populations -evolution by natural selection occurs b/c of consistent differences in survival and/or fertility among different genotypes -genotype associated w/ increased survival and/or fertility tend to increased in frequency -can lead to elimination of deleterious alleles -preserve genetic variation -not all natural selection causes evolution
How is the parent-specific pattern of allele expression set?
-during gamete formation -inherited via mitosis in somatic cells
Model
-each gene comes in 2 forms (alleles) -one allele from mother and one from father
what is Werner Syndrome? what causes it?
-early onset of many age related diseases: skin wrinkling, cataracts, osteoporosis, graying of hair, cardiovascular disease -result of mutation in gene called WRN (helicase that is part of the telomere cap structure) -thought to cause mis capping and premature aging
hybridization
-either using specific primers (PCR) or labeled "probe" homologous to gene of interest usually: -cDNA libraries are spread out on plates and "filter lift" copies are made -genomic BAC libraries can be arrayed in 384 well plates and "pools" made for screening by PCR -hybridization "stringency" can be manipulated (temperature +/or salt concentration)
Isoelectric focusing (IEF)
-electrophoresis -PAGE gel w/ pH gradient, protein move until charge equals 0
Fertilizers
-enabling non-legume species to form symbioses w/ Rhizobium species -reduce nitrate and nitrite pollution
what is involved in genetic recombination?
-endonuclease nicking -strand displacement and pairing with complement -ligation to create heteroduplex molecules -branch migration -duplex separation to generate the charicteristic "Holliday structure"
In general, Mendel observed..
-entire F1 generation resembled 1 of P lines -in F2, recessive phenotype reappeared -3:1 ratio
Whole genome shotgun sequencing method
-entire genome is cut into short, overlapping fragments using restriction enzymes -overlapping fragments collectively form one continuous DNA molecule w/ in a chromosome and are called continuous fragments or contigs
amanoacyl tRNA synthase
-enzyme that charges (activates) tRNAs w/ the appropriate amino acid -20 different ones
HGP Goals
-establish functional categories for all human genes -analyze variation b/w humans -sequence other genomes: E.coli, S cerevisaiae, C. elegans, D. melanogaster, M. musculus -develop new sequencing technologies -disseminate genome information
what is recombinant DNA technology? Give an example
-eukaryotic DNA corresponding to specific genes are isolated and spliced into the bacterial DNA and can be inserted into a bacterial cell and monitored -presence of the eukaryotic gene product in bacteria containing the eukaryotic gene provides direct evidence that this DNA is present and functional in the bacterial cell -has been shown to occur in many instances ie. insulin production by bacteria
Plasmids
-extrachromosomal DNA molecules that replicate independently from chromosomes in bacterial cells -engineered for ease of use in cloning -a number of convenient restriction sites -marker genes to provide: selection for presence in host & visual assessment of incorporation of new DNA -expression vectors often include a leader peptide to facilitate easy purification of expressed proteins
Next generation DNA sequencing
-faster, cheaper sequencing methods are being developed -Archon X PRIZE for Genomics -build and use a device to sequence 100 human genomes in <10 days -w/ <1 error every 100,000 bases (.001%) -need to reassess the reaction: Main speed issue: separating reaction products
How does variation in protein structure provide the basis of biological diversity?
-following translation, polypeptides fold up and assume high order structures, and may interact w/ other polypeptides -3-D conformation is critical to a protein's specific function
DNA sequencing original method
-for last 20 years "Sanger sequencing" has been standard (dideoxynucleotide chain termination sequencing) -DNA polymerase reaction but adding dideoxynucleotides -original method: 4 separate reactions each containing dNTP's and one of the 4 ddNTP's plus a radioactive label
Chaperones
-for some proteins, correct folding is dependent on chaperones -ubiquitous proteins that mediate folding by excluding the formation of alternative, incorrect proteins -misfolded proteins may be nonfunctional and can be detrimental to cells and organisms
Homologous recombinational repair
-form of DSB -fixes a double-strand break by digesting back the 5' ends leaving overhanging 3' ends that pair w/ the undamaged sister chromatid to allow DNA polymerase to copy the undamaged DNA sequence into the damaged strand
PCR primers
-forward primer complementary to 5' end -reverse primer complementary to 3' end of the other strand
Ethical questions related to Somatic gene therapy
-from a population genetics viewpoint is extremely dangerous -increase in prevalence of mutant alleles in our population (many genetic diseases, not cancer) -committed to ever increasing numbers of people needed treatment
How do spontaneous mutations arise?
-from replication errors and base modifications
"The 2010 Project"
-functional genomics and the virtual plant; a blueprint for understanding how plants are built and how to improve them -to understand the function of all genes of a reference species w/in their cellular, organismal, and evolutionary context by 2010 -systems biologists make huge claims that are yet to come true
Mycoplasma mycoides
-functional synthetic genome of mycoplasma mycoides was created in 2010 and successfully transplanted into an existing genome of M. capricolum
Preliminary list of functional categories to which human genes have been assigned, based on..
-functions determined previously -comparisons to known genes from other species -predictions based analysis of protein domains and motifs
sex chromosomes
-gene expression is largely dose dependent -defferent sexes have different numbers of sex chromsomes which encode more than just genes related to sex
Genome sequence is annotated to identify
-genes -their regulatory sequences -their functions *each sequence deposited receives an accession number
Synthesis of "gene chips"
-glass slides onto which single stranded DNA molecules are attached -hybridized to labeled cDNAs -enables analysis of all genes simultaneously -DNA oligonucleotides specific to "every" gene in genome
Spontaneous mutations
-happen naturally, randomly, and are usually linked to normal biological/chemical processes in organisms -rates vary among loci in different organisms
Commercial adoption of GM crops
-has been one of the most rapid cases of technology -80% of processed food contains transgenic food product -usually corn or soybean
Polysomy
-has extra copies of a particular chromosome -often caused by nondisjunction
The Polymerase Chain Reaction (PCR)
-has revolutionized molecular biology techniques -speed, ease of use, sensitivity
Edible vaccines
-have been produced in plants -decreases cost of production -PLUS avoiding needles, need for refrigeration, and need for sterile equipment to administer -bananas would be ideal
What phenotypic consequences can external or internal factors that disturb epigenetic pattern of imprinting produce?
-having only one functional allele makes imprinted genes highly susceptible to the deleterious effects of mutations -mutations in imprinted genes can arise by changes in the DNA sequence or dysfunctional epigenetic changes, called epimutations, both of which are heritable
How is chromatin remodeling facilitated?
-histones have "tails" that are not packed into the folded domains w/ nucleosomes -they have modifications such as acetylation, methylation, phosphorylation and ubiquination - >150 modifications have been found -huge potential information encoding involved in regulation of remodeling (histone code)
Anaphase I
-homologous chromosomes separate -sister chromatids remain attached at centromeres ->Dyads
How do epigenome modifications affect the phenotype of mice?
-homozygous AA mice: gene active only during specific time of hair development. yellow band on black hair shaft resulting in agouti phenotype -nonlethal mutant (A^vy) causes yellow pigment formation along entire hair shaft, producing yellow fur
Prader-Willi syndrome, Angelman syndrome, Beckwith-Weidmann syndrome
-human disorders associated w/ imprinting having their origins during fetal growth and development -in humans, most imprinted genes encode growth factors or other growth regulating gnes
Differences b/w Humans and Chimps
-humans have lost ~80 genes from the LCA -170 genes duplicated in humans (90 different ones in chimps)
Similarities b/w humans and dogs
-humans share 75% of their genes w/ dogs -similar genome size -suffer from many of the same genetic disorders
Mendel's Interpretation
-hypothesized that inheritance is particulate -phenotype is determined by genes at particular locus
Proteomics
-identification and characterization of all proteins encoded by the genome of a cell, tissue, or organism -after everything, potentially most informative "ome" but technically more challenging
Aneuploidy
-imbalanced gene complement -no mitotic issue w/ odd ploides
Reasons for generating transgenic crops include
-improving growth characteristics and yield of crops -increasing nutritional value of crops -providing crop resistance against insect and viral pests, drought, and herbicides -providing substantial sources of energy ie. corps for ethanol production and biofuel
where is crossing over less frequent?
-in heterochromatic regions ie. near centromere -map distance does not reflect physical distance in these regions
Systems Biology
-incorporates data from genomics, transcriptomics, proteomics, and other areas of biology as well as engineering application to attempt to elucidate components of interacting pathways and the inter-relationships of molecules interpreting genomic information in the context of the structure, function, and regulation of biological pathways
Monosomic
-individual missing copy of individual chromosome number 2n-1
What does the A^vy allele result from?
-insertion of transposable element near the transcription start site of the Agouti gene -promoter element in transposon causes change in gene expression
Frameshift Mutations
-insertions or deletions (loss or addition of one nucleotide) -occurs when any # of bases are added or deleted, except multiples of 3, which would reestablish the initial frame of reading
The rate of a mutation (likelihood that a gene will undergo a mutation in a single generation or in forming a single gamete)..
-is exceedingly low for all organisms -varies between different organisms -humans and drosophila have higher mutation rates than Neurospora, viruses, and bacteria -even w/ in the same species the spontaneous mutation rate varies from gene to gene
Method used to develop transgenic mice
-isolate newly fertilized eggs from a female mouse -inject purified clone DNA containing a vector and the transgene of interest into the nucleus of the egg and put the egg back in a female
what is the function of DNA polymerase I?
-it catalyzes DNA synthesis -it removes the primer (made of RNA) -synthesis that fills gaps produced during synthesis -3' to 5' & 5' to 3' exonuclease activity
how is RNAi useful?
-it is now a standard tool used to investigate gene function in a wide range of organisms -mechanism is highly conserved (probably evolved as a viral defense) -can target a single gene or multiple members of a gene family
How can DNA polymerase cause mutations?
-it occasionally inserts incorrect nucleotides, generally due to miss pairing -can proofread and correct these errors, but they may persist if not detected and corrected -predominantly leads to point mutations
What occurs once a gene has been imprinted? In mice?
-it remains transcriptionally silent during embryogenesis and development -most imprinted genes direct aspects of growth during prenatal development -in mice, genes on X chromosome are expressed in the placenta, but genes on the paternal X chromosome are silenced
biological important forms of Tautomeric shifts
-keto-enol forms of C and T -amino-imino forms of A and G -change base pairing as a result of modified H bonding
The C-Value Parodox
-large genomes contain a huge number of repeat sequence ie. 50% repetitive DNA in the human genome -C-value = DNA content in a genome -C-value paradox = lack of correlation b/w genome size and organism complexity ie. an amoeba has one of the biggest genomes known
Tu elements
-larger than IS elements -can introduce multiple drug resistance onto bacterial plasmids (R factors) -can move plasmids onto bacterial chromosomes, spreading multiple drug resistance b/w different bacterial strains
Whole genome shotgun sequencing relative to other methods
-less labor -more sequencing -harder to assemble -decreased sequencing costs -increased computing power
Becker Muscular Dystrophy
-less severe -primarily due to alteration of the protein sequence -full length but amino acids are changed
How to identify clones of interest
-library screening -several possible approaches most based on hybridization
Is sub-cellular localization essential to the biological function of PiCDPK1?
-localizes to the plasma membrane and the cytosol -possesses N-terminal lipid modification sites
Deamination
-loss of amino group (C or A) -changes C to U, A to hypoxanthine: changes pairing
Retinoblastoma 1 tumor suppressor gene
-loss of mutation of both alleles at RB1 contributes to the development of many cancers due to unregulated progression through cell cycle -breast, bone, lung, and bladder cancers
Depurination
-loss of nitrogenous base in the intact DNA helix usually an A or G - cause loss of template in replication
what are the classes of RNA and their basic functions?
-mRNA (messenger RNA) Functional RNA -tRNA (transfer RNA) - amino acid carriers -rRNA (ribosomal RNA) - protein synthesis machinery -snRNA (small nuclear RNA) - mRNA processing -miRNA (micro RNA) - mRNA stability -siRNA (small interfering RNA) - genome defense and DNA methylation)
What does histone acetylation do?
-makes genes on nucleosomes available for transcription
X-linked recessive mutations arising in gametes of a homogametic female may be expressed in hemizygous offspring ie...
-male offspring -this will occur provided that the male offspring receives the affected X chromosome
The cancer stem cell hypothesis
-many scientists believe that tumors are composed of a mixture of cells, many of which do not proliferate -tumor cells that proliferate give rise to cancer stem cells that have the capacity for self renewal -the stem cells divide unevenly, creating one daughter cell that becomes a mature cell type and one that remains a stem cell -every tumor cell has the potential to form a new tumor
Linkage Maps
-map genes based on patterns of inheritance -linear arrangement of genes w/ distances
Between cycles of mitosis, a cell in interphase..
-metabolic activities occur -hard to see individual chromosomes -chromosomes are replicated -sister chromatids -chromatid pairs attached at centromere
Current DNA sequencing methods
-modifications to the Sanger method allow reactions to occur in a single tube w/ each of the 4 ddNTPs labeled w/ a different fluorescent dye -DNA fragments are size fractionated by "capillary" electrophoresis and scanned w/ a laser to stimulate the dyes to fluoresce different wavelengths of light which computer converts into DNA sequence -cheaper and much faster than older method, but still rather slow and expensive
Moloney murine leukemia virus
-modified mouse virus -3 essential virus genes removed and human DNA inserted -unable to replicate in cells, but can integrate into human chromosomes and will hopefully express the human gene inserted
Why are dogs a phenomenal research genetic resource for humans?
-most have a small genetic base -based on selection for phenotype (genetic mutations) -tend to be homozygous: exposes recessive mutations -often genetic diseases are associated w/ particular breeds
Gene-therapy risks
-most have been traced to the vectors used -many viral vectors eventually cause an immune response (has killed patients) -development of leukemia has also been a big problem -also have a cloning capacity too low to deliver many genes -new viral vectors and genome targeting strategies are being developed to help overcome current problems
Palindromic
-most recognition sites are palindromic and are often cleaved sequences in and offset manner to produce "sticky ends"
P elements in Drosophila
-most significant transposable element in Drosophila -P elements can insert themselves into or near genes, causing high rates of mutations -Insertion into the coding region of a gene can terminate transcription and destroy normal gene expression -Insertion into the promoter can affect the level of gene expression -Insertion into introns can affect splicing or cause premature termination of transcription
p53 tumor suppressor gene
-mutated in 50% of cancers -encodes a transcription factor that represses or stimulates transcription of >50 genes -continuously synthesized, rapidly degraded -p53 can arrest the cell cycle at several phases, but cells lacking p53 are unable to arrest at cell cycle checkpoints or enter apoptosis in response to DNA damage
Cancer cells show higher than normal rates of..
-mutation -chromosome abnormalities -genomic instability
What have studies on drosophila by Hermann Muller shown?
-mutation of white gene for red eye color (near the end of the X chromosome) causes white eyes rather than red -he also found mutants with a varied phenotype (mixture of red and white facets on eye) -mutants were x-ray generated
What is usually necessary to fully express the cancer phenotype?
-mutations in other genes ex.) development of familial adenomatous polyposis (FAP)
Mutagens
-natural or artificial agents that induce mutations -wide array: chemicals (natural and man made), many wavelengths of radiation
Inbreeding
-nonrandom mating b/w relatives
Germ-line Interference
-not currently an option -not authorized -would solve some ethical issues, but create others
Most inherited cancer-susceptibility genes are
-not sufficient in themselves to trigger cancer development -at least one other somatic mutation in the other copy of the gene must occur to drive a cell toward tumorigenesis -loss of heterozygosity
Genetic engineering
-novel aspect of biotechnology -alteration of an organism's genome using recombinant DNA technologies to add or remove a gene (from the genome) -produces genetically modified organisms (GMOs)
Pro-metaphase/Metaphase
-nuclear membrane disappears -spindle fibers attach to centromeres -chromosomes begin to move toward metaphase plate -prometaphase: chromosomes are moving -metaphase: after migration
Tautomeric Shifts
-occur in nucleotides and can cause mutations due to anamalous base pairing -single proton changes
Where are CpG regions located? how does their methylation effect transcription?
-often located in/near promoter sequences adjacent to genes -their methylation blocks transcription
polyploids of odd ploidy
-often sterile or low fertility -in meiosis, some chromosomes do not have a homolog to pair w/
What mechanism do mammals use?
-one X in females is silenced (heterochromatin): Barr Body -only reactivated in germ line cells
One-gene;one-enzyme hypothesis
-one gene encodes one enzyme
SWI/SNF
-one of the best studied remodeling complexes -may alter nucleosome structure in several ways
Errors in replication
-one of the most common sources of mutation -99% of errors are corrected by proofreading -bacterial DNA polymerase III recognizes and corrects errors in replication (proofreading) -senses incorrect base pairing, reverse cuts out + replaces the base
Major current focus of the NIH
-organisms that live in or on our bodies -many are beneficial and critical to health
Translocations
-part of chromosome moves to non homologous chromosomes -interstitial vs reciprocal
What did a low protein diet fed to pregnant rats result in?
-permanent changes in expression of several genes of both F1 and F2 offspring -associated w/ hypomethylation of promotor regions
Finding a clone of interest using DNA probes
-plate out library -make filter lifts and denature DNA -hybridize to labeled probe -line up vs. signal w/ plate, excise area and grow clone -usually needs 2 rounds
the product of transcription in eukaryotes require..
-post transcriptional processing to produce mature mRNAs -addition of 5' cap (protects nucleases and may be involved in export from nucleus -addition of poly A tail (aid transport to cytoplasm) -excision of introns through splicing and exons are joined -mature mRNA usually much smaller than initial RNA
Artificial Selection
-used to develop breeds and strains of use to humans
Genetic tests based on restriction enzyme analysis
-prenatal diagnosis of sickle cell anemia: Southern Blot Analysis to detect a restriction fragment length polymorphism (RFLP) in the B globin gene -a point mutation destroys an MstII site, resulting in a single large fragment on a Southern blot -only 5-10% of mutations can be detected by restriction enzyme analysis
Transposons
-present in all organisms, but their function (or if they have one) is still unknown -used in genetic research as mutagens, cloning tags, and vehicles for introducing foreign DNA into model organisms
What are the levels of protein structure?
-primary -secondary -tertiary -quaternary
Centromeres
-primary constrictions along eukaryotic chromosomes -mediate chromosomal migration during mitosis and meiosis
Selective breeding
-prior to genetic engineering, modifying plants and animals to enhance production was restricted to selective breeding of naturally occurring or mutagen-induced variants (mutants)
in situ hybridization
-probe tissues w/ labeled DNA -7 developmental genes visualized by in situ hybridization
Meiosis in animals
-products of meiosis = oocytes in females -spermacytes in males
Meiosis in plants
-products of meiosis = spores -spores are haploid -divide meiotically to produce gametaphites -> gametes -fused gametes form diploid zygotes "sporophite"
Apoptosis
-programmed cell death -when DNA damage is so severe that repair is impossible, the cell may initiate apoptosis -second line of defense -prevents cancer and eliminates cells not contributing to the final adult organism
Allele Frequency
-proportion of alleles in a population of a specific type -an allele frequencies must add to 1 and all genotype frequency must sum to 1 =frequency (homozygous w/ that allele) +1/2 frequency (heterozygote)
What happens to pRB during G0 stage?
-protein is non phosphorylated and binds to transcription factors (E2F) inactivating them -phosphorylation releases bound regulatory proteins and allows cells to traverse S, G2, and M phases
rRNAs
-provide important catalytic functions associated with translation -rRNA gnes (rDNA) are moderately repetitive and tandemly repeated in genomes
BLAST results
-ranked alignments w/ similar sequences -similarity score(identity value) -E value (expert value)
Drosophila
-ratio of the # of X chromosomes to # of autosome sets -ratio>1 for females -ratio<1 for males -XX = females -X0 = males
Sickle-Cell Anemia
-recessive genetic disease in which afflicted individuals are homozygous for the Hb^5 hemoglobin allele -Erythrocytes under low oxygen tension become elongated and curved due to polymerization of hemoglobin -heterozygotes are unaffected carriers of the affected gene
Cyclin-dependent kinases (CDKs)
-regulated by cyclin synthesis and destruction -cyclins bind to specific CDK: active as a complex -complexes selectively phosphorylate (and activate) other proteins to regulate the cycle
Ubiquination steps
-regulates breakdown of many proteins -Ubiquitin chain is degraded -Ubiquitin is attached to a different protein -Ubiquitinated protein -26 S proteosome -Degredation to oligopeptides -Remove Ubiquination chain
Photoreactivation repair
-removes T caused by UV -depends on the activity photoreactivation enzyme (PRE) -in many organisms, but not humans
ADA in MLV
-replacement ADA cloned into MLV -used to transform isolated T cells -1 bn cells cultured and injected into blood stream -some migrate to bone marrow -patient makes 25% of normal ADA levels and has a normal life has restored the health of 20 children
Basic Bacteria
-replicate rapidly -cultural in liquid medium or "plated" on solid surfaces (visualized by clumps of cells to form colony) -abundant and variable -some cannot grow in absence of certain cellular components or energy sources -others resistant or susceptible to antibiotics -some produce distinctive colony shapes
what is the function of DNA polymerase III?
-responsible for the main 5' to 3' polymerization essential in vivo -its 3' to 5' exonuclease activity allows proofreading during synthesis
Enzymes used in Recombinant DNA Technology
-restriction enzymes (sequence specific cutting) -DNA polymerases (copying) -DNA ligases
DNA microarrays
-reveal profiles of gene expression -create visual representations of genome expression -good for studying ie. -tissue/cell type differences -developmental progressions -virulent vs avirulent pathogen -+ or - non diseased tissue -responses to environmental stress *this method is being replaced by next generation DNA sequencing of cDNA
in eukaryotic translation..
-ribosomes are larger than in prokaryotes -transcription and translation are spatially and temporally separated
EPSP synthase
-roundup herbicide (glyphosate) resistance -allows spraying of fields w/ herbicide to reduce competition w/ weeds (weeds account for 10% of crop loss world wide) -glyphosate targets chloroplast EPSP synthase and kills plants -glyphosate resistance EPSP gene was identified in E. coli, cloned and put into plants by using Agrobacterium transfaciens mediated transformation
Non research applications of PCR
-screening for mutations involved in genetic disorders -diagnostic for detection of bacteria and viruses in humans and pathogens ie. E. coli and Saphylococcus aureus in contaminated food -particularly advantageous when studying samples from single cells, fossils, or forensics, where a single hair or even a siliva moistened postage stamp is the source of DNA -used to enforce the worldwide ban on the sale of certain whale products and to determine the pedigree background of purebred dogs
How is the presence of the transgene verified once baby mice are born?
-screening using tissue and PCR is done -integrated DNA will be inherited in all offspring, but they are not homozygous for the trangene -siblings matings of F1 can generate homozygosity
Beadle Tatum experiment
-showed nutritional mutations in the bread mold Neurospora caused loss of enzymatic activity that catalyzes an essential reaction in wild type organisms -provided evidence for the one-gene;one-enzyme hypothesis
Network Map
-shows interacting proteins, genes, and other molecules (interactome) -helps model potential interactions of molecules involved in normal and disease processes -several cancers share interacting genes, despite affecting different organs
RAS family proto-oncogenes
-signal transduction molecules that regulate cell growth and division by transmitting signals from the cell membrane to the nucleus, stimulating cell division -are GTPases that alternate b/w active and inactive states -involved in 30% of all human tumors -enzyme activity is not their primary mode of action-they bind to and activate other proteins -regulation of their own activity involves GTP hydrolysis
zygote
-single cells from which new individuals develop -males and females produce gametes which combine to form zygote
Polymorphic
-sites that differ -genetic mapping involves finding statistical associations b/w polymorphic sites and presence/absence of a disease -DNA difference need not fall in a region that causes disease, rather it is a marker that is closely linked to disease gene
what is the structure of tRNA?
-small and stable -75-90 nucleotides long and contain post transcriptionally modified bases that enhance H-bonding during translation -each tRNA has an anticodon that base pairs with the codon in mRNA -corresponding amino acid is bound to CCA sequence at the 3' end of all tRNAs
Plasmid
-small, circular DNA molecule -plasmid makes a copy of its DNA by rolling circle replication and single stranded copy is passed
microRNAs
-small, noncoding RNA molecules (miRNA) also participate in epigenetic regulation of gene expression -involved in controlling the pattern of developing embryos and in the timing of developmental events and physiological processes such as cell signaling -miRNAs are transcribed as precursor molecules 70-100 bases long that can form a double-stranded stem loop
SDS-PAGE
-sodium docecyl sulphate (SDS) polyacrylamide gel electrophoresis -denatures proteins and coats them w/ negatively charged SDS
What is BLAST (Basic Local Alignment Searching Tool)?
-software application used to compare a segment of genomic DNA to sequences in the major database and identify portions that align with or are the same as existing sequences -identify homologous genes that are evolutionarily related in other organisms
What kind of cells are mutations in?
-somatic cells; only less than 1% of cancers are associated with germ-line mutations -rarely arises from a single mutation, but from accumulation of mutations in many genes
Transformation
-some bacteria absorb DNA from the environment -if absorbed DNA incorporated in hot cells genome
Limitations of PCR
-some info about the nucleotide sequence must be known in order to synthesize primers -minor contamination of DNA can cause minor problems -huge issue w/ human samples w/ implications for medicine and particularly forensics -limit to length of fragment that can be amplified -for must commercial enzymes a few kb -but improved enzymes are constantly being developed (best currently 20 kb)
Post-replication repair
-sometimes the replisome skips over a lesion and requires homologous recombination mediated by the RecA protein -RecA mediates recombination (crossing over) w/ the undamaged strand, providing a template for repair
Trinucleotide repeat sequences
-specific short DNA sequences repeated many times -normal individuals may have 30 repeats, but some have over 200 repeats w/ in and surrounding specific genes -Fragile X syndrome -Huntington's disease -Myotonic dystrophy
Metaphase I of Meiosis
-spindle fibers attached -chromosomes line up at metaphase plate w/ 1 homologous pair on each side -1 centromere on each side
Self medicating cows
-staphylococcus aureus is the main cause of mastitis in cows -causes losses>$2bill/yr -antibiotics not very effective -transgenic cows have been generated incorporating a lysostaphin gene (from staphylococcus simulana) -lysostaphin cleaves S. aureneus cell walls
What are barrier insulators? Give an example
-stop spread of heterochromatin -DNA elements that create a local environment unfavorable for the spread of euchromatin ex.) might bind a histone acetyltransferase, keeping surrounding histones acetylated
Open Reading Frames (ORF)
-stretches of nucleotide that when translated to protein generate a series of amino acids prior to a stop codon, suggestive of a protein-encoding gene -identification of open reading frames requires translation of all six reading frames
Function can be predicted from..?
-structural analysis of protein domains and motifs -structural analysis at the amino acid -many aspects of protein structure can give insight ie. membrane spanning domains, targeting signals, functional motifs: helix turn helix, leucine, zipper, or zinc finger motifs -all can be identified using bioinformatics software
Genomics encompasses
-structural genomics (genomic DNA) -functional genomics (aka transcriptomics) -comparative genomics (>1 genome) -metagenomics (multiple genomes of a population of different organisms)
Transcriptome Analysis (global analysis of gene expression)
-studies expression of genes qualitatively and quantitatively qualitatively - identifying which genes are expressed and which are not quantitatively - by measuring varying levels of expression of genes -reveal profiles of gene expression in cells and tissues *provides information < dependent on epigenetic effects transcriptomes - after transcriptional regulation
Whole-genome transcriptome analysis of pathogens
-used to inform researchers about genes that are important for pathogen infection and replication -different gene-expression profiles of mice following exposure to Neisseria meningtidis (SARS virus) or E. coli
Why was the one gene; one enzyme hypothesis modified?
-studies of human hemoglobin established that the one gene encodes one polypeptide -not all proteins are enzymes and some proteins have more than one polypeptide subunit -because of this, the one-gene;one-enzyme hypothesis was modified to one-gene; one-protein and then to one-gene;one-polypeptide chain
Alfred Sturtevant
-suggested that genes lie in a linear array along chromosome -used r estimates to map genes
Bacterial Conjugation
-the direct transfer of genetic material (DNA) from one bacterial cell to another -exchange is not reciprocal -1 donor cell and 1 recipient cell
why are genes always represented as the DNA sequence of the coding strand (5' to 3' sense)?
-the first codon on the table was made using RNA -consistency makes it easier for us to understand -we always get the data as DNA sequence
Severe combined immunodeficiency disease (SCID)
-the first disease to be treated with gene therapy -caused by mutation in adenosine deaminase (ADA)
What are the risks of GMO organisms and crops?
-the potential creation of "superweeds" -escape of transgenes into crop plant relatives giving them a competitive advantage in the wild -crop to wild species gene flow happens -potential escape of genes which make pharmaceutical or industrial precursors into varieties of the same crop used for food
Types of histones
-there are 5 types of histones -all contain large amounts of positively charged amino acids lysine and arginine -bond electrostatically to the positively charged phosphates of DNA nucleotides -histones H2A, H2B, and H4 occur as a complex of 2 types of tetramers
How can amino acids tell us about unknown proteins?
-there are databases of protein "domains" (distinct regions made up of specific amino acid sequences) are associated w/ unique functions in proteins -50 to 300 amino acids -represent protein modules that fold into stable, unique conformations -different domains impart different functional capabilities -some domains target proteins to specific compartments w/ cells -generally a small amino acid sequence, which is recognized by sorting machinery
what do DNA polymerase I, II, and III have in common?
-they both elongate an existing DNA strand (primer), but cannot initiate DNA synthesis -all three possess 3' to 5' exonuclease activity for proofreading
How do retroviruses cause cancer?
-they cause cancer when they integrate near a proto-oncogene or by integrating a copy of a host proto-oncogene into its genome -the RNA genome of the virus is copied into DNA by reverse transcriptase enzyme, which is brought into the cell by the infecting virus -the DNA copy enters the host-cell DNA and integrates at random
What is true of mitochondria and chloroplasts' DNA?
-they contain their own DNA and are inherited through maternal cytoplasm in most organisms -the nature of their DNA is remarkably similar to viruses and bacteria
How are P vectors used?
-to introduce transgenes into Drosophila - a technique known as germ line transformation -P elements are also being used to generate mutations and to clone mutant genes
What is the most significant environmental carcinogen?
-tobacco smoke, which contains at least 60 mutagenic chemicals -30% of human cancer deaths are associated w/ cigarette smoking
Pesticides
-transfer natural disease resistance from one species to another
Gene therapy
-transferring specific genes into human cells to treat genetic disorders -normal allele of a gene is transferred into a patient's cell that has one or more mutant alleles -only applicable to what type of mutated allele -usually uses a genetically modified retroviruses as a vector
Enviropigs
-transformed w/ a gene for phytase -breaks down dietary phosphorus -reduces phosphate pollution caused by pig farming -phytase produced in salivary glands and secreted in the salive increased the digestion of phosphorus contained in feed grains
Novel Resistance to Viruses
-transgenic papaya, resistant to ringspot virus, was introduced to Hawaii and has saved papaya industry -no natural resistance known -plant expressing viral coat proteins develop immunity
Glofish
-transgenic strain of zebrafish, contain a red fluorescent protein from sea anemones -though marketed as pets, these animals may be useful for assaying heavy metal contamination in water
Ac-Ds system in Maize discovery
-transposons were discovered in Maize by Barbara McClintock (1940s) -fully not accepted until 1960s -Nobel prize 1983 -studies color patterning in Indian Corn -10 chromosomes -found strain in which Chr 9 broke frequently
What are issues of GMOs in biotechnology dependent on?
-type of organism involved -where and how that organism is grown -implications to human biology
Mutation
-ultimate source of genetic variation -very weak evolutionary force
the genetic code is..
-unambiguous -degenerate -commaless -non-overlapping -nearly universal (some exceptions in mitochondria)
aneuploid
-unbalanced karyotype -often have severe abnormalities
RNA interference (RNAi) trials
-underway -one RNAi strategy to treat a form of blindness (macular degeneration) targets a gene called VEGF
All cancer cells share 2 fundamental properties
-unregulated cell proliferation -metastatic spread
Ames Test
-used to assess the mutagenicity of compounds -uses any of a dozen strains of Salmonella typhimurium selected for increased sensitivity to mutagens and ability to reveal the presence of specific types of mutation -measures frequency of reversion to a His synthesis mutations -many known carcinogens have been shown by the Ames test to be strong mutagens
Genetic Knock-Down studies
-uses the natural RNAi system -construct DNA sequence complementary to gene of interest that form a hairpin structure -leads to double stranded RNA, activation of Dicer and RISC systems -degradation of the endogenous mRNA of the gene targeted
Protein folding
-usually a spontaneous process based soley on the combined chemical properties inherent in the amino acid sequence
DNA and RNA electrophoresis
-usually agarose gel electrophoreses, polyacrylamide gels v. small fragments -nucleic acids are always negatively charged, separation based on size
Extranuclear Inheritance
-usually inherited from one parent (mother) -offspring resemble mother mitochondria = mtDNA chloroplasts = cpDNA
what are ideal conditions for PCR
-usually want an annealing temp of 60 degrees C -nucleotide composition matters (50 G/C to 50 A/T ideal)
Moderately Repetitive DNA includes
-variable number tandem repeats (VNTRs) -minisaellites 10-100 nucleotides (used in DNA fingerprinting) -microsatellites usually 1-4 nucleotides
Baculovirus
-virus used to infect insect cells -ideally in plants (cost/efficiency wise)
Types of Genetic Variation
-visible -molecular (ie. blood groups, chromosome variants, RFLPS, SSLPs, SNPs, VNTRs, direct sequencing)
How else can probes be used..
-visualize molecules after electrophoresis -run gel, "blot" to a membrane
Vitamin content
-vitamin A deficiency affects 200 million women in children -golden rice provides a possible solution -daffodil phytoene synthase genes used produce B-carotene -humans use beta carotene to make vitamin A
Human insulin
-was purified from cow/pig intestines -similar but not the same protein -2 subunits produced as fusion proteins, purified and cleaved to release insulin polypeptides -subunits spontaneously unite to form activate insulin -> effective, less side effects
E. Coli Case Study
-we are full of it -genome = 4.6 Mb -similar level of difference to human vs. chimps -strains contain islands of genes specific to each
Ethyl-methane sulfonate
-widely used in mutagenesis expts -alkylates keto groups changes base pairing
Matrix-assisted laser desorption ionization (MALDI)
-widely used mass spectrometry approach for identifying proteins -knowing all the genes in a genome we can search to find out which gene the peptide sequence comes from
Repetitive Sequences
-widespread in eukaryotic genomes -large factor in the difference in genome sizes
Genes on same chromosome
0<r<.5
How many DNA molecules are in each chromosome?
1 DNA molecule
Human Genome size; how many base pairs?
1 meter long, 3.2 billion base pairs
How do individuals differ from one another genetically?
1 of every 1000 base pairs
What forms of phosphate groups do nucleotides have?
1 phosphate group = NMP's 2 phosphate groups = NDP's 3 phosphate groups = NTP's
3 next generation machines on campus
1. 454 Sequencing 2. Ion Torrent 3. SMART (single molecule real time sequencing)
PCR uses
1. Amplifying directly from cDNA (RT-PCR) or genomic DNA 2. Library screening (arrayed genomic libraries) 3. Genotyping (detecting POLYMORPHISM: wide range of uses) 4. Modifying DNA sequence: directed mutagenesis, adding restriction enzyme sites 5. Differential screening 6. Quantifying gene expression (real-time PCR, qPCR)
Presents 2 challenges
1. Bioinformatics: software to analyze the huge amounts of data 2. Bottleneck in research will move from gene discovery to functional analysis
DNA amplification
1. Cloning vectors (DNA replication in cells) 2. Amplification in vitro (thermostable DNA polymerase)
Epigenetics modifications alter chromatin structure by several mechanisms including..
1. DNA methylation 2. histone modification 3. actions of miRNA -all without changing the sequence of DNA
Summary of E.coli replication
1. DNA polymerase III: main synthesis 2. B clamps: keeps pol III's attached to DNA 3. Primase: makes RNA primers 4. Helicase: unwinds DNA helix by breaking H bonds 5. single-stranded binding proteins: keep strands apart 6. DNA polI: replaces RNA primers with DNA 7. DNA ligase: joins fragments together 8. Topoisomerases: relax supercoiling by breaking 1 or both DNA strands allowing rotation ex.) DNA gyrase
steps in PCR
1. Denature 2. Anneal (primers) 3. Extend (synthesis of DNA from primers) -repeated over and over using thermocycler to amplify the DNA exponentially
PCR reactions
1. Double-stranded DNA to be cloned is put in a tube w/ a thermostable DNA polymerase, a buffer containing Mg2+, the 4 dNTP's and 2 oligonucleotide primers
2 transgenes account for almost all of the acreage
1. EPSP synthase 2. BT crops
Basic procedure for producing recombinant DNA involves
1. Generating DNA fragments (often) using restriction enzymes 2. Joining the fragments w/ a vector 3. Transferring recombinant DNA molecules to a host cell to produce many copies that can be recovered from the host cell
Post-translational modifications before proteins become active, for targeting, and recycling include..
1. Glycosylation 2. Lipid modifications 3. Phosphorylation/Dephosphorylation 4. Cleavage 5. Ubiquination
How does RNA differ from DNA?
1. RNA is usually single stranded 2. bases are attached to ribose rather than deoxyribose -an extra OH group facilitates many processes 3. Uracil vs. Thymine 4. Can catalyze reactions (ribozymes) -ability to make shapes; HOW?
How does histone acetylation work?
1. affects interaction b/w DNA and the histone monomer -makes the nucleosome more likely to slide 2. influences binding of regulatory proteins to the DNA
structural chromosomal changes
1. duplications 2. deletions 3. inversions 4. translocations
what are the 5 ways that transcription in eukaryotes differs from transcription in prokaryotes?
1. eukaryotes occurs in the nucleus and is not coupled to translation 2. eukaryotic transcription requires chromatin remodeling 3. in addition to promoters, enhancers and silencers also influence transcription 4. eukaryotic mRNAs require processing to produce mature mRNAs 5. eukaryotes possess 3 forms of RNA polymerase, each of which transcribes different types of genes
Making cDNA
1. extract total RNA from tissue 2. purify mRNA 3. 1st strand cDNA synthesis dNTPs, primers, + enzyme
Linkage & Recombination Mapping
1. linkage maps 2. physical maps
Applications
1. medicine ie. antibiotic resistance 2. agriculture 3. wildlife management and conservation biology 4. forensics and genetic counseling 5. evolutionary biology
Population Genetics deals with..
1. patterns of genetic variation 2. processes that determine or change those patterns
Hardy Weinberg Law states:
1. random mating 2. no consistent viability or fertility difference (no selection) 3. no mutation 4. no migration 5. infinite population size 6. equal frequency in both sexes 7. non overlapping generations
Process of RAS GTP binding
1. receptor senses growth factors 2. receptor autophosphorylates 3. recruits GEF 4. GEF stimulates RAS to exchange GDP for GTP 5. RAS GTP activates a kinase phosphorylation cascade 6. ends w/ transcription factors that cause cell to enter cell cycle -mutations that convert the proto oncogene to an oncogene lock RAS into its active confirmation constantly stimulating the cell to divide
For a molecule to serve as the genetic material it must exhibit what 4 key characteristics?
1. replicate 2. store information 3. express information 4. allow variation by mutation
3 major epigenetic mechanisms
1. reversible modification of DNA by addition/removal of methyl groups 2. alteration of chromatin by addition/removal of chemical groups to histone proteins 3. regulation of gene expression by small, noncoding RNA molecules
what are the 7 key issues that must be resolved during DNA replication?
1. unwinding of the helix 2. reducing increased coiling generated during replication 3. synthesis of a primer for initiation 4. discontinuous synthesis of the second strand 5. removal of the RNA primers 6. joining of the gap filling DNA to the adjacent strand 7. proofreading
We still don't know the function of what percentage of E.coli genes?
12%
How many components do prokaryotic replisomes have?
13
the human genome contains genes that encode how many different DNA polymerases? How many are involved in replication of nuclear DNA?
14 DNA polymerases; only 3 are involved in replication
mini satellite markers
15 to 100 nucleotides long
How many cell divisions in meiosis?
2
yeast genomes contain..
250 to 400 origins called autonomously replicating sequences (ARS)
how many components do eukaryotic replisomes have?
27
n equation for meiosis
2n --> n+n+n+n (4 haploids)
tetraploids
2n=x
frequency (MN genotype)
2pq
How many families of transposable elements are found in Drosophila?
30
Organisms that share genes w/ humans
30% with yeast 80% with mice 98% with chimpanzees
how many genes in a typical human are heterozygous?
30,000 to 40,000 ~3,000 to 4,000 heterozygous
Result of Meiosis
4 cells each w/ n chromosomes
what direction does chain elongation occur? how does this occur?
5' to 3' direction by addition of one nucleotide at a time to the 3' OH end
how many triplet codons assigned to amino acids been yielded using the various techniques combined to decipher he genetic code?
61 triplet codons
Tranposable Elements in Humans
> 50% of the human genome is composed of transposable element in DNA - 1% of the human genome comprises gene coding sequence DNA -Most human transposons appear to be inactive, but it is possible for a mobile element to transpose from one chromosome to another in gamete-forming cells of the mother, causing a genetic disease
differences b/w eukaryotic DNA and prokaryotic DNA
>DNA in eukaryotic cells than prokaryotic cells -chromosomes are linear in eukaryotes -DNA is complexed w/ proteins and nucleosomes
Chargaff's Rules
A = T and C = G, but the % of C+G does not necessarily =A+T
Barr Body
A dense object lying along the inside of the nuclear envelope in female mammalian cells, representing an inactivated X chromosome.
how many hydrogen bonds are present between base pairs?
A-T base pairs forms 2 Hydrogen bonds & C-G forms 3 Hydrogen bonds
Reciprocal Translocation
ABCD -> ABYZ WXYZ WXCD
Deletions
ABCDEFG -> ABF
what is the only initiator/START codon for methionine?
AUG
which codons serve as termination codons and do not code for any amino acids
AUG, UAA, UGA
Purines
Adenine and Guanine
What kind of DNA is the Watson-Crick model of?
B-DNA which is believed to be the biologically significant form
Large insert libraries (genomic DNA)
Bacteriophage (virus) - 15 kb largely obsolete Cosmid - virus/plasmid combo ~50 kb BAC (bacterial artificial chromosomes) 150 to 300 kb YAC (yeast artificial chromosomes) > 300 kb but unstable
What is the location of the phosphate group on a nucleotide?
C-5' position
DNA synthesis at a replication fork involves:
DNA polymerase III (x2) single stranded binding protein DNA gyrase DNA helicase RNA primers
What are the 3 modes of DNA replication possible?
Conservative, Semiconservative, and Dispersive
Pyrimidines
Cytosine, Thymine, and Uracil
What did Hershey and Chase demonstrate? How?
DNA alone directs bacteriophage virus reproduction in the infection of E. coli. By differentially radiolabelling DNA and protein, transfection of viral DNA into bacterial cells proved conclusively that the viral DNA alone contains all the necessary information for production of mature viruses
What is the most common cause of spontaneous mutation?
DNA base damage by depurination and deamination
what enzyme covalently joins the discontinuously synthesized strands?
DNA ligase
what group is DNA gyrase a member of?
DNA topoisomerases
Recipient Cell
F-
T or F: all RNA are involved in encoding proteins
False; RNA has various other important functions
what is gene silencing?
Fire & Mello reported that injecting the synthetic double stranded RNA into C. elegans embryos selectively switches off the gene that the RNA encodes
correlating SNPs
Genome Wide Association Studies (GWAS)
Genomic vs. cDNA
Genomic: -genes in ~1:1 ratio -elones have promoters and introns -huge amount of extra non-coding material cDNA -elone abundance = % transcript in starting tissue -no introns or promoters -less junk
how was evidence favoring DNA as genetic material obtained?
Griffith (1927) showed that avirulent strains of Diplococcus pneumoniae could be transformed to virulence. He speculated that the transforming principle could be part of the polysaccharide capsule or some compound required for capsule synthesis
Nondisjunction
Homologues fail to separate during meiosis I
Interference
I = 1- (observed frequency of double crossovers)/(expected frequency of double crossover)
small ribosomal subunits bind to..
IF1 and then to mRNA
Similarities b/w Humans and Chimpanzees
LCA = 6.3M yrs -mutations have accumulated in both -differ by <2%, share 99% of the same genes -35 M nucleotide changes -5 M insertions/deletions -29% of proteins are identical -most differ by only 1-2 amino acids
what is the origin of replication in bacteria called?
OriC
What do bacteria use to conjugate?
Pili
How do retroviruses replicate?
RNA serves as a template for synthesis of complementary DNA by the RNA-dependent DNA polymerase reverse transcriptase. This DNA can be incorporated into the host cell genome
RNA homopolymers
RNA with one type of ribonucleotide
RFLPs
Restriction Fragment Length Polymorphism
How does the ribosome location in prokaryotes differ from eukaryotes
Ribosomes are not free floating in eukaryotes as they are in bacteria, but are associated with the endoplasmic reticulum (forming rough ER)
SSLPs
Simple Sequence Length Polymorphisms -variation in short DNA repeats
what are the two consensus sequences which all E coli promoters have?
TATAAT located at the -10 region (Pribnow Box) & TTGACA located at the -35 region (35 nucleotides upstream from initial site of transcription) -sequences are similar in different genes of the same organism or genes of related organisms
what is the repeating sequence in humans?
TTAGGG
What other forms of RNA exist?
Telomerase RNA(snRNA): RNA processing Antisense RNA, microRNA(miRNA), and short interfering RNA(siRNA) involved in gene regulation
dosage compensation
Theory that X chromosome inactivation equalizes gene expression between males and females
protein gel
Western blot -detect with antibodies
What is the most widely used method?
Whole genome shotgun sequencing
the haloenzyme (active form) contains..
alpha2, beta, beta prime, and sigma
Turner Syndrome
X
Rosalind Franklin's critical experimental result
X-ray diffraction analysis produced a "3.4 angstrom periodicity" characteristic of a helical structure, which led Watson and Crick to propose a double helical structure for DNA in 1953
homogametic sex
XX
Kleinfelters Syndrome
XXY
heterogametic sex
XY
what are the 2 major requirements of DNA polymerase I?
a DNA template and all 4 deoxyribonucleoside triphosphates (dNTPs)
TATA box
a core promoter element that binds TATA binding protein (TBP) of transcription factor (TFIID) and determines start site of transcription
Superfamily
a group of related multi-gene families ie. an ancestral gene encoding an O2 transport protein was duplicated, producing 2 sister genes, evolving into myoglobin and hemoglobin
what is the rho termination factor?
a large protein that physically interacts with the growing RNA transcript and in some cases termination depends on it
what is the initial amino acid incorporated into all proteins?
a modified form of methionine N-formylmethionine (fmet)
BCR-ABL
a mutant form that is autoactive ie. xoderma pigmentosa
Familial Retinoblastoma
a mutated RB1 allele is inherited causing an 85% chance of developing retinoblastomas and increased chance of developing cancers
What do DNA and nucleotides consist of?
a nitrogenous base, a pentose sugar, and a phosphate group
what does DNA polymerase III require to elongate polynucleotide chains?
a primer with a 3' OH group
ABL
a protein kinase that acts in signal transduction that transfers growth factor signals to the nucleus
Strands of helix are unwound creating..
a replication fork
what is unique of the telomerase enzyme?
a ribonucleoprotein with an RNA that serves as the template for synthesis of its DNA complement (reverse transcriptase)
Monogenic diseases
a single base-pair change in one of the 20,000 human genes can lead to serious inherited disorders
epigenetic trait
a stable, mitotically, and meiotically heritable phenotype that results from change in gene expression w/o alterations in DNA sequence
termination is signaled by..
a stop codon (UAG, UAA, UGA) in the A site
IVF has been associated with..
a three to six fold risk of Beckwith-Weidemann Sydrome (BWS) - an overgrowth disorder -less than 90% of children born w/ BWS after ART had imprinting defects
Secondary Structure
a. alpha helix b. beta pleated sheet
Behavioral Mutations
affect behavior patterns of organisms ex.) the mating behavior of a fruit fly may be impaired if it cannot beat its wings
Metastasis suppressor genes
affect only metastatic tumors, often affected (reduced) by epigenetic factors
Regulatory Mutations
affect regulation of gene expression
What is the impact of DNA base modification ie. Cytosine methylation?
affects whether DNA binding proteins can bind to the DNA sequences (+ or - effects)
Prions; give examples
aggregates of misfolded proteins causing transmissable brain disorders in mammals ex.) -Scrapie in sheep -Bovine spongioform encephalopathy (mad cow disease) -Creutsfeldt-Jakob disease (in humans) -Huntington, Alzheimers, and Parkinsons disease are linked to formation of abnormal protein aggregates in the brain
what is the major biological significance of telomeres and telomerase?
aging and cancer
Complete Penetrance
all individuals w/ genotype have same pheonotype
what is the function of exonuclease activity?
allows proofreading and repair
what subunits does E Coli RNA polymerase contain?
alpha, beta, beta prime, and sigma
what do ARS's contain?
an 11 bp consensus sequence flanked by other short sequences involved in initiation
What is Imprinting? Give an example
an epigenetically regulated process in which genes are expressed in a parent of origin pattern ex.) expression of either maternal or paternal genes (not both)
Epigenetic changes create..
an epigenome that can regulate normal development or generate physiological responses to environmental signals
How was the genetic code deciphered?
an in vitro protein synthesizing system along with the ability to produce synthetic mRNAs using polynucleotide phosphorylase (an RNA polymerase)
What did cytological analysis reveal?
an inversion carrying the white gene
what happens when AUG appears internally in mRNA?
an unformylated methionine is inserted into the protein
Natural Radiation (UV light, X rays), natural dietary substances, and substances in the external environment
can cause DNA lesions, producing mutations that can lead to cancer
DNA methylation
another change in chromatin that plays a role in gene regulation (addition/removal of methyl groups to or from bases in DNA)
cDNA libraries can also be screened by..
antibodies, which recognize 7-20 amino acid sequences
chiasmata
areas of contact, The X-shaped, microscopically visible region representing homologous chromatids that have exchanged genetic material through crossing over during meiosis.
What other structural component provides stabilizing factor?
arrangement of sugars and bases along the axis
how many origins do mammalian genomes contain?
as many as 25,000
histone acetyltransferase enzymes
associated w/ increased transcription
Rate of Deleterious Mutation in humans
at least 1.6 per individual per generation
Transduction
bacteriophage transfer genetic material among cells
Point Mutations
base substitutions in which one base pair is altered ex.) CAT to BAT
What type of direction does DNA occur?
bidirectional instead of unidirectional (there are 2)
Restriction Enzymes
bind DNA at a specific recognition sequence (restriction site) and cleaves the DNA to produce restriction fragments
Histones
bind to DNA in eukaryotic organisms
methylation can repress transcription by..
binding to transcription factors of DNA
Hemoglobin
binds and transports oxygen
Translation
biological polymerization of amino acids into polypeptide chains
What is being done to increase the efficacy and yields of biopharmaceuticals?
biopharmaceuticals are now produced in eukaryotic hosts
What happens to imprints during gamete formation in germ cells
both chromosome sets have their imprints erased and are each reprogrammed to carry a female/male imprint that is transmitted to the next generation through the egg/sperm
cis
both mutant or both wild type product
enhancers
can be upstream, within, or downstream of gene & can modulate transcription from a distance
Elongation requires...
both ribosomal subunits assembled w/ mRNA to form the P(peptidyl) site and A(aminoacyl) site
how do peptide bonds form?
by a dehydration reaction between the carboxyl group of one amino acid and the amino group of another
How is Kozak sequence considered to increase efficiency of translation?
by interacting with the initiator tRNA
How is chromatin remodeled most easily?
by sliding nucleosomes to expose the regulatory sequences
how are introns spliced out?
by spliceosomes in a reaction involving formation of a lariat structure that contains snRNPs (small nuclear ribonucleoproteins) -pre mRNA can be spliced in different ways, yeilding different exons in mature mRNA
How is silencing caused?
by the spread of heterochromatin, but spread varies in different cell lineages
GFP
can be used as an expression marker and to study protein localization
Base-analogs
can substitute for purines or pyrimidines during nucleic acid replication ex.) 5-bromouracil -pairs 5-BU to A -but Br atom increases likelihood of a tautomeric shift causing 5-BU to G
Clonal
cancer cells in primary and secondary tumors originate from a common ancestral cell that accumulated numerous specific mutations ie. Burkitt's lymphoma involves a translocation between chromosome 8 and either 2, 14, or 22, but all lymphoma cells in an individual patient contain identical translocation breakpoints -all cells have the same pattern of X-chromosome inactivation in females (more evidence of being clonal)
What must happen for tumors to metastasize from a primary tumor?
cancer cells must digest components of the extracellular matrix and basal lamina that normally inhibit migration of cells
Donor Cells
carry "fertility factor" (F+) as a plasmid
Meiosis
cell division process resulting in gametes (sex cells)
What occurs at G1/S, G2/M, and M checkpoints of the cell cycle?
cells decide whether to proceed to the next stage of the cell cycle
telocentric
centromere at end of chromosome
metacentric chromosome
centromere near center of chromosome
submetacentric chromosome
centromere slightly off center
Ethical questions related to Enhancement gene therapy
controversial and unapproved issue of whether genetic technology should be used to enhance human potential either of individual on their offspring
Mutation definition
changes in DNA sequence generate variation that results in phenotypic variability in populations, and as a result are vital adaptations to environmental change and evolution BUT they are also the source of genetic changes that can lead to cell death, genetic disease and cancer
what is the first step in elongation?
charged tRNAs enter A site and peptidyl transferase catalyzes peptide bond formation b/w the amino acid at the A site & the peptide chain bound to the tRNA in the P site
Intercalating Agents
chemicals whose dimensions and shapes allow them to wedge between DNA base pairs, causing base-pair distortions and DNA unwinding -Ethidium Bromide -Chemotherapeutic agents -these changes in DNA structure may affect many functions, including transcription, replication, and repair leading to frameshift mutations
eukaryotic DNA is complexed with DNA binding proteins, existing in the cell as...
chromatin
nucleosome
chromatin + histones (beads) nucleosome = histone octomer
What causes position effects?
chromatin environments at different places in the genome
What must happen to chromatin for replication and gene expression?
chromatin must relax its compact structure and expose regions of DNA to regulatory DNA binding proteins (chromatin remodeling)
Karyotype
chromosomal complement
Allopolyploids
chromosome sets derived from different species ex.) wheat
Auto polyploids
chromosome sets derived from same species
GTP dependent release factors
cleave the polypeptide chain from the tRNA and release it from the translation complex
I near 1
closely linked genes
Homologs
closely related genes (high DNA sequence similarity)
What makes cancer dangerous?
combination of uncontrolled cell proliferation and "metastatic spread"
initiation complex
combines large and small subunit
What does the A-T and G-C base pairing provide?
complementarity of the two strands and chemical stability to the helix
what is a replisome?
complex made up of haloenzyme + other proteins at the replication fork
ribosomes
consist of ribosomal proteins and RNAs (rRNAs) -have large and small subunits
Actin and Myosin
contractile proteins found in abundance in muscle tissue
DNA ligase
covalently joins restriction fragments to produce intact DNA molecules
Natural metabolism
creates oxidative and end products that can damage DNA, proteins, and lipids
Three Point Cross
cross between three point testcross of a triple heterozygote with a triply homozygous recessive
Monohybrid cross
crossing for a single trait
methylation is associated with..
decreased gene expression
Chronic myelegenous leukemia (CML)
defects in genes that control DNA repair (involving a structure termed the Philadelphia chromsome)
Interference
deficiency in double recombination, measured by interference I = 1- (observed frequency of double crossovers/expected frequency of double crossovers)
Genetic Information Nondiscrimination Act
designed to inhibit improper use of genetic information in health insurance and employment
Tumorigenesis
development of a malignant growth
What causes allele-specific imprinting and gene silencing?
differential methylation of CpG-rich regions and promoter sequences
2n
diploid chromosome number
centromere
divides each chromosome into 2 arms
Temperate
do not kill host right away
nonsense codons
do not specify an amino acid, but signal STOP
main non-effect of inbreeding
does not change allele frequency
Paracentric Inversion
does not involve centromere
Alkylating agents
donate an alkyl group to amino or keto groups in nucleotides to alter base-pairing affinity
What mechanism does the sex chromosome problem drive?
dosage compensation
How did myoglobin arise?
due to a duplication nucleotide substitution and chromosomal translocations
Law of Segregation; where does it occur?
during Anaphase I of Meiosis, homologous chromosomes separate
Law of Independent Assortment; where does it occur?
during Metaphase I of Meiosis, pairs of homologous chromosomes assort independently of each other
polycistronic mRNA
during transcription in bacteria, this type of RNA is produced and encodes more than one protein (genes in bacteria are called cistrons) -eukaryotes have monocistronic mRNA
Principle of Equal Segregation
each heterozygote produces equal quantities of G and g alleles
Why is DNA replication called semiconservative?
each replicated DNA molecule consists of one "old" strand and one "new"
What is colon, prostate, and breast cancer associated with?
eating red meat and animal fat
What kind of bacteria can exchange DNA?
ecoli
How often is the pattern of imprinting in mammals reprogrammed?
every generation
what is telomerase?
enzyme that directs synthesis of the telomere to repeat sequence to fill the gap
Cancer is now viewed as a disease that involves what two factors?
epigenetic and genetic changes that lead to alterations in gene expression and the development of malignancy
Random Genetic Drift
evolution that occurs b/c of random sampling of alleles b/w generations
T or F: normal somatic cells make telomerase
false; their telomeres get progressively shorter as the cells divide
accrocentric
far off center
F=1/16
first cousins
What enzymes are used now?
fluorescent proteins either on their own or as fusion proteins
Why were algorithm based software programs developed?
for creating DNA sequencing alignments where similar sequences of bases (contigs) are lined up for comparison
combination of SDS-PAGE and Isoelectric focusing
for max separation: 2D electrophoresis, IEF followed by SDS-PAGE
Z-DNA
forms a left-handed double helix
what does the binding of DnaB and DnaC do?
further opens and destabilizes the helix
microsatellites
generally repeating dinucleotide repeats 5'CACACA3'
During Independent Assortment r = .5 when..
genes are on different chromosomes
Orthologs
genes at the same locus in different species inherited from a common ancestor
Proto-oncogenes
genes whose products promote cell growth and division including: -transcription factors that stimulate expression of other genes -signal transduction molecules that stimulate cell division -cell-cycle regulators that move through the cell cycle -cyclin D1 and cylcin E are proto oncogenes
what is homologous recombination?
genetic exchange at equivalent positions b/w 2 chromosomes w/ substantial DNA sequence homology -needs tight regulation
Backwards Genetics
genome --> trait (phenotype)
What is a property of all cancers to date?
genomic hypo-methylation
What are the effects of genetic defects in cancer cells?
genomic stability, DNA repair, and chromatin modifications
Penetrance
genotype may or may not show phenotype
If assumptions hold..
genotypic frequency can be computed from allele frequency
Population
group of individuals that exist together at the same time and place
how are genes placed on chromosomes in bacteria?
groups of genes whose products are related are clustered along the chromosome and all but the last gene lacks termination signals
what kind of complexes are DNA polymerases?
haloenzyme complexes (enzyme made up of multiple subunits)
What does meiosis do to the number of chromosomes?
halves number of chromosomes per cell
What two forms does chromatin take?
heterochromatin & euchromatin
heterochromatin
highly condensed (not acetylated)
Satellite DNA
highly repetitive and consists of short repeated sequences (often used in evolutionary studies)
What do transcription factors recruit?
histone deacetylase complexes and histone acetyltransferase enzymes
main effect of migration
homogenize genetic variation b/w different local populations
BRCA1
hyper-methylated and inactivated in breast cancer and ovarian cancer
MLH1
hyper-methylated in some forms of colon cancer
What does hyper-methylation do?
hyper-methylation at CpG islands and promoters silences certain genes, including tumor-suppressor genes
Histones near active genes tend to be..
hyperacetylated
sister chromatids
identical copy of a chromosome
Alignment
identifies overlapping sequences, allowing reconstruction of their order in a chromosome
How do tumors form metastases or become "malignant"?
if cells acquire the ability to break loose, enter the blood stream, invade other tissues and form secondary tumors
Virulent
immediately lyse and kill host cell
Gene density
in eukaryotes is very low compared to bacteria and varies even b/w chromosomes in a species -large variation in introns size and number b/w and w/in genomes
Trisomy 21
in humans is cause of down syndrome
How has comparative genomics been valuable?
in identifying members of multi gene families including nonfunctional pseudogenes
2n + or - k
in normally diploid species
Introns
intervening sequences: regions of initial RNA transcript that are not expressed in the amino acid sequence of the protein -removed by
Pesiticides and antibiotics
in plants can be carcinogenic but do not diminish the serious cancer risk upon exposure to synthetic pesticides or abestos
how are overlapping genes created?
in some viruses, genes initiate at different AUG positions out of frame w/ one another creating several distinct polypeptides from one RNA
Ionizing radiation
in the form of X-rays, gamma rays, and cosmic rays are mutagenic
main effect of inbreeding
increases frequency of homozygotes and decreases frequency of heterozygotes because individuals can inherit 2 copies of the same ancestor
Diploid
individuals have 2 copies of a gene
Xeroderma Pigmentosum (XP)
individuals have lost the ability to undergo nucleotide excision repair
Mendel's basic insight
inheritance is particulate
Antibodies can be made by..
injecting a protein or small synthetic peptide into an organism (mouse, rabbit, chicken..)
Lethal Mutations
interrupt an essential process and result in death
what is the function of the clamp structure associated with each polymerase unit at the replication fork?
it maintains the prcessivity of the gyrase (the number of nucleotides that may be added during synthesis before the enzyme dissociates from the template -critical to rapid in vivo synthesis
what does the DNA double helix unwinding cause?
it makes the template strand accessible to the action of RNA polymerase
RNA polymerase explores the DNA until..
it recognizes a promoter region
what would happen if the ends of germ cells were degraded?
it would be catastrophic; a special mechanism has evolved to repair telomere ends in gametes
okazaki fragments
lagging strand is synthesized as okazaki fragments, each with an RNA primer
what is a replicon?
length of DNA replicated following one initiation event at a single origin
euchromatin
less condensed (acetylated)
Expressivity
level of expression or intensity of expression of a phenotype for a given genotype
Where is the information for making proteins found?
linear sequence of deoxyribonucleotides in DNA
Bioreactors or biofactories
living factories that continuously make milk containing therapeutic protein (herd of goats or cows)
Locus
location of the gene on the chromosome
Lod Score
logarithm of ratio of linkage (r<.5) to no "log of odds" linkage
what are telomeres? (structure and fx)
long stretches of short repeating sequences (10 to 15 kb) that preserve chromosome integrity and stability
q arm
longer arm
23 and Me
looks at all chromosomes to get info about risk of certain diseases
Duplications and Deletions can be visualized by..
loops that develop in Meiosis I
I near 0
looseley linked genes
What causes benign tumors?
loss of genes controlling cell growth
Children born after IVF are at risk for..
low or very low birth weight (due to abnormal imprinting)
Initiation requires...
mRNA, small and large ribosomal subunits, GTP, charged initiator tRNA, Mg2+, initiation factors
polysomes (or polyribosomes)
mRNAs with several ribosomes translating at once
Nutritional quality
making amino acid composition of seeds more appropriate for human/mammalian nutrition (grains and legumes)
euploid
normally there is a balance b/w different genes in diploid genome
what does it mean to say that the genetic code is degenerate?
many amino acids specified by more than one codon
Kozak Sequence
many eukaryotic mRNA's contain (A or G) 3 bases upstream from the AUG initiator codon, which is followed by a G -analogous to Shine-Dalgarno sequence in prokaryotes -sequence is recognized as translational start site, NOT the ribosomal binding site which is usually the 5' cap on the mRNA
Alcohol
may cause inflammation and lead to liver cancer
In vitro fertilization and other assisted reproductive technologies (ART) in humans
may cause problems with imprinted genes
cells used in sexual reproduction form through..
meiosis
What are the 3 classes of RNA DIRECTLY involved in protein synthesis?
messenger RNA(mRNA): coding template ribosomal RNA(rRNA): ribosomal components (80% of total cellular RNA) transfer RNA(tRNA): carries amino acids
CEN region
minimal region for centromere function
zygotes develop through..
mitosis and cell division
Aflatoxin
mold on bread is one of the most carcinogenic chemicals known
x
monoploid # of chromosomes
Translation in eukaryotes generally requires
more factors for initiation, elongation and termination than in bacteria
polymorphic gene/marker
more than 1 allele present in the population
How many base pairs exist in the human genome?
more than 3 billion
Transport proteins
movement of molecules across membranes
non-sense mutation
mutation to a termination codon w/in the protein sequence (protein gets shortened)
RNA gel
northern blot -detect w/ probe
Each gamete contains how many chromosomes?
n
Each zygote gets how many chromosomes from each parent?
n (haploid chromosome number)
diploids
n=x
Nitrosamines
naturally occurring, used as meat preservative, known to cause cancer
HIV
nearly 1000 gene therapy trials are actively underway in the United States alone
Deleting the 6 N-terminal amino acids to remove the acylation sites..
negates plasma membrane localization and the phenotype
Incomplete Penetrance
not all individuals w/ a given phenotype have same phenotype
Genes on same chromosome of male Drosophila..
never cross over (r=0)
nucleoside
nitrogenous base + pentose sugar
I=0
no interference
Robertson Translocation
non reciprocal w/ 2 non homologues joined at centromere
Tumor Suppressor Genes
normally regulate cell cycle checkpoints and initiate apoptosis
blastn
nucleotide vs nucleotide database
How can environmental agents alter gene expression?
nutrition, chemicals, and physical factors such as temp can affect the epigenome
Somatic Mutations
occur in any cell except germ cells and are not heritable, but not unimportant
Germ-line Mutations
occur in gametes and are inherited
X-linked Mutations
occur within genes located on the X-chromosome
Autosomal Mutations
occur within genes located on the autosomes
Oligonucleotides vs polynucleotides
oligonucleotides are short chains of up to 20 nucleotides or so, longer chains are called polynucleotides
Trisomy
one extra copy of some chromosomes n = haploid #, trisomic 2n+1 chromosomes
GenBank
one of the most important databases maintained by the National Center for Biotechnology Information (NCBI)
Each step in tumorigenesis appears to result from..
one or more genetic alternations that progressively release the cell from the normal controls on cell proliferation malignancy
Subunit vaccines
one or more surface proteins from a virus or bacterium developed by genetic engineering ex.) hepatitis B virus surface protein has cloned into yeast expression vector and purified from yeast
the entire bacterial chromosome = ?
one replicon (4.6 million bp)
Interstitial
one way movement of genes ABCD ->AD WXYZ WXBCZ
monomorphic gene/marker
only 1 allele present in the population
What portion of the eukaryotic genome constitutes functional protein encoding genes?
only a small portion (2-10%)
where does protein synthesis occur? what does this imply?
outside the nucleus (RNA must be transcribed from DNA templates)
frequency (MM genotype)
p^2
explanation for trans product
parental genes associated due to physical linkage
Dispersive Replication
parental strands are dispersed into 2 new double helices following replication, therefore each strand consists of both old and new DNA
Specialized Transduction
phage carries specific parts of bacterial genome
what is Conservative Replication?
polynucleotide chains are synthesized like in semiconservative replication, however following synthesis, the 2 new strands come together & the parental strands redissociate. The original helix is thus "conserved"
Wobble Hypothesis
predicts that though the first two base positions are critical, H bonding between codon and anticodon at the 3rd position is subject to modified base pairing rules, therefore the anticodon of a single form of tRNA can pair with more than one triplet in mRNA
What does mitosis do to the chromosome #?
preserves chromosome number
Mitosis
process of nuclear division that results in 2 "daughter cells" each containing same chromosomal complement as the parental cell (2n --> 2n + 2n)
Biopharming
production of proteins
what is the sigma subunit responsible for?
promoter recognition; there are many different sigma subunits, each recognizing promoter regions, which provide specificity to the initiation of transcription
what is specific about RNA polymerase II (RNP II) during transcription?
promoters have a core promoter element that determines where RNP II binds to the DNA and where it begins copying the DNA into RNA
what is the function of the RecA protein in E.coli?
promotes exchange of reciprocal single stranded DNA molecules and enhances H-bond formation during strand displacement
Genotype Frequency
proportion of a particular genotype
what are nucleosomes?
protein spools that DNA is wound around, which needs to be disassembled before and reassembled after replication
blastp
protein vs protein database
What was previously thought to be the genetic material? Why?
protein; DNA was thought to be too simple with only 4 types of nucleotide vs 20 different amino acids in proteins
How was the initial insight that proteins are important in heredity discovered?
provided by the study of inborn errors in metabolism
Base pairing in DNA in terms of purines and pyrimidines structure
pyrimidine + pyrimidine = DNA too thin purine + purine = DNA too thick purine + pyrimidine = thickness compatible with X-ray data
frequency (NN genotype)
q^2
Genes far apart on same chromosome
r = .5
F=0
random mating
fraction of polymorphic genes
ranges from 24-50%
Map Distance b/w genes
rate of recombination
What is the focus of epigenetic therapy?
reactivation of genes that have been silenced by methylation or histone modification
The strongest direct evidence for DNA as the genetic material comes from..
recombinant DNA technology
What mechanism do C. elegans use?
reduction of X expression in females
epigenome
refers to the epigenetic state of a cell
Hormones and their receptors
regulate various activities
Paralogs
related genes w/in a species (come from duplication events in a genome)
what does alternative splicing produce?
related proteins from a single gene, increasing the number of products derived from a single genome
Structure of viral and bacterial chromosomes vs eukaryotic DNA
relatively simple DNA molecules: -a single nucleic acid molecule -few if any associated proteins -much smaller than eukaryotic chromosomes -viral chromosomes consist of single or double stranded DNA or RNA -bacterial chromosomes are circular, double stranded DNA and compacted into a nucleoid (DO NOT compact DNA like eukaryotes)
DNA double strand break (DSB)
repair is activated when both DNA strands are cleaved
general transcription factors
required for all RNP II mediated transcription and help RNA polymerase II bind to the promoter and initiate transcription
Sporatic Retinoblastoma
requires 2 independent mutational events of RB1 w/in the same cell
Induced Mutations
result from the influence of an external factor, either natural or artificial
Muscular Dystrophy
results from mutation in the gene encoding dystrophin
Most retroviruses that cause cancer are..
retroviruses (RNA) known as acute transforming retroviruses because they transform cells into cancer cells
What does DNA hypo-methylation do?
reverses inactivation of genes leading to unrestricted transcription of many gene sets including oncogenes (cancer causing genes)
C-DNA, D-DNA and E-DNA
right handed forms of DNA that are less compact than B-DNA
Transgenic Salmon
salmon expressing human growth hormone grow 400-600% faster than wild type apparently w/ no adverse health affects
Duplications
segments of chromosome occurs twice tandem: ABCDECDEFG reverse: ABCDEEDCFG displaced: ABCDEFCDEG (transposable elements)
Copy Number Variations (CNVs)
segments that are duplicated or deleted
What are other properties of cancer cells?
selective hyper-methylation and gene silencing
what did the Tyler-Woods-Hughes experiment demonstrate?
semiconservative DNA replication in eukaryotes
What is the accepted model for replication in viruses, prokaryotes and eukaryotes?
semiconservative replication
What must be known to amplify?
sequence of fragments you want to amplify
Repetitive DNA
sequences are repeated many times w/in eukaryotic chromosomes and there are a number of categories of repetitive DNA, making Eukaryotic chromosome organization complex
Telomeric DNA
sequences consist of short tandem repeats that contribute to stability and integrity of chromosomes
There is a large number of single copy noncoding regions, some of which are pseudogenes. What does this mean?
sequences that have undergone significant mutational alterations (insertions and deletions) and are not transcribed
Caspases
series of proteases that are responsible for initiating apoptosis and digesting the intracellular components
Complementarity of DNA strands allows each strand to..
serve as a template for synthesis of the other
tRNAs
serve as adaptor molecules to convert the codons in mRNAs to the correct amino acid ie. translation of one chemical language to another -have anticodons that complement mRNA codons -covalently attached to corresponding amino acids
p arm
shorter arm
Hardy-Weinberg Law
shows how Mendelian inheritance affects allele frequencies
what happens when initiation has been completed w/ synthesis of the first 8 to 9 nucleotides?
sigma dissociates and elongation proceeds with core enzyme
Single Nucleotide Polymorphisms
single base changes in the genome, many associated with disease conditions
Genomic alterations that are associated with cancer range from..
single nucleotide substitutions to large-scale chromosomal rearrangements, amplifications, and deletions
DNA gel
southern blot -detect w/ probe
triplet codons
specify one amino acid & contain "start" and "stop" signals, non-sense codons necessary to initiate and to terminate translation
what is the function of single strand binding proteins (SSBPs)?
stabilize the open confirmation
E value
statistical analysis comparing result w/ random chance -smaller the better
What do newer approaches involve to create transgenic animals?
stem cells rather than fertilized eggs
Function of oligonucleotides polynucleotides
store vast amounts of genetic information and give rise to extraordinary variation
r<.5
strong evidence of linkage
Collagen and Keratin
structural proteins
Functional Genomics
study of gene function based on the RNAs or possible proteins they encode as well as regulatory elements
Similarity Score
sum of identical matches/total # of bases or amino acids aligned
what does unwinding of DNA produce?
supercoiling (added twists & turns of DNA)
what is the function of primase?
synthesizes an RNA primer that provides the free 3' hydroxyl required by DNA polymerase III
2nd step of elongation
tRNA bound to the peptide chain moves to the P site
consanguine
term describing individuals descended from a commen recent ancestor
what happens when RNA polymerase stop traversing the gene?
termination of the nucleotide sequence is encountered when transcription terminates due to hairpin formation in the RNA, in which an enzyme falls off DNA
remaining 3 triplets are..
termination signals and do not specify any amino acid
What is age related cancer an indication of?
that cancer develops from accumulation of several mutations for epigenetic changes
what happens when a nucleotide is added?
the 2 terminal phosphates are cleaved off
why is the code degenerate?
the Wobble Hypothesis
the Genome
the complete set of DNA in a single cell of an organism
methylation occurs most often on..
the cytosine of CG doubled in DNA
what happens to somatic cells through out the life of an organism?
the ends of somatic cells are degraded through out life, which appears to play a major role in aging
Phenotypic expression of conditional mutations depends on..
the environment
Why is a recessive autosomal mutation in a somatic cell of a diploid organism unlikely to result in a detectable phenotype?
the expression of these mutations would likely be masked by expression of the wild-type allele within a cell
What has been approved to treat some forms of lymphoma?
the first HDAC inhibitor, vorinostat (Zolinza)
What does the formation of nucleosome represent?
the first level of packing, where the DNA helix is reduced to about 1/3 of its original length by winding around the histones, nucleosomes are condensed several times to form the intact chromatids
Mutator Phenotype
the high level of genomic instability in cancer cells
Enzymes
the largest and most diverse group of proteins are involved in biological catalysis, a process where the energy of activation for a given reaction is lowered
as the replication fork moves what strand can serve as a template for continuous DNA synthesis?
the leading strand
in prokaryotes, the initiation codon of mRNA (AUG) calls for..
the modified amino acid formylmethionine (fmet)
Where is DNA found in eukaryotes?
the nucleus and organelles
heterogeneous nuclear RNA
the product of transcription in eukaryotes
what is transgene silencing dependent on?
the site of integration into a genome
Genomics
the study of genomes
epigenetics
the study of the ways in which these changes alter gene expression
the site where transcription begins is called..
the transcription start site
Genome transplantation
the true test of the functionality of a synthetic genome
Biotchnology
the use of living organisms to create a product or a process that helps improve life for humans or other organisms
Metagenomics (Environmental Genomics)
the use of whole genome shotgun approaches to sequence genomes from entire communities of microbes in environmental samples of water, air soil
Neural Mutations
the vast majority of all mutations, occur in the large portions of the genome that do not contain genes and therefore have no effect on gene products
what did studies by Nirenberg and Matthei lead to?
they added RNA homopolymers to the in vitro translation system to decipher which amino acids were encoded by the first few codons based on which amino acids were incorporated into the polypeptide
Why do eukaryotic chromosomes contain multiple origins of replication?
they are large with many origins of replication to allow the genome to be replicated in a few hours
What is done after antibodies are made?
they are purified, detected, using a secondary antibody fused to an enzyme
What does indirect evidence about changes in nutrition and exposure to agents tell us?
they can affect the developing fetus and have detrimental effects during adulthood
What happens to chromosomes during metaphase? Give example in human genome
they condense by a factor of 10,000 ex.) human genome shrinks 1 m to .1 mm; mile fishing line into pen
What controls metastasis?
thought to be controlled by many genes, including those that encode cell adhesion molecules and proteolytic enzymes ex.) metalloproteinases are present at higher than normal levels in malignant tumors and are not susceptible to normal controls conferred by regulatory molecules such as tissue inhibitors of metalloproteinases (TIMPs)
How is the epigenome transmitted?
to daughter cells by mitosis and future generations by meiosis
Fragile X Syndrome
trace trinucleotide repeats
Forward Genetics
trait -->genome
Generalized Transduction
transducing phage can carry any part of the bacterial genome
Transposable elements
transposons or jumping genes are DNA sequences that can move or transpose w/in and b/w chromosomes, inserting themselves into various locations w/ in the genome
which nucleotides are encoded by a single codon?
tryptophan and methionine
RNA heteropolymers
two or more different ribonucleoside diphosphates are added in combination to form the artificial message (used to decipher more codons employing the same method as homopolymers)
How do viruses with RNA rather than DNA core replicate?
via RNA replicase
3rd step in elongation
uncharged tRNA moves to the E (exit site)
Chromatin
uncoiled nucleoprotein structure in which eukaryotic chromosomes are complexed into (looks like string of beads under E.M.)
what is a reason that cancer cells can proliferate forever? ("immortal")
unlike most somatic cells, malignant cancer cells have telomerase activity
What kind of sex chromosome mechanism does Drosophila use?
up-regulation of X in males RNA protein complex (MSL) binds X chromosome + doubles gene expression (acetylates histones)
Competence
uptake of DNA only possible in cells that are in state of competence
Basic approach for determining linkage
use binomial distribution assuming range of recombination frequencies: compare to probability with no linkage (r=.5)
what is the function of helicases?
use energy from ATP to break H bonds and denature helix
Fluorescent in situ hybridization (FISH)
used to identify the location of DNA or RNA of interest
2 Dimensional gel electrophoresis (2DGE)
used to separate hundreds to thousands of proteins w/ high resolution
Nucleic Acid Electrophoresis
uses an electric field to separate DNA and RNA fragments by size, such that smaller fragments migrate through a gel at a faster rate than large fragments (molecular sieving through gels of various pore size)
Bioinformatics
uses computer based approaches to organize, share, and analyze data in relation to gene structure, gene sequence and expression, and protein structure and function
what did research by crick show?
using T4 phage and frameshift mutation showed that a triplet code provides 64 codons to specify the 20 amino acids
Primer Design
usually 16-30 bases long, one forward, one reverse
Encyclopedia of DNA Elements (ENCODE)
was created w/ the aim of using both experimental approaches and bioinformatics to identify and analyze functional elements that regulate expression of human genes
what is the problem with lagging strand synthesis at chromosome ends?
when RNA primer is removed, there is no free 3' OH group for elongation lagging strand synthesis requires primers upstream of synthesis
Transversion
when a purine and pyrimidine are exchanged
Transition
when a pyrimidine replaces a pyrimidine or when a purine replaces a purine
maternally imprinted
when genes are expressed only if inherited from father
paternally imprinted
when genes are expressed only if inherited from mother
trans
wild type with mutant product
Ethical questions related to Germ line therapy
would stop increase in mutant alleles BUT brings other ethical issues: affects genomes of future generations
Typical Results of monohybrid cross
yellow: total = 6022 % = 75% ratio = 3:1 green: total = 2001 % = 24.9%
Length of genes linked together
~10 map units long
