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MMBIO 240 Unit 2
Starting at topic 10
| Question | Answer |
|---|---|
| What are genes | Old definition: DNA that codes a protein New: DNA that encodes for the function of an RNA or protein. |
| Gene expression | Old: production of a protein via central dogma (DNA-->RNA-->protein) New: old still applies, but we know too that some RNA's function w/o protein and once a cell starts making a protein it can choose to stop. |
| Why use Reverse transcription | RNA is unstable, so convert it to DNA to work with. Complementary DNA (cDNA) is antiparallel. Done with reverse transcriptase. |
| Reverse transcriptase | RNA dependent DNA polymerase, discovered in retroviruses. |
| How reverse transcription works | Anneal primer (like oligo dT). Reverse transcriptase+ dATP (or other type of triphosphate like dGTP), alkaline digest of RNA, leaving the cDNA. (First strand synthesis. 1 copy of each RNA) |
| The 3 types of priming in reverse transcription | 1. Oligo (dT)/poly dT: Matches poly A tail. TTT... Will prime with any mature RNA with a poly A tail. 2. Random hexamers: 6 nucleotides. Makes cDNA of ALL RNAs 3. Gene specific: makes cDNA of specific sequences |
| Why make cDNAs? | Normal DNA is huge because it has so many introns/exons. Making cDNA's helps you reduce that size and study alternative splicing of exons. |
| Cloning cDNAs purpose | Insert cDNA in expression vector Makes recombinant DNA Transcribed to mRNA, translated to protein Makes recombinant protein Can use to make therapeutic proteins/hormones like insulin |
| Reverse transcriptase PCR (RT PCR) | Use reverse transcriptase enzyme to amplify mRNAs by making cDNA out of them, then use Polymerase Chain Reaction to amplify. |
| Quantitative PCR | Each cDNA produced creates a fluorescence. Quantify the amount of light. |
| Microarrays | A way to look at gene expression in samples by testing RNA levels. Test thousands of genes at a time. |
| How to make a microarray | 1. Isolate mRNA from 2 samples. 1 red 1 green 2. Convert mRNAs into cDNA w/ reverse transcriptase and fluorescent deoxyribonucleotides 3. Add cDNA to microarray containing complementary seq that anneal 4. Glowing spot=more gene expression in sample |
| Polymorphism | Variations that exist in the genome of a population |
| Allele | Same gene, different chromosomes (1 dad 1 mom, same gene) |
| The 3 types of polymorphisms | 1. SNP (single nucleotide polymorphism) 2. Insertions/deletions 3. VNTRs (variable number of short tandem repeats, non coding areas of genome) |
| Single nucleotide polymorphisms | Substitution: nonsense (codes stop), missense (codes different acid), silent (diff nucleotide same acid) Frameshift from insertion/deletion |
| Wobble Hypothesis | Multiple codons for each amino acid. Alanine coded by GCU, GCC, GCA, GCG. Allows for silent mutations. |
| Frameshift mutations | Whenever insertion or deletion happens in groups of anything but 3s. |
| Use of studying polymorphisms | Disease prediction/prevention, identify in crime scenes, etc. |
| Restriction Fragment Length Polymorphisms | Everyone has rare and unique palindromes in their VNTR. When cut, gives you different size fragments. |
| Steps to RFLP | Extract DNA from blood Restriction enzymes cleave @ palindromes Electrophoresis separates fragments by size Transfer fragments to membrane (southern blot) Radioactive Probe binds Wash unbound X-ray film Compare crime scene pattern w/ suspects |
| Fastest way to detect polymorphisms | Microarray with SNPs of interest. If match, will bind and release flourecence. |
| What is an assay | Measuring enzyme activity |
| Do prokaryotes and eukaryotes have chromatin? | No, only eukaryotes, bacteria doesn't care. |
| What are the levels of DNA? | 2nm naked DNA 11nm nucleosomes 30nm filaments 300nm extended form 700nm condensed section of chromosome Mitotic Chromosome |
| Active form of DNA | Ramen bowl easily read by proteins so it can express genes |
| Metaphase chromosome | Condensed with a centromere (DNA seq repeats where spindle fibers attach) and telomere (DNA seq repeats at ends to protect genetic info) |
| Level 1 DNA | 2nm Naked DNA, Plain ol nucleotide structure bonded together 5' to 3' antiparallel strands. |
| Level 2 | 11nm DNA wraps 1.6-2 times around each histone in the octamer. |
| What charge are histones mostly | Positive to attract DNA |
| How to know how much DNA wrapped around histones? | Micrococcal nuclease cuts the DNA between nucleosomes. |
| Purpose of H1 domain on histone | Higher compaction between histones, releases after nuclease digestion. |
| Level 3 | 30nm Nucleosomes compact because of H1 interactions. |
| Level 4 | 300 nm 30nm associates with proteins for further compaction, making a skeleton where the 30nm fills in to make the 300nm. |
| Acetylation | Changes histone more neutral, activating genes. Acetyl coA added by histone acetyl transferase (HAT). HDAC removes it from the lysine tail. |
| Methylation | Makes it more positive, attracting DNA closer, deactivating genes. |
| The CHIP Assay: what is it and what does it show? | Chromatin immunoprecipitation Shows what proteins associate with what DNA (transcription factors) and if histones are modified (methylation) |
| Steps for CHIP assay | Cross link protein+DNA w/ formaldehyde Sonication breaks into usable fragments Antibodies bind, pull down fragments in centrifuge Reverse cross linking PCR Analysis |
| Transcription factors | Regulate transcription by recruiting/repelling RNA polymerase. |
| How to analyse a CHIP assay | 3 columns in PCR Analysis: Control antibody that shouldn't bind to any DNA Control on non-pure DNA BCR1 (or other) input. Rows mean those seq associated. |
| Goals of DNA replication | 1. Accurate 2. Keep ends from shortening 3. Complete new copies for cell division |
| The models of DNA replication | Conservative: original/original, new/new Semiconservative: original/new, orignal/new Dispersive: orignal and new mix on strand with another mixed strand |
| General steps of a pulse chase experiment | Add a "pulse" (label) of a substance (radioactive nucleotides/amino acids) Newly synthesized molecules in cooperate the label "Chase" (remove) the labeled substance Observe the labeled molecules over time |
| What pulse chase experiments tell you | Where things go in a cell How things move in cell How things made in cell and at what rate How long before thing degrades |
| Generic pulse chase with IRP2: how quickly will it degrade in the presence of a drug? | Grow cells in the presence/absence of a drug Pulse cells with S35 Chase S35 after 2 hrs Purify IRP2 w/ affinity chromatography SDS PAGE Detect radioactivity w/ x-ray Quantify amount per band |
| Meselson-Stahl pulse chase experiment RESULT | Shows that DNA is semiconservative |
| Meselson-Stahl pulse chase experiment PROCEDURE | Grow bacteria in heavy nitrogen (HN) until all DNA made w/ HN: pulse Switch bacteria to regular N for 1rnd replication Centrifuge separates DNA w/ Heavy/light/mix of N in cooperated in strand Showed some DNA ended w/ 1 heavy 1 light strand |
| OriC | Origin of replication. Lots of A:T's. Eukaryotes need many, bacterial plasmids only need 1. |
| Initiator protein | Binds to oriC |
| Helicase | Breaks hydrogen bonds between bases to unzip the DNA along each replication fork |
| Single stranded binding protein | Stabilize the ssDNA, makes sure it doesn't re-anneal to original strand. |
| Primase | Lays down RNA primers to start synthesis because it creates a free 3' end. |
| DNA polymerase | Adds nucleotides to 3' end based off of template. III- does most of the work. Dimer with 1 working on the lead strand, the other on the lagging strand. Proofreads mismatch+fixes I-Fills in where RNA primers are on lagging strand |
| RNAse H | Removes the RNA primers |
| Ligase | Glues the newly synthesized DNA together. Connects okazaki fragments with phosphodiester bonds. |
| Topoisomerase | Ahead of replication fork it prevents super coiling of DNA by breaking it for relief. |
| The problem with linear DNA | Primase doesn't work at the end of a strand, so overtime it shortens. |
| Telomerase | Has RNA template inside Cuts lagging Extends lead strand w/ RNA repeats Makes enough space for primase and polymerase to work It is still uneven at the ends, but ends are junk DNA so its ok. Protects the important stuff. |
| What are the environmental causes of DNA damage and how we are exposed to them | UV light: sunlight X-rays: dentist, space Gamma rays: space, radioactive isotopes Hydrolytic cleavage: water Oxidation: normal cell stuff Alkylation agents: chemicals Chemical cross-linking agents: chemicals DNA insertions: viruses+transposons |
| What are the types of DNA damage and how are we exposed to them | Pyrimidine dimers: UV light Hydrolytic cleavage: water (depurination/deamination) Alkylation: enzymes that add alkyl groups Added -OH/-O to base: reactive o2 species Interstrand cross links: mustard gas DNA insertions: virus+transposons |
| Pyrimidine dimers | T:T, T:C, C:C (on the same strand). From UV light UV destabilizes bonds, reform improperly, kink DNA, hard for protein to work with. Photoproduct covalent: IRREVERSIBLE! |
| Hydrolytic cleavage | Hydrolysis of: Phosphodiester bonds: easily fixed by ligase N-glycosyl bonds: depurination. Cuts base Bonds on A/C with amine group NH2: deamination, may change base |
| Alkylation | Add alkyl group to hydrocarbons on cytosine |
| Effects/possibilities of mutation | Can be: Fixed or it could go undetected and pass onto daughter Silent mutation or it could code new amino acid Be in non-coding region or in a gene |
| Reactive oxygen species | Oxygen with unpaired electron is desperate and reactive to fix it. Natural biproduct from mitochondria in cellular respiration. Antioxidants soak them up to prevent DNA Damage. (Fruit+veg) |
| Types of mutations caused by ROS | Extra -O/-OH groups Interstrand bonding (diaganol) |
| Ionizing Radiation on DNA | From x-rays and gamma rays. Double strand breaks without sticky ends so randomly rejoins, screwing everything up. |
| The cells most susceptible to DNA damage | The ones that divide quickly |
| When to intentionally damage DNA | Chemotherapy, immunosuppression, cisplatin drugs make cross links in tumor to prevent replication. |
| Insertional mutagenasis | Retrovirus in cooperates DNA into host, irreversible. Transpoon: enzyme moves nucleotides into smack middle of a gene. |
| When mutations have an effect on the genome it can be: | Loss of function: makes a useless protein Gain of function: once useless protein gains function |
| The Ames test | Looks for gain of function mutations. Why? Identify carcinogens that cause base changes before shampoo can be used on humans. |
| Ames test procedure | Salmonella strain that needs histidine to grow Add wild NOT need His to grow to bacteria Add mutagen/shampoo Add histidine Monitor development of colonies that grow in Histidine (inactive his gene mutated by shampoo to be active and restore His gene) |
| Types of DNA repair | Direct: for minor damage, just reverses Excision: For base errors/bulky stretches of mistakes removed and replaced Mismatch: for base mismatches, short insertions/deletions, similar to excision |
| How do cells detect DNA damage | Damage causes change in structure: DNA repair enzymes detect DNA Replication/RNA transcription enzymes discover damage as they use template to transcribe. |
| Direct DNA repair | Photolyase reversal of thymine dimer: uses energy in light waves to change bonds, or like a methyltransferase group removing an alkyl group |
| BEP Base excision repair | Fixes depurination/deamination/oxidation/alkylation Removes damage, sugar flips out, glycosylase recognizes+removes base AP Endonuclease cleaves backbone on 5' Fill in nucleotides: short w/ pol B, long w/ pol Ligase seals |
| When BEP mechanism is late | RNA polymerase stalls at lesion, recruits repair enzymes to fix |
| Xeroderma pigmentosum | Disease when genes that code for BEP don't work. |
| Linked vs unlinked genes | Linked are close to each other on the same chromosome and usually move together if crossing over occurs. Unlinked are not on the same chromosome, and crossing over does not occur |
| How does crossing over occur | Probably the Holiday junction, in meiosis prophase 1 |
| Steps of Holiday Junction | Sister chromatids align in prophase 1 Nick DNA Strands breathe apart and re anneal to other strand because sister has similar sequence DNA displace each other (branches migrate+resolve) |
| Proteins involved in holiday junction | RuvA/B/C |
| The ways to resolve the Holiday junction | 1. Branch reaches end of linear molecule (crossing over 2. Double strand break 2a. Gene conversion, dispersive 2b. Crossing over |
| Gene conversion vs crossing over | Crossing over is a big segment, usually reaches end of molecule/junction Gene conversion is small exchanges of genes |
| Homologous recombination | When sister chromosomes recombine in crossing over events before cell division. Makes children have gene combos. |
| The 3 ways DNA recombination repairs DNA | Repairs double strand breaks When DNA skips section, other strand can cross over to fill gap when DNA polymerase stops at lesion, can cross to other to get past it |
| How is homologous recombination used in studies? | To study large genomes by manipulating small fragments. Promote 2 crossing over events that happen spontaneously. |
| Knockout mice | Delete portions/all of a gene to study lack of it. Lack of gene "x" results in "y" disease. Test drugs and model human disease. |
| Examples of common knockout mice | SCID mouse: Lack of the gene that codes T+B cells so no immune system. Dystrophin: muscular dystrophy gene |
| Knockin mice | Add genes to genome. |
| Breeding mice to get the homozygous allele | +/+ is wild type allele +/- is 1st round, heterozygous recessive allele of 1 wild 1 inserted gene -/- is the knockout mouse with the homozygous inserted mutation. |
| How to make a knockout mouse | Clone DNA w/PCR-->in plasmid Restriction enzymes cut out gene/parts of it put in bacteria. Track w/selectable marker gene resists hostile envmt:NeoR bacteria in stem cells. Recombination swap some Implant success stem in female Breed success babies |
| Nuances of knockout mice | If the knockout/in is so lethal it kills mouse before born/developing/breeding, can't study it. So, use conditional knockouts that trigger when developed: cre-lox: Code between loxP sites is forced out by the cre enzyme recombining lox P together |
| Steps to conditional knockout | Mouse A w/ seq of interest+loxP sites Mouse B w/ recombinase gene+promoter that only works to initiate transcription at certain period of development Breed until offspring are -/- As promoter activated, Cre recombines loxP to delete the gene |
Created by:
lilelise
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