11725208
Description
Flashcards by Candice Young, updated more than 1 year ago
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Created by Candice Young
over 6 years ago
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| Question | Answer |
| gene | segment of DNA that encodes a functional product along with the regions that regulate its expression |
| Open Reading Frame (ORF) | stretch of DNA from start to stop codons that encodes a protein |
| mutation | permanent, HERITABLE alteration in base sequence of DNA |
| allele | one of many alternative forms of a gene, think WT vs Mutant type |
| genotype | nucleotide sequence of genome/gene in question |
| phenotype | visible or measurable property conferred by a genotype |
| If asking: What GENES cause this PHENOTYPE? | take WT cells with normal phenotype X --> mutagenize cells --> Isolate X- mutants --> locate mutations in genome (yfg-Z) --> infer genes yfg-Z involved in generating normal phenotype X |
| Selectable mutations | confer a distinct GROWTH ADVANTAGE on mutant strain under some condition Ex: antibiotic or bacteriophage resistance |
| Non-selectable mutations | do NOT confer a growth advantage, though may result in some other clear change Ex: Histidine auxotrophs, Lac- mutants that cant grow with only lactose in medium, Mot- mutants that can't swim, differently colored mutants |
| auxotroph | mutants that can't make a certain product and rely on this product to grow (must be fed it) Ex: His- mutants |
| Selection | establish conditions where only the MUTANTS you want will GROW --> parent strain and all unrelated mutants die Ex: antibiotic/bacteriophage resistance mutagenize --> spread 10^8 survivors on plate --> 1-100 colonies will grow in selective conditions |
| selection: pros and cons | Pros: very efficient! can select desired mutants from HUGE pops of undesired Cons: not always possible, can't select for something that causes a growth disadvantage or death |
| Screen | after mutagenesis, grow ALL survivors under PERMISSIVE conditions --> then test all survivors for growth/behavior under NON-PERMISSIVE conditions --> replica plate to find mutants with desired trait Ex: can't grow without specific nutrient, loss of motility, can't grow at a specific temperature |
| Screen: pros and cons | Pros: Only option if phenotype involves a LOSS of normal abilities Cons: ALL survivors of mutagenesis must first be grown as independent colonies before they can be tested (takes a LOT more plates and time), generally less powerful evidence |
| Replica plating | use in a SCREEN to identify mutants with desired phenotype 1) grow all mutants as single colonies in permissive conditions --> 2) print colonies on non-permissive plate and permissive plate --> 3) blank spaces on NON-permissive represent desired mutants!! |
| essential gene | Gene whose functions is required under all known GROWTH conditions Ex: DNA replication, tx, tl, cell division, chromosome segregation --> can only isolate mutations in essential genes by SCREENING for conditional loss-of-function mutants |
| How to screen for a conditional loss of function mutant | screen for temperature-sensitive (ts) mutations: protein has an amino acid substitution (caused by a missense mutation) that causes it to work CORRECTLY at the LOW (permissive) temperature and FAIL at the HIGH (nonpermissive) temp --> then examine ts mutants for desired phenotype |
| mutation rate | probability that a given gene will acquire a mutation in one generation can be increased 10-1000X by MUTAGENS (ie: chemicals/radiation) |
| spontaneous mutations | arise from errors in DNA replication; each gene has about a one-in-a-million chance of being mutated each cell division --> bacterial cultures can have >108 cells/ml --> many mutants likely present |
| microlesions | base pair substitutions, small insertions/deletions |
| macrolesions | large deletions, insertions, duplications, or inversions (Ex: TRANSPOSONS) |
| Mutagenesis | increase the rate of mutation using mutagens --> increase our chances of finding desired mutants Mutagens can be chemicals, radiation, or transposons |
| Chemical Mutagens: Nucleotide Base Analogs | create mutations when incorporated into DNA during replication look like A/C/T/G --> create single-base substitutions in rounds of replication 5-bromouracil: looks like T, bond w/ A or G 2-aminopurine: looks like A, bond w/ T or C |
| UV radiation | ATAT pyrimidine dimers form from UV light --> UV damage detected by RecA which induces SOS response! |
| SOS response system | translesion synthesis: polymerase synthesizes DNA from a template containing pyrimidine dimers (which can't base-pair normally) --> UmuCD polymerase adds RANDOM bases across from the lesion --> mutations |
| transposons | mobile DNA elements, occur naturally move from place to place in the genome --> can be used to create random mutations target DNA doesn't matter, it jumps RANDOMLY will cause a loss of function mutation wherever it jumps |
| transposase | recognizes inverted repeat sequences (IR) --> cuts at ends of transposase gene + cuts the target DNA --> ligates into new site *each transposase is specific to own IRs, movement will not affect other transposons!* |
| Engineered Transposons: Requirements | 1) Transposase OUTSIDE mini Tn 2) Antibiotic resistance gene INSIDE mini-Tn 3) Plasmid cannot replicate in the host strain to be mutagenized must be able to only jump ONCE, can't replicate in offspring |
| Why can't the inserted mini-Tn not move again? | the transposase was only encoded on the plasmid!! this isn't transferred to cells after division |
| advantage of Tn mutagenesis | mutated gene is easy to identify: since we know the mini-Tn sequence --> design DNA primers to locate site of insertion, will read out whatever DNA you want |
| genetic exchange | How bacteria acquire new genes from the environment/from other bacteria |
| horizontal gene transfer | NATURAL genetic exchange among bacterial strains/species Use to: 1) to identify affected genes in mutants (complementation) 2) engineer bacteria w/ desired properties |
| transformation (natural) | transfer of genetic material from one bacterium into another DNA acquired by transformation, binds to protein and becomes ss --> RecA nicks chromosome, makes a ss segment --> ss DNA combined with ss chromosome --> homologous recombination --> integrated DNA replaces similar genes |
| Griffith experiment | S cells = virulent; R cells = avirulent heat killed S-cells --> healthy mouse live S-cells --> dead mouse live R-cells --> healthy mouse Live R + killed S cells --> dead mouse --> something could transfer the genes of dead S cells to living R cells!! |
| F plasmid | used during conjugation; plasmid that undergoes homologous recombination tra = genes involved in conjugative transfer, transferred last oriT sequence = origin of transfer during conjugation |
| homologous recombination | physical exchange between two highly similar DNA molecules can NOT be used to integrate entirely foreign DNA into bacterial chromosome, because no homology! ssb, RecA, RecBCD, RuvABC proteins mediate process |
| competence | ability of cells to take up free DNA, can be INDUCED by incubating cells w/ high conc. of Ca++ or by electroporation --> increase cell permeability + allow uptake of DNA --> can introduce non-homologous, replicating plasmids to any cell |
| Plasmid | circular genetic element that replicates independently of the chromosome; contain NO essential genes; small; replicates by normal cellular machinery |
| conjugation | DNA transfer requires cell-cell contact (using a F PILUS); mediated by F plasmid --> encodes proteins that catalyze its own transfer F+ cell donates to F- cell --> both become F+ |
| tra regions of F plasmid | contain genes necessary for F plasmid to be self- transmissible transferred last |
| oriT region | origin of replication, where mobilization of F plasmid starts |
| oriV region | allows plasmid replication within the host, without transfer |
| transposons/IRs in F plasmid | help mediate integration into host chromosome |
| Why would we use bacterial conjugation? | 1) Transfer a plasmid that WILL REPLICATE 2) Transfer a plasmid that WILL NOT REPLICATE (intending some of DNA transferred is integrated into chromosome by homologous recombination 3) Transfer plasmid w/ engineered transposon --> mutagenize recipient strain |
| transduction | phage-mediated (often specific) transfer of genes between bacterial chromosomes 1) Phage attacks, injects its DNA 2) Host chromosome cut into pieces 3) New phage proteins/DNA made -->DNA packaged into phage particles 4) RARE packaging of host chromosome fragment; creates a transducing particle |
| transducing particle | when host chromosome fragment is packaged into a viral transducing particle- **RARE** this fragment will be transferred to recipient + incorporated into recipient chromosome by homologous recombination IF donor & recipient bacteria have similar enough DNA |
| transductants | the product of homologous recombination in a recipient cell with a transducing particle --> this REPLACES some old part of the host chromosome |
| Why use transduction? | to transfer mutations from one bacterial chromosome directly to the chromosome of another strain |
| How to combine two mutations in the chromosome of one bacterial strain | USE TRANSDUCTION --> take one cell w/ KmR tn insertion in yfgA gene, another w/ point mutation in yfgB gene --> infect yfgA::Tn strain w/ phage and harvest all phage after cell lysis --> tiny fraction has original host DNA w/ the tn --> infect yfgB mutant w/ phage harvested from yfgA --> plate cells on medium w/ antibiotic to select for double mutant --> observe consequences of both mutations |
| Why would we want to express a particular gene from a plasmid in a bacterial host? (think: how could we understand a particular gene's function?) | 1) To try moving the gene around to a different species or type of mutant 2) To express a different allele of the gene than the WT 3) To complement genomic mutation with the WT allele of the gene on a plasmid 4) Change the regulation of the gene (turn it off/on, increase its expression level) |
| complementation | getting a mutant phenotype to be expressed using some sort of gene carried on a plasmid |
| How to find affected gene when using UV or chemical mutagenesis | Make a genomic library: isolate genomic DNA from WT parent & break it up using restriction enzymes --> ligate fragments into plasmid that CAN replicate in strain of choice --> have mixture of plasmids, each one containing a diff seg of chromosome THEN : Select for presence of the plasmid --> select/screen for colonies that have WT phenotype RESTORED --> means this corresponds to mutated site in chromosome |
| When to use RANDOM mutagenesis | when I don’t know AT ALL what genes are involved in my process of interest OR when I have no sequence information --> use chemical mutagens, UV radiation, transposon |
| When to use TARGETED mutagenesis | when you have candidate genes to test --> use sequence information of genes you are targeting or the whole genome sequence |
| targeted gene disruption | 1) PCR some known gene (orfA) + surrounding region, clone into a plasmid 2) Cut gene (orfA) with restriction enzymes 3) Replace internal region w/ antibiotic resistance gene (Kan^R) (leave 500-1000 bp of sequence at each end so that homologous recombination can occur!) |
| How to transform some competent bacterium with a knockout plasmid | 1) linearize modified plasmid with some restriction enzyme OR 1) use a modified plasmid that doesn't replicate in the host you are making the KO --> antibiotic resistance conferred by recombination of disrupted gene/tn into chromosome |
| When does targeted gene disruption NOT work? | --> when the gene you disrupt is ESSENTIAL |
| How would we learn the null phenotype of an essential gene? | make plasmid w/ essential gene driven by inducible promoter (ts) --> transform plasmid into host strain and keep gene ON using some inducer --> w/ plasmid gene ON, knock out chromosomal copy of the gene using targeted gene disruption --> when plasmid gene the only copy, turn OFF by removing the inducer --> wait for the existing protein to be turned off --> observe cells to see what happens in the absence of the essential gene/protein |
| inducible promoters | encode proteins that are needed in the cell only under specific conditions EX: Caulobacter senses xylose --> Pxyl promoter turns on genes (xylXABC) to use xylose as C source can be put in front of any gene of interest to turn on/off and observe consequences! |
| Forward genetics | Make RANDOM mutations (chemical mutagens, UV radiation, transposons) --> select or screen through many mutants to find ones with correct phenotype "what gene did we affect to get some trait?" |
| Reverse genetics | Make TARGETED mutations in some gene of choice --> test each mutant in a set of not too many mutants for the desired phenotype "what trait will we affect if we change this gene?" |
| To mutate a known gene using a transposon | select mutants that had received a transposon --> select or screen for mutants with your desired PHENOTYPE *no homology required, no need for RecA protein (transposase is the catalyst instead)* |
| Transposon Mutagenesis vs Targeted gene disruption | both cause a knock out mutation BUT tn: RANDOM, know the phenotype, no homology required, uses transposase, have IRs at ends TGD: NOT random, don't know the phenotype, |
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