Karteikarten
Lectures 4 & 5 & 6 PMB
Karten in diesem Stapel (63)
- 1genesegment of DNA that encodes a functional product along with the regions that regulate its expression
- 2Open Reading Frame (ORF)stretch of DNA from start to stop codons that encodes a protein
- 3mutationpermanent, HERITABLE alteration in base sequence of DNA
- 4alleleone of many alternative forms of a gene, think WT vs Mutant type
- 5genotypenucleotide sequence of genome/gene in question
- 6phenotypevisible or measurable property conferred by a genotype
- 7If 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
- 8Selectable mutationsconfer a distinct GROWTH ADVANTAGE on mutant strain under some condition
Ex: antibiotic or bacteriophage resistance - 9Non-selectable mutationsdo 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 - 10auxotrophmutants that can't make a certain product and rely on this product to grow (must be fed it)
Ex: His- mutants - 11Selectionestablish 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 - 12selection: pros and consPros: 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 - 13Screenafter 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 - 14Screen: pros and consPros: 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 - 15Replica platinguse 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!! - 16essential geneGene 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 - 17How to screen for a conditional loss of function mutantscreen 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
- 18mutation rateprobability that a given gene will acquire a mutation in one generation
can be increased 10-1000X by MUTAGENS (ie: chemicals/radiation) - 19spontaneous mutationsarise 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 - 20microlesionsbase pair substitutions, small insertions/deletions
- 21macrolesionslarge deletions, insertions, duplications, or
inversions (Ex: TRANSPOSONS) - 22Mutagenesisincrease the rate of mutation using mutagens --> increase our chances of finding desired mutants
Mutagens can be chemicals, radiation, or transposons - 23Chemical Mutagens: Nucleotide Base Analogscreate 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 - 24UV radiationATAT pyrimidine dimers form from UV light --> UV damage detected by RecA which induces SOS response!
- 25SOS response systemtranslesion 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 - 26transposonsmobile 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 - 27transposaserecognizes 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!* - 28Engineered Transposons: Requirements1) 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 - 29Why 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
- 30advantage of Tn mutagenesismutated 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
- 31genetic exchangeHow bacteria acquire new genes from the environment/from other bacteria
- 32horizontal gene transferNATURAL genetic exchange among bacterial strains/species
Use to:
1) to identify affected genes in mutants (complementation)
2) engineer bacteria w/ desired properties - 33transformation (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 - 34Griffith experimentS 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!! - 35F plasmidused during conjugation; plasmid that undergoes homologous recombination
tra = genes involved in conjugative transfer, transferred last
oriT sequence = origin of transfer during conjugation - 36homologous recombinationphysical 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 - 37competenceability 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 - 38Plasmidcircular genetic element that replicates independently of the chromosome; contain NO essential genes; small; replicates by normal cellular machinery
- 39conjugationDNA 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+ - 40tra regions of F plasmidcontain genes necessary for F plasmid to be self- transmissible
transferred last - 41oriT regionorigin of replication, where mobilization of F plasmid starts
- 42oriV regionallows plasmid replication within the
host, without transfer - 43transposons/IRs in F plasmidhelp mediate integration into host chromosome
- 44Why 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 - 45transductionphage-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 - 46transducing particlewhen 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 - 47transductantsthe product of homologous recombination in a recipient cell with a transducing particle --> this REPLACES some old part of the host chromosome
- 48Why use transduction?to transfer mutations from one bacterial chromosome directly to the chromosome of another strain
- 49How to combine two mutations in the chromosome of one bacterial strainUSE 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 - 50Why 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) - 51complementationgetting a mutant phenotype to be expressed using some sort of gene carried on a plasmid
- 52How to find affected gene when using UV or chemical mutagenesisMake 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 - 53When to use RANDOM mutagenesiswhen 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 - 54When to use TARGETED mutagenesiswhen you have candidate genes to test
--> use sequence information of genes you are targeting or the whole genome sequence - 55targeted gene disruption1) 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!) - 56How to transform some competent bacterium with a knockout plasmid1) 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 - 57When does targeted gene disruption NOT work?--> when the gene you disrupt is ESSENTIAL
- 58How 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
- 59inducible promotersencode 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! - 60Forward geneticsMake 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?" - 61Reverse geneticsMake 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?" - 62To mutate a known gene using a transposonselect 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)* - 63Transposon Mutagenesis vs Targeted gene disruptionboth 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,