Topic 9 - Genetics Flashcards
microbial genetics grew from ____, led to ___ ____
microbiology
molecular biology
when did microbial genetics start
1940s-1950s
required development of 2 model systems for genetic investigations:
- Escherichia coli and Salmonella typhimurium
- E.coli K-12 = not pathogenic, studied
organization of bacterial genomes
single chromosome and plasmids
bacteriophage DNA can also be present
what is replicon?
chromosomes + plasmids in cell
plasmid copy # in cell is ___ regulated
CLOSELY
- diff plasmids can be copied diff times
plasmids typically ____ than genomes
smaller
T/F plasmids usually do not encode housekeeping codes
T
T/F antibiotic resistance genes uncommon in plasmids?
F
what is plasmid copy number governed by?
plasmid-encoded genes
plasmids with similar replication controls =?
incompatible (Inc)
why are bacteria ideal genetic research candidates
- one chromosome for easy detection of mutations
- early studies, nutritional mutants were used
– allowed study of one gene based on its inability to use or produce a particular nutrient
wild type meaning
- strain most like that found in nature
- original isolate
- source for deriving mutants
mutant meaning
- strain carrying a mutation, relative to wild type
mutation meaning
- change in a gene that disrupts/alters functions
allele meaning
variant of a gene
- may be gain of function
- may be loss of function
- may be change of function
auxotroph meaning
- mutant that is unable to make a specific compound
- often a mutation in AA biosynthesis
prototroph meaning
- strain capable of making all required organic compounds
how are genes/proteins named generally?
genes
- three-letter abbr. in italics, followed by a capital letter to separate genes in same pathway
proteins
- given same three-letter designation, first letter capitalized, no italics
why do microbial geneticists compare wild-type strains and mutant strains?
goal: to identify differing alleles of genes
mutant selection
- isolation of cells with a particular genotype on basis of growth
- can select for His+ on basis of growth
- canNOT select directly for His-
mutant screening
- identification of cells with a phenotype
- colour, morphology, “no growth”
- CAN identify His- by screening
difference between selection vs screening
selection: cannot directly identify auxotroph on basis of growth
screening: can directly identify auxotroph through phenotype
what makes a mutation selectable or non-selectable?
selectable mutations generally give a growth advantage under specific conditions
- e.g., conditions that kill wild-type
- useful in genetic research
non-selectable mutations confer NO advantage or confers a DISadvantage
- detection requires screening of a large # of colonies
phenotypic selection
- use a growth medium that inhibits microbes lacking desired gene
- antibiotic selection commonly used
screening - replica plating
- more tedious than selection
- can facilitate screening with replica plating
- duplicate plates are created (one lacks particular nutrient)
- a mutation has occurred where a colony grows on full support plate but doesn’t grow on partial support plate
screening - patching
- transferring colonies (w/ toothpick) to a gridded plate
- usu more accurate and reproducible than standard velvet replica plating
types of mutations (4)
silent - no change in AA seq of protein; usu third position of codon
missense - change in codon that results in a diff AA
nonsense - early STOP
frameshift - shifts reading frame (insertion or deletion of nucleotides)
reversion mutation meaning (why problematic? how to avoid?)
reversion: a mutation that “corrects” a metabolic abnormality back to wild-type form
- problematic when trying to determine mutation rates of a chemical or DNA exchange rates between two microbes
- to avoid this problem, double and triple auxotroph mutant strains were studied (decreases possibility of a spontaneous reversion mutation)
spontaneous mutations (2 experiments - Lederberg + Luria-Delbruck)
- experiments by Esther Lederberg used replica plating to illustrate spontaneous mutation without selective pressure
(mutations did not adapt in real-time, were always there) - Luria and Delbruck showed variable resistance to phage infection arises in bacteria without selective pressure (random)
Richard Lenski E. coli experiment
cultures given an extended generational time w/ selective pressures -> compared to original cultures w/o selective pressures
- culture grown under selective pressure displayed higher fitness
restriction enzymes
- name explains what organism they came from
- cut DNA at a specific recognition site
- usu palindromic (sticky/blunt ends)
- similar ends of cut DNA can be paired together and ligated
modification enzymes
- REs ALWAYS paired with modification enzymes
– often in a single operon
– referred to as “R/M systems” - recognize the same site as the paired RE
- methyltransferase activity protects DNA from endonuclease activity (e.g., EcoRI doesn’t cut E.coli)
cloning vectors
- REs allow researchers to stitch together DNA fragments into recombinant molecules
- recombinant molecules can be used to clone a bacterial gene of interest
- vectors are used to insert a recombinant DNA molecule into a recipient host bacterial cell (plasmids, phages, cosmids)
plasmid cloning vectors
- first used in 1970s by Cohen
- cut fragments from 2 plasmids carrying antibiotic resistance genes w/ same RE
- transformed strain exhibited traits from both plasmids
oriV = ?
origin of replication
(v = vegetative growth/replication)
what does the ori control? (2)
(1) what types of organisms the plasmid can be in
(2) # of copies of plasmid that will be made
desirable plasmid traits for easier gene cloning (5)
- origin of replication (ori)
- selectable marker gene
- multiple cloning site
- small size
- high copy #
X-gal screening for transformed cells
- no beta-galactosidase = white, meaning insert successful!
- X-gal = analog of lactose (cleaved by beta-galactosidase)
X-gal = white (yes insert)
X-gal = blue (no insert)
shuttle-vector plasmids
- shuttle-vector plasmids have MULTIPLE types of ori
- expands range of host cell types that the plasmids can be inserted into
(replication in certain hosts is restricted by ori)
phage vectors
- mix viral DNA with fragment of interest
- lysogenic lambda phage can carry ~20kb fragments -> take this genetic material out, add gene of interest (needs to be same length)
- Cos (abbrv of “cohesive end site”
phages infect cells, replicates and lyses cells, then we can recover recombinant phage from plaques
Cos =?
cohesive end site
cosmids
- phage genome that omit nearly all the phage DNA, leaving more room for the fragment
- only the critical phage cos packaging recognition sites remain
- other elements incl a multiple cloning site and an antibiotic selection marker
- cosmids can typically carry 35-45kb fragments
- NO LYSIS, viruses just added to inject genomic data
list cosmid vector components
- cos site
- cloning site
- oriV
- antibiotic resistance
transformation
- introduction of extracellular DNA into organism (from env)
- doesn’t require cell-to-cell contact
- a part of SOS reaction to genetic dmg***
- some bacteria naturally competent for transformation
- other bacteria can be artificially induced to become competent
– treatment w/ Ca2+ cations (double charged, attracts DNA ss and cell membrane to get the physically close)
– electroporation
during transformation, the foreign DNA comes into the cell in ss/ds?
single strand!!! (activates SOS response)
transformation of what response? AKA?
SOS response (to genetic damage)
ALA homologous recombination
conjugation (what is it, mechanism)
- transfer of DNA from cell to cell via DIRECT CONTACT/SEX PILUS formation
mechanism:
- F plasmid carries gene to form sex pilus bridge” between 2 cells
- F plasmid can be copied and sent across the bridge (origin of transfer (oriT) first) into recipient cell
- turns an F- cell into an F+ cell capable of conjugating with another F- cell
- note: tra genes
big difference between transformation and conjugation
transformation: no direct cell-to-cell contact
conjugation: requires direct cell-to-cell contact
F plasmid =?
fertility plasmid
F-plasmid conjugation steps
- sex pili pull cells together, forms mating bridge, ss nick made at oriT
- one strand of F plasmid is transferred into recipient (one strand kept behind), replication occurs in both donor/recipient -> makes ds F plasmids
- results in two F+ bacteria
how does the F plasmid integrate into host chromosome?
homologous recombination
what does oriT need?
origin of transfer needs tra (tra operon codes for essential proteins needed in conjugation)
what does “F plasmid is an episome” mean?
F plasmid is DNA that can integrate into chromosome or exist autonomously
Hfr =? explanation
high frequency of recombination strain DNA transfer
- incorporated F plasmid sends host cell DNA next to its incorporation site across mating bridge over time
- can be used to “map” location of genes in host chromosome
Hfr cell vs F+ cell
Hfr cell = integrated F plasmid (big host DNA w/ F plasmid combined)
F+ cell = F plasmid separate from chromosome, attached through homologous DNA seq (SITE-SPECIFIC recombination)
Hfr gene transfer is like F plasmid but…?
they both use sex pili but Hfr uses the integrated F factor to insert genes into F- cell.
by stopping conjugation at diff time intervals, gene location can be mapped (genes closer to site of integration are transferred first)
generation process of F’ plasmids
- an incorporated F plasmid excises itself
- excision is inaccurate, some host DNA is excised too
- when F’ plasmid conjugates, it sends the HOST cell DNA to recipient
triparental conjugation
- conjugation can still occur using recombinant plasmid lacking the required tra genes and a helper plasmid w/ tra genes
- more room in the recombinant plasmid for a desired DNA fragment
helper plasmid conjugates into donor strain, encodes proteins for transfer of recombinant plasmid from donor strain, conjugates into recipient
transposition (what is it, discovered by who)
- movement of DNA via mobile genetic elements
- transposable elements can move within and between genomes
- first detected in corn by Barbara McClintock
transposition can be subdivided into:
- insertion sequences: encode only proteins needed for transposition
- transposons: contain other genes in addition to those needed for transposition
transposons can be non-replicative or replicative, meaning?
non-replicative - cut and paste
replicative - copy and paste
mechanisms of transposition:
- requires transposase and resolvase genes
- replicative transposition: copies element and moves copy to another location
- non-replicative transposition: cuts and pastes element into new location
transpositions can be used to ___ functional genes and observe phenotypic changes
disrupt
____ vector plasmid carrying the transposon
- details
suicide
- recipient cell gets the plasmid, but plasmid can’t replication
- transposition can still occur at random
- screening and/or selection for desired disruption follows
transduction
- virus accidentally packages a fragment of host cell DNA (=transducing particle)
- virus delivers that fragment instead of viral DNA to next cell
- virus unable to replicate bc lacking viral genome
historically, co-transduction frequency was used to?
map bacterial genomes
- genomes that were closer to a known marker gene would be transduced with that marker more frequently than ones farther
co-transduction can also be used to modify bacteria (example?)
ex. specialized transduction: Shigella dysenteriae DNA in E.coli causing romaine lettuce poisoning (Shiga toxins)
