Bacterial Genetics Flashcards
Advantages of microbes for genetics (6 advantages)
Simple to maintain and cultivate
Reproduce rapidly
Large populations are produced quickly, allowing for spontaneous mutants to arise
Selection techniques can be used on bacteria
Bacteria are haploid so phenotype mutations are seen immediately
Small genome
How did they create the first synthetic bacterium? (5 steps)
- Designed bacterium sequences on a computer
- These sequences were synthesised and assembled and ligated to form a circular chromosome
- The chromosome was then propagated into yeast to clone it
- The chromosome was then isolated and transplanted into an existing bacterium
- The original bacterium DNA was digested, leaving the synthetic chromosome
Forward vs Reverse genetics?
Which is easier and why?
Forward - Identification of genes responsible for a given phenotype
Reverse - Identification of phenotypes caused by a specific gene
Forward is easier as you already have the phenotype, so you can do screens for genes that affect that phenotype
Explain complementation
Forward or reverse genetics?
When is it a particularly useful method?
(refer to image on notes)
Forward genetics
Useful if the gene is an operon; Polarity effects
Mutation in gene A could knock out whole operon
If we insert each gene separately, and the phenotype reverts to wild type, the gene introduced is the one that is mutated in the mutant phenotype
Advantages and disadvantages of complementation (1 for A; 2 for D)
Advantage - Emphasis is on the desired phenotype (e.g. loss of pathogenicity or growth on media lacking arginine)
Disadvantage – Slow; May be impossible to find all the genes in a species for a given phenotype
Process of reverse genetics (4 steps)
- Mutate gene in vitro
- Over-express the gene
- Use RNA-seq to determine changes in the transcriptome
- Determine phenotype of resulting mutant strains
What is a BiOLOG phenotype array?
Disadvantage of reverse genetics?
20 x 96 well plates, all with a defined media that can be altered (e.g. 96 different carbon or sulphur sources; Some wells may contain antibiotic)
Can be costly and may not reveal the phenotype
What are some uses of mutants? (5 uses)
Mutants help to identify genes involved in a particular function
Mutant phenotypes can be informative about pathways (e.g. blocks in pathways allow accumulation of intermediates)
Permit understanding of metabolic regulation (e.g. mutants of transcription factors that lead to upregulation of a gene)
Identifying the site of action of an antibiotic (e.g. rifamycin inhibits RNA synthesis; An insensitive mutant had a change in the RNA polymerase, proving that the antibiotic works by binding to the RNAP)
Conditional lethal mutations
- E.g. Temperature sensitivity
- Permissive temperature (e.g. 30°C) – Phenotype not apparent (behaves like wild type)
- Restrictive temperature (e.g. 37°C) – Mutant phenotype expressed
- Often mis-sense mutations destabilise protein structure/function only at the higher temperature; Allows us to study genes essential for viability
- We also use temperature sensitivity when constructing mutants in the lab
What is the name for a point mutation?
Types of point mutation? (2 types)
SNP - Single Nucleotide Polymorphism
Transition - Change from one pyrimidine/purine to another
Transversion – Change from a purine to pyrimidine (vice versa)
Other types of mutation? (3 types)
Larger mutations – Insertion of a section of DNA into chromosome e.g. transposon
Deletion of a portion of the chromosome
Inversion – Flipping a portion of a chromosome; Can be used to turn gene ‘on and off’
Other types of mutation? (4 types)
Silent – Change in codon/sequence causes no change in resulting amino acid
Missense mutation – Change one codon to another
Nonsense mutation – Change a codon to Stop
Frameshift mutation
What happens in a frameshift mutation?
Insert or delete a single base
Changes bases read by ribosome
Alters all codons downstream of mutation
What happens in slip strand mis-pairing?
Errors in DNA replication cause slippage of a codon so it is not base pairs to another codon but its neighbouring codons are base paired (see image on notes)
Sources of mutations? (5 sources) (elaborate a little bit)
Spontaneous mutation - Occur at the same rate in a cell over time
Electromagnetic radiation
Chemical - Analogues of bases; Base-modifying chemicals
Intercalators - Insert between bases of each strand of DNA; Cause frameshifts
Biological - Transposons
When does recombination/crossing over occur?
How long must the homology be?
When linear/plasmid DNA is homologous to another piece of DNA (non-homologous recombination can occur but it is rare)
Homology must be 500bp-100bp in length
How does site-specific recombination occur in bacteriophages?
The att system
Phage genome codes for integrase enzyme; Promotes recombination between att site of phage and att site of bacteria
What are the 4 types of DNA repair?
Methyl mismatch repair (MUT) – Mis-paired base cut out of strand
Nucleotide excision repair (NER) – Thymine dimers; Induced by UV
Base excision repair – Damaged bases; Excised by specific enzymes
Recombinational repair
What occurs in Methyl mismatch repair (MUT)? (refer to image on notes)
DNA becomes methylated at GATC site
Incorrect base inserted during replication, so no base pairing at a certain site
MutS binds to region of incorrect base pairing
MutS then recruits 2 enzymes, MutL, and MutH
MutL only recognises hemi-methylated DNA (DNA that’s methylated on the parental strand, but is missing on the new strand)
- MutL is globular and has 2 domains; These domains are brought together to drag DNA into a loop
MutH is an endonuclease, so it will make a nick in the DNA at the GATC site
The 2 strands are then unwound from eachother with UvrD (helicase) and an exonuclease removes damaged strand
DNAP I fills in the gap and a ligase ligates the ends
What occurs during Nucleotide excision repair (NER)? (repair of thymine dimers)
UvrA and UvrB enzymes come in as they recognise the thymine dimer
They bend the DNA, and UvrC (endonuclease) recognises the bend, and makes a nick on both sides of the dimer
Helicase unwinds and removes nicked part of strand
DNAP I comes in and fills gap and ligase ligates the ends
What occurs during Base excision repair?
DNA glycosylase comes in recognises base damage, and removes it; This is called an AP site
AP endonuclease nicks backbone of AP site
Helicase unwinds and removes region
DNAP I fills in gap and ligase seals the nick
What occurs during recombination repair?
Occurs just after DNA strand replication
Uses an enzyme called RecA which lines up regions of homology from sister strand to the gap
Strand invasion – Sister strand invades top strand
Gap filled in using DNAP I once the thymine dimer removed
When does SOS repair occur?
It is ______-prone?
When there are multiple mutations all across the chromosome
It is error-prone; Rapid but high error rate
