Microbial genetics Flashcards
What are microbes often exploited as by geneticists and biochemists ?
Model systems.
What are microbes often exploited as by geneticists and biochemists ?
Model systems.
What can you perform easily on microbes?
Mutant screens.
What are three advantages in using microbes to study model systems?
- Reproduce rapidly.
- Cheap to maintain and cultivate.
- Large number of individual cells.
Why can the mutant phenotype be seen immediately when studying microbes?
They are haploid.
What are the genome sizes of Mycroplasm, Ecoli and yeast?
Genetic manipulation, such as gene knockouts are not straightforward in microbes. True or false?
False.
From a microbiologists perspective, what are 5 reasons to study microbial genetics?
- Ecology.
- Cell biology.
- Pathogenicity.
- Evolution.
- Biotechnology.
What is the classical genetic approach also known as?
The forward approach.
What are the six steps of the classical/ forward approach?
- Random genome wide mutagenesis.
- Phenotypic screening.
- Biochemical/ physical extraction of mutants.
- Genetic analysis.
- Gene isolation.
- Gene sequence determination.
What are the six steps of the classical/ forward approach?
- Random genome wide mutagenesis.
- Phenotypic screening.
- Biochemical/ physical extraction of mutants.
- Genetic analysis.
- Gene isolation.
- Gene sequence determination.
What can you perform easily on microbes?
Mutant screens.
What are three advantages in using microbes to study model systems?
- Reproduce rapidly.
- Cheap to maintain and cultivate.
- Large number of individual cells.
Is it always possible to find all the related genes for the phenotype in the classical approach?
No.
What are the genome sizes of Mycroplasm, Ecoli and yeast?
Genetic manipulation, such as gene knockouts are not straightforward in microbes. True or false?
False.
From a microbiologists perspective, what are 5 reasons to study microbial genetics?
- Ecology.
- Cell biology.
- Pathogenicity.
- Evolution.
- Biotechnology.
What is the classical genetic approach also known as?
The forward approach.
Through answering the question ‘What genes make an organism pathogenic?’ Are you using forward or reverse genetics?
Forward.
What are the six steps of the classical/ forward genetic approach?
- Random genome wide mutagenesis.
- Phenotypic screening.
- Biochemical/ physical extraction of mutants.
- Genetic analysis.
- Gene isolation.
- Gene sequence determination.
Can you find mutants with defects in essential genes in the classical method?
Yes.
Apart from being able to find defects in essential genes, what is another advantage of using the forward/ classical pathway when studying microbial genetics?
The empathies is on the desired phenotype.
Is the classical genetic approach slow?
Yes.
Is it always possible to find all the related genes for the phenotype in the classical approach?
No.
How would you describe reverse genetics?
Gene to biological function.
How was the reverse genetic method historically done?
Protein product used to find gene in the library via N terminal sequence ( defect colonies whose DNA hybridises to a degenerate oglionucleotide probe.)
How were most cell cycle genes discovered?
Conditional mutants.
What is now available to be used in reverse genetics?
The entire microbial sequence.
What does modern reverse genetics focus on?
One gene of a particular interest (e.g. it may have homology to a known gene in another microbe.)
In reverse genetics the role of each gene is often asked. What three steps allow this to be answered?
- DNA mutated in vitro.
- Substitute the mutated DNA for the WT.
- Determine the phenotype of the resulting mutant strain.
What allows genes to be defined to a particular function?
Mutants.
What can mutants allow the accumulation of?
Intermediates.
What do allow regulatory mutants allow?
The definition of regulatory proteins and there site of actin in the DNA.
What is the permissive temperature for E.coli?
30 degrees.
What is the restrictive temperature for E.coli?
42 degrees.
Why is the mutant phenotype expressed in conditional mutants at the higher temperature?
Often because of missense mutations which destabilise the protein structure.
What time of mutation does tautorism cause?
Transition.
What was shown in the 1980’s in regards to CDNA?
That complementation may be possible from a cloned gene of another species.
Where is the cdc2 gene found?
S.pombe.
Where is the CDC28 gene found?
S.cerevisiae.
Where is CDC2 found?
Humans.
What are the 8 types of mutations?
- Point.
- Insertions.
- Deletions.
- DNA rearrangements.
- Transposon mediated mutations.
- Epigenetic mutations.
- Chromosome translocations.
- Chromosome loss.
Increasing in complexity and severity.
What is base tautorism an example of?
A point mutation.
What is base tautorism?
When a base is changed by the repositioning of a hydrogen ion thus altering the position of the hydrogen bond. This results in incorrect base pairing in replication.
What is an imino tautorism?
A-C.
What is an enrol tautorism?
T-G.
What time of mutation does tautorism cause?
Transition.
What is described below?
Hydrolysis changes a normal base to a atypic base containing a keto group instead of an amino group.
Spontaneous deamination.
What example of spontaneous deamination is easily spotted by DNA repair mechanisms and why?
C-U and HX.
Uracil is not normally found in DNA.
What example of spontaneous deamination is not easily spotted by DNA repair mechanisms?
5MeC -T
Thymine is already in DNA so this is less easily detected.
What results in slipped strand mispairing?
Local denaturation of hydrogen bonds.
When does slipped strand mispairing happen?
When there is a run of identical codons.
What does slipped strand mispairing result in?
A new strand longer than the parent strand and a frame shift.
What is used in some pathogenic bacteria to switch expression of surface proteins meaning they can evade the immune system?
Slipped strand mispairing.
Some bacteria use slipped strand mispairing to switch their expression of surface proteins in order to evade the immune system. What is this called?
Phase variation.
What is illegitimate recombination?
Recombination between DNA sequences that are only part homologous.
What do mismatch repair mechanisms recognise?
The pre-existing dam-methylated strand.
Some bacterial repair systems are error prone. What is an example of one of these systems?
SOS repair system.
What are the four types of chemical mutagens?
- Base analogous.
- Base modifications.
- Intercalating agents.
- UV light.
What do base analogous mutagens cause?
Substitutions.
What do base modifications mutagens cause?
Mispairing.
What do intercalating agent mutagens cause?
Framseshifts.
What do UV light mutagens cause?
Thymine dimers.
What is 5-bromouracil an example of?
A base analogue of thymine.
Only the keto form of 5-bromouracil acts as a thymine analogue. True or false?
False, the enol form also does.
How does 5-bromouracil cause a AT-CG transition?
5-Bu keto incorporated in place of T. 5-bu enol in DNA then pairs with a incoming G causing a AT-CG transition.
How does 5-bromouracil cause a GC-AT transition?
5-BU enol paired with G restores to its normal kept form. pairing with A in the text round causing a CG-AT transition.
What does nitrous acid do to DNA in vitro/ in vivo?
Deaminates DNA.
What is adenine deaminated into and what does this pair with?
HX which pairs with Cytosine.
What is cytosine deaminated into and what does this pair with?
Uracil which pairs with adenine.
Cytosine can be deaminated into uracil by nitrous acid. This has a serious impact on the DNA. True or false?
False as uracil is recognised by DNA repair mechanisms.
How does hydroxylamine modify DNA and what effect does this have?
It adds an hydroxyl group onto cytosine which then behaves like thymine.
Do alkylating agents modify DNA in vitro or in vivo?
In vivo.
Name three alkylating agents…
NTG, FES, EMS.
What do alkylating agents do to DNA?
They add an alkyl group (methyl to ethyl) to guanine residues causing mispairing or depurination.
Why do alkylating agents prevent one H bond forming?
As the H bond should usually form from double bond O which is bond to an alkyl group meaning the H bond can no longer form.
What are ICR191, acridine orange, ethidium bromide and benzopyrene all examples of?
Intercalating agents.
Interacting agents are planer three ring molecules. What are they similar in size to?
Purine-pyridine base pair.
What do intercalating agents result in?
+1 and occasionally +2 frameshift mutations.
What are the two ways in which enzyme mechanisms can respond to intercalating agents?
- Effected region chopped out.
2. Random based insertions.
When enzymes respond to intercalating agents it always results in a frame shift. True or false?
True.
What part of the DNA absorbs UV light?
The bases.
What is the wavelength of UV light?
260nm.
What photo product does UV light result in?
A thymine dimer.
What is a thymine dimer?
When two thymine residues on one strand covalently link..
DNA repair mechanisms can not repair thymine dimers accurately in eukaryotes but can in bacteria. true or false?
False. DNA repair mechanisms can repair DNA accurately in both bacteria and eukaryotes.
What are the three methods present to repair thymine dimers?
A1. Photo reactivation.
- Dark repair.
- SOS repair.
What does photoreactivation involve?
Visible light cleaves dimers.
Dark repair does not result in any errors. True or false?
True.
How many bases do the UVR genes remove in dark repair used to fix thymine dimers?
12.
What form of repair mechanism is the last resort in terms of thymine dimers?
SOS repair.
SOS repair is the last resort in repairing thymine dimers but it is not error prone. True or false?
False. It is error prone. Mutations can be added but these may not be lethal.
Are endo or exonuclease used in dark excision repair?
Both.
What is the role of endonuclease in dark excision repair?
It cuts the damaged DNA allowing it to be removed but he exonuclease.
What three Uvr genes are used in dark excision repair?
A, B, C.
SOS repair changes DNA lesions into residual but with error. True or false?
False, this stage is not error prone.
In sos repair what does residual lesions cause?
Unscheduled arrest of DNAP.
What does recA normally act as?
A co-protease.
What activates recA in SOS repair?
Unscheduled arrest of DNAP.
What causes LexA repressor cleavage?
Activated recA.
What does LexA switch on?
30+ SOS genes including pol2, pol4 and pol5.
Pol2 is involved in SOS repair. What is its role?
RecA feedback.
Pol4 is involved in SOS repair. What is its role?
Encodes sfi (cell division inhibitor.)
Pol5 is involved in SOS repair. What is its role?
Signals inducible error prone repair.
What does Pol4 encode and what are they?
Umm C,D which are polymerases.
What is the consequence of SOS repair?
Increased cell survival and mutagenesis of phage repressors.
What do Umu DC genes add to the DNA in SOS repair?
Two random bases opposite thymine, these residues however are not correct hence why it is error prone.
Why is it often difficult to find direct selection of a mutant phenotype?
Because mutations are often lethal.
Instead of detecting a mutant phenotype what is it easier to detect?
A WT phenotype in the presence of a vast access of mutant cells.
What does looking a WT mutant in a presence of a vast amount of mutant cells allow you to do?
To look at mutants which reverse the effect of an original mutation.
What is something that directly reverses the original mutation called?
A true revertant.
What is something that indirectly reverse the original mutation called?
Supressor.
What is special about the Salmonella used in the Ames test?
It is histidine requiring.
What does the first plate in the Ames test show?
The natural revertants.
What does the second plate in the Ames test show?
Natural and chemical revertants- if there is more than the chemical is causing revertants.
What sort of plate is used in the Ames test?
Minimal media without histidine.
What percentage of known chemical carcinogens were mutagenic?
95%.
What does the Ames test reduce?
Animal testing.
If there are more colonies in the second Ames plate what is your chemical causing?
Mutations.
Will A+B bind if there is also a suppressor mutation?
Yes.
What can suppressor mutations help define?
Protein- protein interactions.
When is random mutagenesis useful?
When the gene defining the phenotype is unknown.
What three types of genes can be identified by random mutagenesis?
DNA replication, pathogenicity and cell cycle control.
What simplifies random mutagenetic processes?
Genomic sequences and fast (deep) nucleotide sequencing.
What does specific mutagenesis allow?
Targeted changes that let you look at a protein in detail..
When doing targeted substitutions what do you normally need to use?
Naturally occurring amino acids as unnatural ones are difficult to incorporate.
What are the six key steps in site directed mutagenesis?
- Anneal a mismatched synthetic DNA primer to a target strain.
- Synthesis of complementary strand in vitro.
- Remove template strand.
- The synthetic duplex is introduced into E.coli
- Direct selection of mutants.
- Selection of mutants by phenotype.
What are the three steps of site directed mutagenesis?
- Mutant strand synthesis.
- DpnI digestion of template.
- Transformaion.
What does DpnI only cleave?
Methylated DNA.
Mutant strand synthesis involves thermal cycling, why?
- Denatures DNA template.
- Anneal mutagenic primers.
- Extend and incorporate primers with PfuUltra DNAP.
What is the definition of a transposon?
DNA sequences that can move (transpose) from one genetic element to another and which contain genes additional to those required for transposition. They insert into DNA in an (almost) random manner using terminal insertion sequences.
What are antibiotic resistant cassettes?
Cloned genes used for targeted gene inactivation.
Who discovered transposons in the 1940’s?
Barbara McClintock.
What did Barbara McClintock find transposons to be responsible for?
Variation in maize cornals.
In what types of organisms are transposons fond in?
Bacteria, archaea and many eukaryotes.
What are transposons an important source of?
Spontaneous mutation.
In what two ways do transposons effect genome evolution?
- Mediating interactions between plasmids and the bacterial chromosome.
- Very important in antibiotic resistance by horizontal transmission of drug resistance.
What do insertion sequences encode?
ONLY functions required for transposition.
How many copies of IS1 does E.coli have?
5-8.
How many copies of I2 does E.coli have?
5.
What is phage mu?
A bacteriophage within a transposon which can behave as either (i.e. it can still independently replicate as a phage.)
Can IS independently replicate?
No they must become part of the target regulon.
What are transposon target sites made of?
Inverted repeats.
How many base pairs are in IS1?
23bp.
What does the transposase enzyme do?
Binds to inverted repeats at the end of the transposon. It recognises, cuts and ligates DNA.
What type of cuts does the transposase enzyme make?
Staggered.
How many types of transposons are there in bacteria?
100s.
Chloramphenicol resistance is encode for by what type of transposon?
Simple.
How many genes are in a simple/composite transposon?
1 or a few.
What type of transposon is Tn9 an example of and how big is it?
Simple transposon. 2600 bp.
Tn5 is a simple/ composite transposon that contains an operon. What genes are found in it?
KmR, BleoR, SmR.
What is the size of the Tn5 operon?
51kb.
What are conjunctive transposons responsible for?
Wide spread antibiotic resistance.
What type of transposon contains ITR (Inverted terminal repeats?)
Complex.
What are conjunctive transposons?
Large, complex transposons with addition tra genes allowing the transposon to be transferred as a plasmid that can not independently replicate. Instead it conjuncts into additional cells and into the recipient chromosome.
Why do conjunctive transposons have a very broad host range?
Their hosts can be heterologous.
What are conjunctive transposons responsible for?
Wide spread antibiotic resistance.
Transposons target accurate spots in the genome. True or false?
False, they target random, approximate sites.
Why transposons cause polar effects on downstream genes?
Because they can interrupt the coding sequence and operational units.
What is a hot spot?
WHatA specific sequence that a transposon prefers to jump into.
What does the ‘polar effect’ help you to distinguish?
What genes are in a certain operon/ find if the genes are transcriptionally linked.
What can some Insertion sequences, including IS2 do?
They contain promote reading outwards so can switch on downstream genes.
In E.coli what does the IS provide homology between allowing occasional integration and occasional aberrant excision?
Between the F plasmid and the chromosome.
What plasmid is lost at 42 degrees?
ts plasmid.
How many cells will the ts plasmid integrate into?
1 in 10^4.
The ts plasmid can not grow at 42 degrees. What temperature can it grow at?
30.
How can you see where the ts plasmid is integrated?
42 degrees which the antibiotic corresponding to the selectable markers.
If you plate 10^8 cells how many mutants do you expect?
10^4.
What four reasons make transposons useful mutagens in genetic engineering?
- Mark gene with selectable antibiotic resistance.
- Inactivates genes completely.
- Relatively stable interactions.
- Modify for more functions, including reporter genes.
Why are reporter genes incorporated into vectors?
Their products are easily detected.
What are three examples of reporter genes?
- Lacz.
- Green florescent protein.
- Protein Z-GFP fusion.
What is the purpose of Z-GFP fusion?
Shows the position of protein Z in the cell.
What is a real life example of Z-GFP fusion?
MinC and MinD GFP proteins in E.coli. These oscillate from one pole to another and inhibit cell division at the poles.
What do transposons only work with?
Genetically tractable bacteria.
Name three examples of non genetically tractable bacteria.
- Mycobacterium tubercolosis.
- Yersina pests is (Plague)
- Treponema pallidum (Syphillis)
With genome sequences and PCR you can clone and express genes and use reverse genetics to make mutants without transposons, provided what is available?
A way to introduce mutated DNA into that species.
What are the four steps involved in making antibiotic resistant cassettes?
- Use the genome sequence of bacterium X to design primers for PCR. Clone into a plasmid E.coli vector that will not replicate in bacterium X.
- Use restriction enzymes to cut out and delete most of the gene.
- Replace with gene encoding resistance to an antibiotic that X is normally sensitive to.
- Transform plasmid into bacterium X and select for resistance.
What does direct selection procedures allow growth of?
Mutant colonies.
Is direct selection a powerful method?
Yes.
What four things can be used in direct selection?
- Antibiotics.
- Toxic analogues for carbohydrates.
- Amino acids.
- Nucleotide metabolism.
What is ONPG an analogue for?
Lactose.
When is ONPG toxic?
When it is transported/ metabolised.
What is 5 methyl tryptophan an analogue to?
Tryptophan.
How can mutants be resistant to 5 methyl tryptophan?
Either fail to transport it or over produce tryptophan (regulatory mutants).
What are the three steps for screening for motality or chemotaxis in bacteria?
- Inoculate cells from single colonies from a mutagenesis experiment into soft agar plates.
- WT cells will form rings as the cells can swim outwards.
- Mutants defective in motality will form smaller rings or no rings.
What are fla mutants?
Mutants with no flagella.
What are mot mutants?
Mutants with flagella that do not work.
What are che mutants?
Flagella move but cells can not respond to the concentration gradient.
What are the steps for enrichment techniques?
- Kill WT but let mutants survive.
- Add something like penicillin which only kills living cells.
- Wash cells and transfer survivors to medium allowing growth of mutant and WT.
- Mutants enriched 100 fold.
