GENE221 Molecular and Microbial genetics

What are some important features within the ColE1 plasmid which is also found within E.colI? - oriV - Imm - ColE1 - mob - rom - OriT - car

6.4kb (small) - high copy number OriV - origin of replication at which the copy number is maintained at 15 per cell. imm - gene that encodes immunity to Colicin antibiotic ColE1 - synthesis of Colicin antibiotic mob - Nuclease required for mobilisation in order for the use of machinery within other plasmids to occur (saves energy) rom - Protein required for copy number control OriT - origin of conjugative transfer (however doesn't have machinery for this process so hijacks it from other plasmids within the cell = a mobilising plasmid. cas - for site-specific recombination to resolve multimers

What is the structure of CI proteins and binding affinity?

95% of CI in the cell in dimerised via C-terminal interations (protein/protein interaction) - CI dimers use their N terminal domain to bind DNA within operator sites as it is able to recognise their sequence (protein/DNA interation) - CI has the highest binding affinity to the OR1 site which inhibits the binding of RNA polymerase to the PR for Cro synthesis. - It then binds to OR2 which increases the ability of RNA polymerase to bind to the weak promotor of PRM which increases the synthesis of CI itself. - Finally when concentrations of CI are very high it will bind to OR3 as well which turns off the transcription of both genes until concentrations of CI decreases again (may be due to cell division).

What is an apurinic site?

A site without a purine (a base).

How can hybridisation be used to identify a gene of interest? (sequence)

Attach labelled probes which have complementry sequences to the desired gene sequence. 1. Take double stranded DNA and seperate it into 2 single strands. 2. Place DNA on a filter to which it will bind to. 3. Add labelled DNA probes and incubate them together 4. Hybridisation of complementary DNA will occur. 5. Wash away the labelled DNA that did not hybridise to DNA bound to the filter. 6. Autoradiograph the labelled radioactive hybrid DNA to find the desired clone for amplification *need to know the target gene to identify the clone of interest using this method as an oligonucleotide is designed to hybridise with a short fragment of the gene. (usually radioactively labelled to identify clones with a similar DNA sequence).

How does genome annotation work?

Automated bacterial genome annotation (BASys) This method is able to identify genes based on a preexisting database of sequences. - can lead to bad annotations propagating through sequences due to incorrect annotations within the database. Manual genome annotation: Yield higher quality data. Genes can be located by the identification of start and stop codons.

How are results of GWAS reflected?

Can use this information to create a P value (estimates the likelihood to see this mutation by chance). A smaller P value (<0.05%) indicates that a particular mutation is related to the characteristic of interest as it is a more significant mutation. Larger P values indicate that the mutation is not significant. - you often see many SNPS next to each other that are significant which may just be due to linkage via recombination. Those that are closer together are more likely to be recombined together. *the results should be plotted on a Manhattan graph.

How did the acquisition of Mycolactone associated plasmid result in the KEY EVENT OF SPECIALISATION in the evolution of the M. ulceran bacteria?

Acquisition of mycolactone production (174kb plasmid) what the key even in the speciation of M. ulcerans from M. marinum as it is: 1. essential for the carriage/ replication in some host insects 2. Caused the divergence of strains that cause disease in frogs/fish to those that have a narrow host spectrum of humans. Molecular targets of Mycolactone: Targets are not well known but concentration determines its function. Low concentrations - causes immunoreppressant activity High concentrations - after accumulation it has cytotoxic effects on tissues which leads to its destruction = ulcers

Effective population size?

Can be estimated using the within species diversity (H) and a mutation rate and lots of other assumptions. - If there is very little variation within a species there is a small effective (original) population size as they tend to be very homogenous and lack diversity.

How did Aflatoxin cause mutations in Jeffery Miller's experiment?

Aflatoxin is naturally produced by fungi. Caused mostly GC to TA mutations but also more of other substitutions too 1. Aflatoxin reacts with the guanine base which destabilises its bond to the DNA backbone (sugar and in turn phosphate) 2. The normal base pairing of Guanine to Cytosine still occurs like normal 3. The weakened bond results in the dissociation of Guanine later on creating an apurinic site. 4. When DNA synthesis occurs error prone DNA polymerase reaches the apurinic site and puts an Adenine opposite to this blank location by default = pre-mutagenic lesion. 5. When a second round of DNA synthesis occurs the mutation occurs as the Adenine is paired with a thymine instead of the G-C base pair that was meant to be there (substitution mutation).

What is canonical and non-canonical base pairing of RNA?

Canonical (Watson-Crick) = C and G or A and U - Strong G and C and weaker A and U bonds. Non-conical (Wobble) = G and U - Other non-canonical base pairing can occur but this is extremely rare The base pairing of RNA determines its structure and in turn function.

How is the direction of transfer by a particular Hfr strain determined?

Chromosomal gene transfer by a particular Hfr strain in unidirectional from a fixed origin. The orientation of that the F factor inserts into the chromosome depends on the orientation of the IS2 or IS3 that is recombines with. - due to there being 18 sites of possible transfer there are several Hfr strains each with a characteristic origin and direction of transfer. *watch a video to understand how the recombination effects direction of transfer ****

What is the difference between a core genome and an accessory genome in bacteria?

Core genome: Contains the important chromosomal DNA that is vital for survival. - contains essential genes - Vertical gene transfer via binary fusion so both daughter cells get these genes however can be horizontally transferred too. Accessory genome: Contains many non-essential genes such as those found in plasmids, islands and transposons. - Usually beneficial to the cell but are usually selfish. - Horizontally transmitted between bacteria.

How can the significance of a variation in non-coding RNA be determined?

Does the variation presenve or break an otherwise conserved secondary structure of non-coding RNA. - mutations in loop regions tend to contain more variation than bases that are involved in structural base pairing. - mutations in base pairing nucleotides may also support co-varriation in another nucleotide in order to preserve the secondary structure.

What are plasmids? - one of focus for cloning?

Double stranded DNA molecules that replicate in cells independently of the host chromosome. They are inherited by daughter cells and are non-essential for growth. - They are good cloning vectors as we know their sequence well and they are small and easy to purify and manipulate. - pUC plasmid is going to be inserted into E.coli for cloning.

What are some general features of bacterial genomes?

Double stranded DNA which is usually circular (but can be linear such as in Streptemyces bacteria that we use to make aloof our antibiotics. Usually only contain one chromosome but can contain 2 such as in V, cholera Size of genome ranges between 0.112Mb to 14.7Mb (130x larger) - they can also contain plasmids (small or very large such as megaplasmids)

What are some examples of bacteria that produce antibiotic and bacteriocin?

Ecoli (ColE1) - is 6.kb in length and is able to produce Colicin antibiotics that fight off closely related bacteria within the environment = less competition as it contains imm genes too that give it resistance to colicin. - regular circular plasmid Streptmyces (SCP1) - produces many of the antibiotics that we use in daily life. - 350 kb in length so very long and linear in arrangement therefore telemeres aid its replication. *bacteria that produce antibiotics need to also contain antibiotic resistant genes in order to survive. = resistance is a significant medical issue.

How does control of plasmid numbers cause the out competition of incompatible plasmids?

Incompatable plasmids share the same regulatory mechanism therefore are subject to each other's inhibitor. - one will eventually gain the upper hand and outcompete the other

What is the lamda bacteriophage?

Lamda is a bacteriophage that infects E.coli. It has a long flexible tail and an isocohedral head. - lamda is able to enter either pathway and therefore is a TEMPERATE

Why do we care about mutations?

Mutations generate diversity which drives evolution. It gives us insight into how genes and their products work. - Mutations can lead to cancer and other diseases. - Can result in resistance to antibiotics in microbes. - Can give commercial properties to animals and plants.

Is there an optimal frequency for mutation?

Mutations in humans occur at a rate of 1/1,000,000,000 which Is good for maintaining the integrity of out DNA. - However, in some situations bacteria have higher mutation rates which gives them an adaptive advantage (HIV) - Strains have also been found to have a lower mutation rate than usual.

What are common replacements of amino acids in non-synonymous coding variation?

Order from most to least common: - WYF - MILV - HRK - DEQN - STAGP * non-synonymous replacements are most common within each of these groups due to them sharing biochemical properties and other replacements being negatively selected due to large differences in their biochemical properties.

Why is proof-reading of DNA so important? - bacteria - mice

Proof reading has been found to reduce the frequency of spontaneous mutations in bacteria compared to those who didn't have this method. - 1/100,000 normally but with proof reading only 1/1,000,000,000 gets through DNA proof reading protects mitochondrial DNA from mutation in mice which prevents premature ageing.

What are the pros and cons of hybridisation?

Pros: - Easy - Cheap - Gene doesn't need to be expressed Cons: - Need some of the sequence information of gene you are trying to isolate.

What are the pros and cons of using antibodies to locate a gene of interest?

Pros: Will detect genes that are translated into proteins Cons: Need antibodies that are specific tot he protein of interest (which is often not available)

How are EMS pre-mutagenic lesions repaired?

Repair proteins called alkyltransferases recognise the ethyl group that has been added to the Guanine and transfers/binds it to itself instead. - takes it away to be broken down which allows for normal base pairing to occur.

How is genomic DNA fragmented for a genomic library? - restriction digestion - partial restriction digest - sequence independent fragmenting

Restriction digestion: Generates a wide range of fragment sizes and needs different vectors for the different size fragments. - the genes are not always on the same fragment therefore there is overlapping of the genes. - Uses multiple restriction enzymes to obtain a complete library. Partial restriction digest: Limits the time that a frequent cutting restriction enzyme can act or the amount of restriction enzyme (frequent cutting Sau3AI) in order to obtains DNA fragments of different sizes and cuts. - creates overlapping genomic information that can be inserted into vectors. Sequence independent fragmenting: Uses a Nebulizer machine which can be calibrated to shear DNA into desired sizes. 1. DNA passes through a tube 2. Solution speeds up and is forced through a constriction 3. Acceleration of the solution stretches DNA causing it to snap 4. Flow rate and constriction determines the size of DNA fragments. - fragment sizes differ based on flow rate.

What did they find from the MRSA GWAS? - SNPs and indels

Results: - found 3,060 SNPs in total and only 100 SNPs were deemed to have statistical significance ( P = <0.05) after quality control - 22 indwells were identified to be associated with toxicity and was present in at least 5 strains of MRSA. The reason for so many of the SNPs identified being falsely identified as positive in the GWAS system is because: - allot of SNPs and indwells may be due to to phylogenic linkage (when horizontal gene transfer occurs it may transfer non-virulent genes along with virulent ones therefore all are deemed to be positive signals but only the virulent ones are =false positives)

What is the Smith-waterman algorithm?

Smith-waterman algorithm: An algorithm that is used to identify regions of similarity between genomes and determines where to put gaps to optimally align the genome. - those that are matched are given and score. *when looking for conserved locations - can identify high scoring diagonal sequences of match.

What is Tautomerisation and how does it occur? - When does this become a point mutation.

Tautomerisation the process by which mismatched bases are created to form Tautomers. - This pairing happens as one of the bases are correct and the other isn't therefore H bonds within one of the bases moves to allow H bonds to form between the incorrect base pairs. - This can happen with different bases but it only becomes a fixed point mutation if replication of this genome occurs again (one cell will contain point mutation a each strand is used as a template)

What did the 3 SNP loci encode in beta-lactam resistant strains?

The 3 loci encoded three penicillin binding proteins which therefore increased beta lactam resistance. - of the 600,000 SNPs located majority had no association to antibiotic resistance because most of them were (nearly neutral) and had no effect on phenotype. Also may have been phylogenically linked to virulence which created false positives.

What is enzyme induction in relation to the lac operon?

The bacteria are exposed to different environments they produce enzymes that are required for growth on a particular substrate when it is present. - when lactose is in higher concentrations enzymes are induced that are responsible for its breakdown (beta galactocidase) *when glucose concentrations are high they will use this over glucose to conserve energy.

What was discovered about the tRNA 2˚ structure and how was this confirmed?

The cloverleaf structure can be adopted by many different RNA which have various sequences Confirmed by RNA analysis: RNA are difficult to locate within the genome as there are no ribosomal binding sites or open reading fragments. There structure is determined by finding the conformation of the MINIMUM FREE ENERGY STRUCTURE which is the most stable structure. - There are 10^30 different structures that can arise but using comparative genomics you can determine which related sequences can adopt the same structure = in this way one structure that can be adopted by call RNA can be determined which is the cloverleaf.

How does the genetic code allow for many neutral mutations to occur?

The genetic code allows for many neutral mutations such as synonymous mutations which encode for the same amino acid OR for non-synonymous mutations that encode biochemically similar amino acids. - amino acids that are similar are more likely to be replaced by eachother as the function of the protein is maintained and various codons can encode the same amino acid (due to redundancy)

What is the minimal genome and why is it relevant?

The minimal number of genes required to support cellular life independently of a host. - Some pathogens have got smaller since diverging from a common ancestor and adoption to pathogenic lifestyles.

What can result from transformation?

The processes used within DNA cloning are not completely efficient including transformation. As a result there can be many resulting E.coli populations including: - nothing is taken up into the cell - plasmids that join back together - plasmids that were never cut - insert only - plasmid with ligated insert - cut plasmid that hasn't rebound together.

How does the lysogenic pathway occur?

The prophage integration into the lysogen requires: - an attP site (attachment site on phage) - an attB site (attachment site on bacterial genome) - Integrase enzyme 1. The integrase enzyme is able to recognise the attP and attB sites and stimulates crossing between these locations 2. Crossing over produces the attL and attR (left and right) sequences on each side of the inserted phage genome. - excision enzymes can be used to reverse the reaction. 3. Now the prophage can be replicated along with the host genome.

How does cutting sticky ends work?

The restriction enzyme EcoRI is able to recognise short sequences of DNA within the plasmid called restriction sites (4-8bp long) and cut DNA at each end of the gene of interest. - by doing so it creates sticky ended cuts that are complementary to each other.

What is a genome?

The total complement of genetic information of cell or virus. - There was a trend in genome sequencing over time in which genomes that were sequenced were larger as time went on and technology advanced. *also found the the human genome is smaller than a lot of plant genomes.

How did they measure the MRSA toxicity phenotype of the various isolates?

The toxicity phenotype of the MRSA was measured by collecting the cell contents of the bacteria when it was in its exponential phase of growth and mixing it with vesicles containing fluorescent dye (toxin-mediated vesicle lysis). - if the vesicle was lysed by the bacterial contents then the dye would be released and could be measured using a fluorometer. More dye released = higher toxicity measurement.

How did Griffiths experiment prove the theory of transformation?

There are (r) and (s) strains of the Streptococcus Pneumonia bacteria. The (s) strain is known as the smooth strain as it has a protecting polysaccharide outer capsule that makes it pathogenic. The (r) strain is rough and doesn't have this protective capsule making it non-virulent. Griffiths experiment: He found that: - If mice were injected with (r) strain they lived - If injected with (s) strain they died - If injected with heated/dead (s) strain mice lived - If injected with heated/dead (s) strain + (r) strain the mice died. Conclusion: The results indicated that there was some transfer between the dead and alive bacteria known as transformation. However, he did not know that DNA was responsible for this process.

What did they find from the MRSA phylogeny?

There are 2 clades of MRSA that are not that closely related. - however both clades contain highly, medium and low toxicity MRSA strains

How does recombination occur at a molecular level? - enzymes involved (5()

There are a few recombination enzymes involved in the process. RecBCD - responsible for the unwinding, nicking and degradation of DNA in one chromosome to generate single stranded DNA RecA - catalyses the base pairing of the single stranded DNA with the target double stranded molecule in the other chromosome. RuvA and RuvB - cause branch migration RuvC protein - resolves the the Holiday junctions by DNA cleavage

How is DNA extracted for from E.coli?

There are many different types of DNA mini prep procedures the most common one being ALKALINE LYSIS. - many companies sell kits for extraction but they generally are variations of the alkaline lysis procedure.

When does Transformation specifically occur in bacteria?

Transformation occurs NATURALLY is some bacteria such as Step. Pnemoniae and Influenza bacteria (some of the most pathogenic bacteria are as a result of transformation) but in some other bacteria such as E.coli it doesn't. Transformation only occurs in a specialised cell state called COMPETENCE which generally only occurs when the bacteria are in their stationary phase and only for a short period of time. - occurs when under internal or external stresses.

How can induction of lytic pathway occur to lamda prophage?

UV radiation can trigger phages to exit the lysogenic state and become lytic. 1. RecA enzyme is able to detect damage to the DNA which causes it to become a co-protease. 2. The protease then cleaves CI which causes the dimerised structure to dissociate from DNA. 3. Dissociation of CI means: - OR2 is unbound so activation of the PRM is lost and CI isn't synthesised anymore - OR1 is unbound so repression of RNA polymerase binding to the PR is lost so Cro and other lytic genes are transcribed - also dissociates from PI to stop repressing the synthesis of integrase and exisionase

how can you make a synthetic minimal cell?

Used Gibson assembly to synthesise and assemble and entire genome. - used watermark labeled DNA fragments of synthetic genome. - inserted the genomes into closely related species that did not contain DNA which allowed the synthetic organisms to grow. - able to remove fragments of synthetic DNA to determine which genes are essential. = first ever synthetic organism was the JCV1-syn30 (synthia) which contained 473 genes for growth of which we don't know the function of 150.

How did the 3 scientist determine that the inducer interacts directly with the repressor?

Used LacIs mutants which don't express the LacZ, Y or A gene as they encode a repressor than can't bind to an inducer (SUPER REPRESSOR) - Is and I+ heterozygotes both expressed nothing therefore Is is dominant. * DISCOVERED THAT THE INDUCER DIRECTS DIRECTLY WITH THE REPRESSOR TO ALLOW TRANSCRIPTION OF OPERON.

What is vertical gene transfer? - impact on diversity?

Vertical gene transfer is the A-sexual reproduction of bacteria via cell division. - the daughter cells are identical to the parent cells therefore the population gains no diversity.

How are apurinic site pre-mutagenic lesions repaired?

Via EXISION REPAIR 1. The enzyme AP endonuclease recognises the apurinic site and cuts the strand of DNA that contains it. 2. The site and adjacent bases are removed by excision endonuclease enzymes 3. The complementary strand is undamaged so it is used by DNA polymerase as a template to replace the missing bases with the correct sequence.

What are virulent and temperate phages?

Virulent phages: Can only enter the lytic pathway and are unable to undergo the lysogenic pathway. Temperate phages: Able to undergo both lytic and lysogenic pathway.

How can we determine when a particular promotor is induced?

We can clone a promotor of Gene X in front of a reporter gene (e.g. LacZ or GFP). - can be used to visually determine under what circumstances the promotor is induced

How do we know that the results of the Leaderberg and Tatum study wasn't just revertants?

We know they aren't revertants as multiple genes were missing from both strains therefore the chance of revertant mutation occurring to create the wild-type was 1/100 million. - Multiple colonies formed therefore some sort of genetic transfer must have occurred?

How do DNA repair systems work?

When a pre-mutagenic lesion occurs DNA replication needs to occur in order for a mutation to result. - DNA repair mechanisms can occur BEFORE replication so mutation isn't established.

When can crossing over be seen?

When meiosis occurs CHIASMATA can be seen which is the physical appearance of crossing over.

What did they discover about the mutagenesis of Caulobacter crescentus?

There was hyper-saturation of transposon mutagenesis throughout the Caulobacter crescentus genome. - there were 428,700 T5n insertions that were unique from the total that were isolated. - 80% of the genome shows no gap greater than 50bp which is where the essential genes must be. - average gap distance of 7.65 bp. Findings: - longer the gene the gene the more IS sequences found within it. - there were 3240 non-essential genes. - Those with no or few transposons are essential (480 genes) - there are 156 fitness genes which may help the bacteria in some way but aren't essential (have more IS than essential but less than non-essential).

What are expression libraries? - how is this an issue for eukaryotic DNA

These are libraries that contain DNA that can be transcribed into mRNA and in turn become a functional protein. eukaryotes contain introns within their genome unlike prokaryotes. - therefore their genes are large and the genes can't be expressed within the cell as functional mRNA or proteins.

What are the differences between M. ulcerans and M. marinum?

These species are closely related and are thought to have evolved from a common ancestor. They have very different lifestyles and pathogenicity. Also less closely related to M. tuberculosis. - M.ulcerans = 5.6Mb and M.marinum = 6.6Mb Growth rate: M. ulcerans - Slow (1 division in 48 hours) M. Marinum - Fast Mycolactone synthesis: M. ulcerans - No M. Marinum - Yes Pigment: M. ulcerans - No M. Marinum - Yes Niches: M. ulcerans - Aquatic plants and INSECTS M. Marinum - Aquatic frogs, fish and protozoa. Disease: M. ulcerans - Cases an EXTRACELLULAR infection on humans hosts only and causes BURULI ULCERS M. Marinum - Is generally non-pathogenic by opportunistic and it can cause intracellular infections with the host range of frogs and human macrophage. results in skin dermal lesions much like TB does

What are conjugative transposons?

These transposons are able to excise and transpose from one cell to another via conjugative intermediates - steal conjugative machinery to transfer to other cells. - Are able to transfer between cells however are unstable (unlike plasmids) and therefore randomly insert into recipient chromosomal DNA or reinsert into donor DNA at IS sites. - important in the dissemination of antibiotic resistance (spread)

What is the structure of RNA within the cell?

It has a linear 1˚ structure which folds into a cloverleaf 2˚ structure which contains: - A D loop - An anticodon loop - A variable loop - A TΨC loop - An acceptor stem The 3˚ structure is an L shape which contains helical twists. *this structure can be modified by covalent methylation and pseudouridine addition.

What is Mycobacterium ulcerans? - causes - growth rate and location - progression?

It is a 5.6Mb bacteria that causes BURULI ULCERS that was first identified in rural Australia (1935) It is a slow growing bacteria (1 division every 48 hours) that causes a necrotising infection of the skin and surface soft tissues which are surprisingly painless and often are left untreated until very late as a result. - it is an endemic in tropical regions due to its optimal growth conditions (esp. in central and west Africa) - OP%TIMAL TEMPERATURE OF 32˚C. - its occurences are increasing in Australia too Progression: Begins as a pre-ulcerative lesion that gets infected with the bacteria. Then proliferates and causes a build up of molecules that cause aviodence of host immune system and the degradation of surface tissues There is sometimes spontaneous healing but it often requires treatment (Surgery, skin grafts, antibiotics) - there is no vaccine for it.

How has reductive evolution been used to identify the differentiation between Mycobacterium ulcerans and mycobacterium Marinum? - plasmids

It is known that M. ulcerans contains a smaller genome and number of genes that M. marinum (4160 compared to 5,424), more insertion sequences and as a result have a higher number of pseudogenes (771 compared to only 65 in marinum). Which indicated that there has been a reduction in the genome of M. ulcerans which has contributed to its evolution. - M. ulcerans and M. marinum is homologous (same) for 98% of its genome which indicated that its divergence occurs about 1 million years ago. The genome has underwent: rearrangements, inversions and deletions which is due to: IS SEQUENCES.

What is a heteroduplex?

heteroduplex is a double-stranded molecule produced by genetic recombination of single strands from different homologous chromosomes.

What are some key theories about evolution and how is this useful for creating a phylogenic tree? - nearly neutral theory - the molecular clock theory

(Nearly) neutral theory of molecular evolution: Most genetic variation between species is either neutral or slightly deleterious and become fixed in the population due to drift - as a result of many mutations leading to unsustained life only silent mutations will become fixed. The molecular clock: the mutation rate of biomolecules can be used to deduce the time when two or more species diverged i.e. closely related species have similar genomes (DNA, RNA & protein) sequences, more diverged species have dissimilar sequences - those that diverged at similar times will contain similar number of mutations. *DNA, RNA and protein sequences can be optimally aligned Evolutionary models capture the variation between sequences

What are the main candidates associated with beta-lactam non-suseptability in S. pneumonaie?

- 3 penicillin binding proteins - 2 cell wall biogenesis genes - 2 cell division pathways - 2 heat shock/ chaperones - recombination

What are some ways of making a GWAS better?

- A large sample of case and control groups - These samples should be of sub-populations (different ethnicities) to make sure some groups aren't overrepresented. - DNA sequencing or SNP arrays are used to identify the geotypes and genetic variation is checked for association with a trait. - Results should then be plotted in a MANHATTAN PLOT which determine the significance of the association with the genetic variation on the y-axis.

A successful GWAS will have?

- A very clear and measurable phenotypic which is not due to external factors. - An adequate sample size and a control group. - Corrected for population structure and or phylogenetic relationships as you may identify mutations that reflect the phylogenetic relationship rather than the actual characteristic so need to remove these variables.

What are the features of the E.coli chromosome?

- About 4.5million base pairs that were sequenced in 1997 - about 4200 genes that 2/3 have had their function determined - 48 transposable elements - Genes realated to function are often clustered together - Good correlation with maps from genetic experiments - Pathogenic isolates differ from benign isolates due to the presence of plasmids and horizontally transferred virulence genes on their chromosomes (pathogenicity islands) Each E.coli is only 40% similar to the next one due to core chromosomal features. The other 60% has been acquired via plasmid and chromosomal transfer of genes.

What has horizontal gene transfer in M. tuberculosis caused?

- Acquired genes that allow for hypoxic growth (growth in low oxygen) - useful for intracellular infection - Prophage genes which encode important virulence factors - Sulfolipids on the cell wall which mediate host/pathogen interaction. - CRISPR provides the M. tuberculosis with phage resistance.

What are the common features of genetic exchange mechanisms via horizontal gene transfer?

- All transfer is unidirectional from one donor cell to one recipient cell. - Only part of the donor genome is incorporated into the recipient cell. - In most cases the transferred DNA is linear fragments and can't be replicated. As a result the transferred genes need to be recombined into the recipient chromosome (circular). - The recipient is permanently changed by incorporated DNA.

What are the limitations of the Salvador Luria and Max Deldruk experiment?

- Bacteria are killed rapidly so there wasn't enough time to adapt - It can take several generations for resistance mutations to give rise to resistance phenotypes.

What are some generalisations that can be made about transduction?

- Bacteriophage acts as a passive carrier of bacterial DNA which is injected into a recipient cell and incorporated by double recombination. - Any bacterial gene can be transacted at a low frequency (generalised transduction) - The amount of DNA that can be transferred is limited by the size of the phage head (about 100kb) - Generalised transduction can be used for high resolution mapping of genes as the distance between markers can be determined based on the frequency of co-transduction. - Very useful for strain construction.

What does the tree of life show overall?

- Eukaryotes are dull (not phylogenetically diverse) - there is a huge amount of bacterial diversity - Bacteria contains many more major lineage than the other domains. - Candidate Phyla radiation is huge.

How can Transformation and recombinant technology be used to induce transformation in bacteria in which it doesn't occur (E.g. E.coli)?

- In E.coli transformation doesn't naturally occur but COMPETENCE can be induced to take up circular DNA with treatment with Calcium Chloride (CaCl) in cold conditions and then heat shock at 42˚C - Electroporation can be uses to create transient pores in the membrane of bacteria using an electric pulse and DNA can be inserted into the pores using a gene gun. * Most organisms can be transferred using one of these methods.

What are the names of the 4 types of transposons?

- Insertion sequences - composite transposons - Non-compisate transposons - Conjugative transposons

Why do we study genetics on micro-organisms?

- Microbes (bacteria and fungi) allow genetic screens that are not possible with higher eukaryotes. - Rapid life-cycle. - Mechanisms and nature of mutation is pretty much the same as in eukaryotes.

What are some features of RNA base pairing within rRNA?

- Only 71.3% of rRNA contacts are canonical or non-canonical G and U base pairing. - There is a high 9.4% of G and A base pairing which tend to be at the end of stems. - 10.6 G and A bonds formed too

What accessory genome components are the large contributors to microbial diversity through gene acquisition?

- Plasmids - Transposons - Bacteriophage - Integrative and conjugative elements *All can contain important phenotypes.

What are some issues with GWAS?

- The studies are frequently under powered as sample sizes are too small to identify small effects. - Correlation does not equal causation as: Multiple testing can falsely link SNP to traits Linkage is associated with variation and phenotypes Significance may be due to population stratification or phylogeny rather than due to the phenotype of interest.

What are some similarities between bacteriophage and eukaryotic viruses?

- They are genetically simple (10-100 genes) - Genomes are single or double strands of DNA or RNA - They range from structurally simple to complex - They rely on the host cell to provide nutrients and chemicals needed to make more of the virus - They can kill the host cell (lytic cycle) or become integrated into its genome (lysogenic cycle).

What are some features of transposons?

- able to move from one site in the genome to another which is independent of host recombination systems (uses transposase enzymes that are sight specific) - Cause large fractions of spontaneous mutations by inserting into genomes as it can result in deletions, inversions, replicons and fusions. - Enables the RAPID ACQUISITION OF MULTIPLE ANTIBIOTIC RESISTANCES and facilitates the flow of of genes between species. - Provides powerful tools for molecular genetics studies.

What is the general structure of an F plasmid? - how is this significant in relation to E.coli?

- about 100 kb in size - contains 1 copy of Is2 and 3 copies of Is3 that - One of the Is3 interrupt a regulatory region that represses transfer and therefore increases the rate of conjugation between cells. E.coli has 12 copies of Is2 and 6 copies of IS3 which gives rise to 18 locations of homology at which a double recombination can occur to give rise to an Hfr strain.

What features can be identified when comparing mRNA and protein alignments?

- codons that are conserved implies importance in function. - More differences in the amino acid or genome sequence between species can be used to position them on a phylogenetic tree. = E.coli is more similar to Stypani than to P aeruginosa. - The 3rd position in codons tends to differ more than other positions. This is generally due to redundant/synonymous mutations occurring which maintains the function of the protein. - MOST MUTATIONS ARE (nearly) NEUTRAL. This is due to either synonymous mutations occurring OR non-synonymous mutations encoding for amino acids with similar chemical properties = maintained function - Insertions and deletions (indels) tend to maintain the frame. (usually occur in groups of 3)

How would you go about building a phylogenic tree? - Hug et. al method

- collect representative high quality genome sequences for all of the known genera (16 rRNA as they are conserves across all life) - Use single cell methods from uncultivable organisms. -Align and concatenated 16 rRNA or Small ribosomal subunit protein sequences that are conserved across all life and use these comparisons to construct a phylogenic tree (those that are more similar are closer together).

What does sequencing cloned genes allow us to do?

- determine the organisation of the gene - Find out what the gene encodes (function of the RNA or protein it encodes) - We can compare the sequence with different organisms. - we can manipulate the gene (mutate it, insert gene into cells of an organism or make large amounts of protein)

What species barriers of bacterial conjugation able to cross?

- diverse bacteria (gram +ve and -ve) - F factor and yeast can mate - Agrobacterium can mate with plants and yeast to genetically modify them to produce nutrients for the bacteria. - The pathogen Bartonella hensalae can mate with human cells.

What are the major differences between the Holliday model and the current model?

- double stranded break in one chromosome rather that a single stranded break in both chromosomes. - Degradation and synthesis of some DNA in current model due to Invasion and displacement.

How do you count co-varrying sites?

- draw lines between where the brackets are (these bases are base paired together) - sites that are not conserved between the compared sequences are co-varrying sites and therefore count both sides. * the most variation within non-coding RNAs is within the loops as they are not involved in structure formation. E.g. there is an AC difference 4 base pairs into the first section which is matched with a UG on the other side. - this would be counted as 2 co-varying sites as a base change in one side resulted in a compensatory mutation on the other side to maintain the 2˚ structure of the RNA = co-variation

What do you make a library from?

- eukaryotic genome (human) - prokaryotic genomes (bacterial) - viral genomes

What features do we have to be aware of when identifying synonymous and non-synonymous mutations?

- must remember that synonymous mutations do not count identical sequences as they are not mutated. - indels are generally not counted. - non-synonymous mutations are mutations that encode a different amino acid. MUST COUNT ALL MUTATIONS WITHIN THAT CODON.

How were essential protein coding sequences identified?

- non essential genes would have Is sequences throughout them and at any point. - essential genes may have some IS sequences either at the beginning (promotor allows transcription anyway) or at the end because there is enough sequence to produce a functional protein. - the bigger the gap between transposon insertion sites the more likely it is to be essential. * can then map known coding genes to this sequence to determine which are essential.

Why is E.coli the workhorse of molecular biology? - why do we study E.coli so much?

- non-pathogenic - Supports the growth of a range of bacterpharges which in turn makes viral studies easier to conduct.

END OF TERMS TEST CONTENT

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How is a plasmid clone made? (4)

1) Restriction- the plasmid vector and the DNA fragment of interest must be digested with the same restriction enzyme to produce 'sticky ends'. 2) Ligation- the DNA fragment of interest must be ligated into a plasmid vector 3) Transformation- the ligated plasmid vector must be transformed into a bacterial host (usually E. coli) 4) Selection- Bacterial colonies carrying the plasmid with the DNA fragment of interest must be selected from those that do not. - bacteria containing the plasmid and the insert of interest also need to be selected for (usually done via blue/white selection)

How is the lac operon regulated?

1. Allolactose which is a side product of the beta galactosidase enzyme produced by the lac operon acts as a inducer molecule to remove the repressor protein that is encoded by the lacI gene upstream from the lac operon. - allows transcription of the lac operon. *negative regulation 2. A starvation signal of ATP being converted to cAMP and forming a complex with CRP allows it to bind to the weak promotor to allow RNA polymerase to bind and initiate transcription of the operon. *positive regulation

What are the 3 ways of finding a gene of interest within a genomic library?

1. Complementation (phenotype / function) 2. Hybridisation (sequence) 3. Antibodies (proteins)

What are the 2 mechanisms of transposition?

1. Conservative (cut and paste) - Moves from one site to another. Only 1 copy. 2. Replicative (copy and paste) - results in 2 copies. One in original location and the other in a new location.

What are some types of analysis you can do with whole sequenced genomes? (6)

1. Construct a phylogenetic tree by determining conserved regions and the relationships between species (close or divergent species) 2. Pangenome analysis - determines which genes are shared (core) and which ones are not (accessory genes) 3. Estimation of effective population size 4. Transmission mapping - geographic tracking of different liniages on global or locations. 5. Recombination hotspots - identify regions under diversifying selection 6. Genome wide association studies - look at trait of interest and try link them to genetic variation.

What is the process of making a plasmid clone?

1. Cut a plasmic at a restriction site 2. Cut DNA to be clone with the same restriction enzyme 3. Use DNA ligase to insert DNA into a plasmid 4. Transform plasmid + insert into the E.coli 5. Screen for cells containing plasmid by plating of antibiotic secretion plates and for those containing an insert of interest (blue/white selection)

What are the 2 names of methods to prevent point mutations?

1. DNA proof-reading by DNA polymerase 2. The Mismatch repair system.

What are the 2 types of methods developed to test for mutagens? - pros and cons of both

1. Direct: The most direct approach is in the laboratory using animals. (+) animals are good lab models for humans. (-) there is a high cost (-) ethical issues 2. Rationale: Genes in microorganisms work in the same way as in eukaryotes so things that cause mutations in eukaryotes should also cause mutation in microorganisms. (+) can screen very large numbers of organisms at once (+) able to screens are available for detecting occurrence of mutation (+) no ethical issues.

What are the uses of transposons?

1. Generate mutations: Antibiotic resistance genes allow for easy selection. 2. Tag genes for subsequent isolation/identification

How did Avery McLeod extend of Griffiths theory?

1. He took the (s) strain and separated out all its components being: RNA, DNA, Proteins, Lipids and Polysaccharides. 2. He streaked each of the components on seperate agar plates containing the (r) strain. 3. He found that only the (r) strain grew on all of the agar plates except the one containing the (s) strain DNA in which both the strains grew. Conclusion: DNA is the transforming principle/factor.

What 6 discoveries where Francois Jacob, Jacques Monod and Andre Lwoff able to make about the lac operon?

1. Induces regulate the synthesis of beta gal. 2. Inducers differ from substrates 3. The genes are controlled together (operonic) 4. LacI encodes a diffusible repressor 5. Inducer interacts directly with the repressor 6. repressor interacts with the operator.

How is copy number of plasmids controlled by plasmid replication? (2 ways)

1. Inhibitor secretion Plasmids copy numbers are controlled by plasmid-encoded inhibitor molecules that act at the OriV. - As cell size increases this inhibition molecule concentration decreased and plasmids are able to replicate from oriV. - Replication causes the secretion of more inhibitor genes and in turn inhibitor molecules which limits plasmid replication again. 2. Control via rom/rop genes: At high concentrations of plasmid: RNA 2 is able to bind to the oriV site which acts as a stable RNA primer to begin the replication of of the plasmid. - The rop gene encodes an antisense RNA 1 structure that is able to from an RNA-RNA hybrid to change the configuration of the RNA-DNA interaction which inhibits the replication of of the plasmid *rop is synthesised more when plasmid concentrations are high At low concentrations of plasmid: RNA 2 is able to bind to the oriV site which acts as a stable RNA primer to begin the replication of of the plasmid. - the RNA 1 antisense sequence is in low concentrations and doesn't bind to the RNA 2 primer therefore replication of the plasmid occurs.

How are DNA rearrangements mediated by transposons?

1. Insertions: Transpositions or homologous recombination due to movement of transposons or similar sequences within genome due to transposons. 2. Replicon fusions: E.g. Hfr formation due to recombination at identical transposon sites on a plasmid and bacterial genome 3. Deletions: Homologous recombination between two copies of a transposon present in DIRECT orientation can result in the loss of sequence between the two transposons. 4. Inversions: Homologous recombination between two copies of transposon present in INVERTED orientation causes the sequence in between to invert into the other direction.

What stages does the lamda bacteriophage have to go through?

1. Invasion of host cells 2. Decision between lysogenic and lytic pathways 3. Maintenance of lysogen 4. Induction (switch from lysogenic to lytic)

What do we do with libraries once we have created them?

1. Isolate a single clone by screening the sequence of our gene 2. Sequence the whole library

What happens in both life cycles up to when a decision needs to be made by lamda virus?

1. Linear genome is injected into the cell 2. Lambda virus circularises within the cell as the cos sites (cohesive sites) on each end bind together like sticky ends. 3. Synthesis from both promotor sites (PR and PL) 4. Decision needs to be made about pathway

What are 4 approaches used to define the minimal genome?

1. Look in nature for the smallest genomes 2. Compare distantly related genome computationally to identity conserved (essential? genes 3. Take small natural genome and find out which genes are essential by mutating it. 4. Creating a synthetic organism.

What are the 2 life pathways that bacteriophages can undergo?

1. Lytic pathway: The phage binds to specific receptors on host cell. Injects its DNA into the cell and hijacks the host cell machinery to create its own components resulting in the lysis of the cell and release of many more phages. 2. Lysogenic pathway: Phage in integrated into the host genome (now called prophage and lysogen) and is replicated along with host genome for generations.

What is the process of conjugation? - What structures form - What enzyme is used

1. Male (F+) cells put out a pilus structure which binds to receptors of an F- cell. 2. The pilus retracts to bring the cells closer together and allows a SEXUAL BRIDGE to form between them. 3. The RELAXASE enzyme nicks the plasmid at the OriT site (Ori transfer site). 4. The ROLLING CIRCLE MECHANISM begins as a single strand of the plasmid is pealed back and transferred through the mating bridge. * The mating bridge is a fragile structure therefore it often breaks before the whole strand has been transported into the recipient cell. 4. The single stranded F factor in the 2 resulting F+ cells is synthesised to become a double strand again.

What were the 2 strategies that existed for sequencing genomes using cloning?

1. Ordered clone sequencing - Francis Colllins 2. Whole genome shotgun sequencing - Craig Venter

What 3 ways can you find a gene of interest within the thousands of clones from a genomic library?

1. Phenotype (function) 2. DNA (sequence) 3. Protein (what it encodes)

What are 3 examples of spontaneous mutations?

1. Point mutations which are substitution mutations of a single base pair in a genome. (A becomes a G) 2. Indels: Indels include insertions or deletions within the genome which causes frame shift mutations and is often as a result of slippage during DNA replication. 3. Large scale rearrangements of chromosomal DNA or insertion of mobile genetic elements.

How do plasmids of the accessory genome survive? (life cycle)

1. Replication of the plasmid from the OriV 2. Segregate - must ensure that each daughter cell receives at least one copy upon cellular division. 3. Must keep host cell happy: - Constrain metabolic load by controlling COPY NUMBER (not too energy expensive) - Large plasmids such as F plasmid have 1-5 copies/cell (low copy number cells) - Small plasmids such as ColE1 can contain 10 to 50 copies/cell (high copy number) 4. Keep host cells under control: - has a mechanism to kill off host cells that exclude plasmids following replication (no plasmid = death) 5. Spread: - Conjugation or non-conjugative plasmids are often mobilisable.

how do cosmids work with cloning?

1. Restriction enzyme will be used to cut insert DNA and restriction sites on the cosmid. 2. The insert will base pair with the sticky ends on the cosmid and it will be ligated together by DNA ligase (ligation) 3. Invitro packaging: Linearised cosmids will be packaged into phages by "stuffing enzyme" which recognises DNA of specific lengths if 2 cos sites are present within it. 4. Phage will inject its genome into E.coli = high efficiency transformation. 5. Within the E.coli the cosmid will circularise and act as a normal plasmid would do.

How do we determine the genome sequence of a bacterium? (3)

1. Sanger sequencing: Involves the addition of dNTPs and ddNTPs that are labeled with different florescent tags. When added to a sequence a laser is used to detect a flash of light in a particular colour. Can be used to produce peaks on a computer that can be used to decode the sequence. Next generation sequencing: - don't need cloning step - shorter reads (generally) - more sequence reads/time 2. Illumina technology: A DNA sample is taken and adaptors are ligated to each end. Cell channels comtain single stranded sequenced that are complementary to the adaptors which causes them to bind. BRIDGE AMPLIFICATION OCCURS in which unlabelled nucleotides are bound the sequence. The newly synthesised strands are then denatured to seperate the and all 4 of the labeled reversible terminators are added. The colour that lights up determines the base that was added. (done in 150 cycles to determine the base order of the entire fragment. 3. 454 sequencing

What are the 4 stages of bacterial genomics and comparative genomics?

1. Sequencing: Determine the genome sequence of a bacterium (DNA sequencing, assembly, gap closure/finishing) 2. Annotation: Identify genes / features of the bacterial genome. (automated and manual annotation) 3. Biology: Biological interpretation of the genome content (relationship of bioinformatics to biology of an organism) 4. Comparison Comparison of the genome content of two or more bacteria. (bioinformatic comparisons and their relationship to biology of organisms)

What are some issues with defining the minimal genome based on genome comparison?

1. Some proteins share no sequence or structural similarity but perform the same function (miss out essential functions) 2. More than one different metabolism/ nutrient can support life which will cause differences in the essential genes between species. 3. As you incorporate more genomes in the comparison less genes are conserved across every one of them.

What is the current model to explain recombination?

1. The RecBCD enzyme cuts both strands on one chromosome and surrounding DNA is degraded from the 5' end forming regions of single stranded DNA. 2. RecA enzyme allows the INVASION AND DISPLACEMENT of other chromosome DNA toward the single strands as they don't like being single stranded. 3. The invading DNA is used as a template strand to create new DNA in the degraded locations = 2 holiday junctions and 2 fused chromosomes. 4. Ruv A and B cause branch migration 5. Resolution occurs using the RuvC enzymes as one of the 2 holiday junctions are cut and DNA is rejoined together. 6. The chromosomes unwind to form recombinant DNA.

How does UV light / radiation cause mutation?

1. The UV light causes Thymines that are adjacent to each other to form double bonds between themselves forming photodymers 2. These bases fail to base pair correctly during DNA replication as DNA polymerase inserts a random base in the complementary position (may not be an A). 3. If DNA replicated again the pre-mutagenic lesions will become permanent lesions.

How does Whole genome shotgun sequencing work?

1. The genome is cut into many randomly sized genome fragments 2. The each fragment is sequenced. 3. Overlap sequence reads of specific contigs are found. 4. Overlap the contains to sequence the complete sequence. 5. There tends to be gaps between contigs but they can be joined together based on which contigs are present within the same vectors (indicates they are close to each other). - sequence from ends of a large number of random clones are assembled on a computer. - Overlapping regions indicate continued sequence. - fill in gaps with targeted sequencing later - FASTER AND CHEAPER THAN THEN ORDERED APPROACH - Automation using Sanger sequencing made this approach excellent.

How are genomic libraries created?

1. The genome of interest is fragmented using various techniques (restriction digestion, partial restriction digest or sequence independent shearing). 2. At the same time Plasmids/cosmids/Bacs are restriction digested with the same restriction enzyme to create compatible ends with he digested genome. 3. Ligation occurs between the fragments and the vectors 4. BACs and plasmids will be transformed into E.coli and cosmids will be transducted into E.coli. 5. Screening of the colonies will occur.

What is the function of some non-coding RNAs? - thermosensors - peptide leaders - frameshift elements - riboswitches

1. Thermosensors - which have specific melting temperatures which can change the RNA strucute to expose the Shine of Dalgarno = transcription of gene at differed temperature. 2. Peptide leaders - are short peptide sequences which are enriched in amino acids that alter the accessibility of DNA via attenuation. 3. Frameshift elements - RNA structures that bind to alter the reading frame of DNA (e.g. push it to +2) 4. Riboswitches - bind to small metabolites to change the structure of the RNA and in turn control its expression

How did they determine the genome of the Caulobacter crescentus neucleotide by nucleotide?

1. They were able to determine the the fine position of sequences by inserting Tn5 transposons within it which had been genetically engineered to contain a promotor region at one end = allow to determine insertion location and orientation (forward or reverse). - this meant that transcription os genes could still occur even if the Tn5 transposon was inserted within the promotor or early on in the sequence. (more will survive). 2. illumina sequencing: was done following the addition of transposons and specific PCR primers. - photographs are taken and colour of light is used to determine base sequence/ - based on where the transposons are in the DNA you can determine where on the genome it sits. = high throughput sequencing which lead to LARGE amounts of data.

How is Transduction limited?

1. Transduction is limited by the size of the bacteriophage head. Generally fragments only as big as 100 Kbs can fit within it. 2. Phage host specificity occurs in which surface receptors on the head is able to recognise self/same species DNA to specifically only package viral DNA within itself.

How does transformation occur (2)?

1. Transformation: Cells are induced to become competent to take up DNA by treating E.coli with ice cold CaCl2 mixed with DNA and then heat shocked at 42 degrees celsius. 2. Electroporation: Cells are made competent to take up DNA by treatment with an electric shock which creates pores in the membrane that DNA can be inserted through via a gene gun. (2500V).

How does competence specifically occur in response to the environment?

1. When environments change occurs such as stresses or detection of population increase this triggers the onset of the natural competence in bacterial cells (machinery made and pilus extends from cell membrane). - induction of competence also triggers lysis and DNA release from a subtraction of cell populations (5-20%) 2. This pilus is used to capture and bring in foreign DNA into the cell. The DNA is often specific as the pilus is able to bind to specific short sequences that are from same species DNA. 3. This DNA is degraded into a single strand beforehand by EndA enzyme. 4. Once inside the cell homologous recombination occurs and the DNA is incorporated in the genome of the recipient bacterium which replaces the original genome. - linear fragment remains which is unviable

What are the CORE non-coding RNAs? - all cells - bacteria

1. rRNA such as the 5S, small subunit and large subunit RNA which are the most conserved structures in biology. 2. tRNA which is important for the process of translation. 3. RNase P RNA which chops tRNA 4. SPR RNA Bacteria: 1. 6S RNA which was the first non-coding RNA to be located and involved in the transition from exponential and stationary phase of bacterial growth. 2. tmRNA which mimics tRNA to rescue stalled ribosomes. 3. Cis-regulatory elements which are regulatory elements for transcription that is encoded for within the genome.

What are some important features within the genetic map of the F plasmid within E.coli? - tra - RepF1A - OriV - Par - res/fcr - ccdA/ccdB - hok/sok - pif - TN1000, IS2 and IS3

100kb in size (large) - low copy number tra gene: Regulates the mating pore and DNA mobilisation functions. RepF1: Determines the vegetative replication and incompatibility between cells. It contains: oriV - Which is the origin of vegetative replication which produces only 1-2 copies per cell. par - Partitioning loci res/fcr - Site specific recombination system that resolves dimers (dimers are plasmids that have recombined together to become a larger plasmid) ccdA/ccdB - A host killing system. hok/sok: Another host killing mechanism (toxin-antitoxin method) which is an example of killing method following host segregation. - toxin is more stable therefore if no plasmid is within cell there is no antitoxin gene to protect it from death. pif - Protection against phage T& by introducing abortive infection. Tn1000, IS2 and IS3 - Transposonable elements that facilitate interactions between F and other DNA molecules.

How does cooperatively of CI binding occur?

2 factors contribute to CI binding: 1. Intrinsic affinity to bind to DNA 2. Cooperativity - CI will bind to the OR1 operator due to a high intrinsic affinity to bind to this location - CI will bind almost immediately after to OR2 operator as the protein-protein interaction between the C terminal domains which cause the dimers to lean toward each other to form a very stable structure. = increased binding affinity - Binding of CI to OR3 is weak as there is no cooperatively and affinity to bind to DNA is low.

Which coding genes did they find to be essential within Caulobacter crescentus?

480 total essential coding elements: - transcription factors (13) includes: 2 sigma factors, 5 cell cycle master regulators and 5 transcriptional regulators of unknown functions - proteins of unknown function - metabolism - ribosome function - other processes - cell structure (wall/membrane) - cell cycle/devision and replication genes. THERE ARE 60 with unknown functions

What is mycobacterium leprae?

A bacteria that causes disease of the peripheral nerves and is the causative agent of Leprosy. - it is almost uncultivable as it doubles ever 14 days. - Obligate intracellular parasite - Infects Schwann cells of the nerves and proliferates causing a loss of myelin. = sensory loss and disfiguaration and is a major problem due to the immune system.

What is a pre-mutagenic lesion?

A change in DNA that may lead to mutation if not corrected before a second round of DNA replication.

What are genomic libraries? - what is its aim?

A collection of all the DNA fragments of a given species that have been taken from one organism and inserted into a vector for transport (cloning) into a host Aim: To have a panel of bacteria containing individual clones that represent all the DNA in an organisms genome.

What is Mycobacterium leprosy?

A disease of the peripheral nerves - it causes skin lesions (granulomas) - if left untreated can cause damage to skin nerves, limbs and eyes. - can remain latent without any symptoms for 5-20 years. - 2-3 million are disabled due to leprosy and there are 250,000 new cases each year.

What is the CRISPR-Cas system in bacteria?

A genetic component of the accessory genome of a bacterial cell that acts as an adaptive immune system in prokaryotes. - Series of DNA fragments from broken down invaders are incorporated into the CRISPR-Cas region which gives the cell memory of the invader for immune responses to occur. = multimillion dollar business

How did a later study redefine the mycoplasma minimal genome?

A later study involving M. genitalium reduces the factor of cross-feeding - mutants that were growing side by side may have been sharing diffusible nutrients that the other couldn't produce. - they took all grown colonies and determined that they could grown independently. = there are at least 382 essential genes (larger than pervious).

How is F plasmid replication controlled?

A low concentrations of the F plasmid: RepA is a gene within the F plasmid that produces proteins that only bind to the replication sites as they have a high binding affinity to initiate replication of the plasmid. At high concentrations of F plasmid: RepA is transcribed more so more proteins are produced causing a higher concentration of this molecule. As a result, the proteins bind the the high affinity replication sites as well as lower affinity iterons (repetitive sequences) within the plasmid which causes plasmid dimerisation/handcuffin - replication can't occur when this dimerisation takes place until separated at cell division. * ParM is able to push these dimerised plasmids apart to the seperate poles of the cell.

What is the BLOSM62 matrix?

A method of scoring the similarity of amino acids in order to compare them and determine the frequently at which specific amino acids will replace each other. - did this with a 20x20 comparison of all the amino acids. = they found that there were 5 blocks of most frequently replaced amino acids (shown to have +ve values) *most random mutations have a negative score therefore they are not viable for survival or function of that protein.

What is a mutagen?

A mutagen is an agent that is capable of increasing the mutation rate of an organism. - Cant cause mutation in organisms including people. These mutations are associated with hereditary diseases and with cancer.

What is Gibson assembly?

A way of joining together multiple fragments of DNA in one reaction. - use PCR to amplify the fragments but design overlapping ends of products so the 5' end can be chewed back and DNA can anneal together to DNA polymerase to complete the fragments.

What is Horizontal gene transfer and 3 examples of it?

Also called lateral gene transfer and is a process by which a bacteria gains genes directly from another cell and incorporates it into the their genome. - can spread fitness-enhacing traits such as antibiotic resistance. - A great mechanism for ongoing adaptive evolution. 1. Transformation - uptake of NAKED DNA into a COMPETENT recipient cell which naturally occurs in a limited number of bacteria. 2. Conjugation - DNA transfer (mating) through cell to cell contact via a sexual pilus which is mediated by a plasmid or excised transposon. 3. Transduction - DNA transfer mediated by a bacteriophage containing previous host bacterium DNA.

What is the structure of the switch region of the prophage that can be used to maintain lysogeny or induce lytic pathway?

An 80bp region between the CI and Cro genes contains 2 promotors being (PR) promotor right that goes toward Cro and (PRM) Promotor repression maintenance. In between these promoters are 3 operator sites (OR1, OR2 and OR3) that are about 17 bp in length that Cro and CI are both able to bind to at different affinities. (Different concentrations of a protein determines the ordered changes in sites that are bound to by it) - RNA polymerase can bind to either of these promotors at the -10 or -35 location but not at the same time. - The promotor regions don't overlap - The OR2 operator spreads across both promotors.

Define a mutagen - when mutations do they increase most?

An agent capable of increasing the frequency of a mutation. - they tend to increase substitution mutations the most but also can result in indels (insertion or deletion)

Define Auxotroph

An auxotroph is a mutant organisms which requires a particular additional nutrient to grow, which the wild-type strain doesn't (as it is able to synthesise it).

What is transposase?

An enzyme that is most abundant within the cells that is responsible for transposon insertion into the genome. - Transposons were first discovered in bacteria and now make up 50% of the human genome and 70-90% of maize genomes.

How do we use BACs to clone really big fragments?

BACs are bacterial artificial chromosomes which have the features of: - based upon F plasmid in E.coli - LOW COPY NUMBER (1-2 copies) - Contains genes to stabilise the plasmid - selective marker of Antibiotic resistance (chloramphenicol) - CAN CARRY LARGE DNA INSERTS of about 75 to 300kb (much more than plasmids and cosmid vectors) - may contain colour selection (Lac alpha). * this allows packaging of much larger DNA inserts from large genomes such as humans.

How do we identify homology?

BY SEQUENCE COMPARISON: Usually 2 or more genomes are aligned and matched characters are considered to be homologous . - these matches identify functional, structural and evolutionary differences between sequences. *Sequences can be 87% similar but not 87%homologous

What is a bacteriophage plaque?

Bacteriophage plaques = holes that are formed in a lawn of bacteria that have been killed by the phage. (each plaque is a spot where a single cell has been infected by the T4 virus).

How can recombination frequency be used to do high resolution mapping on the genome?

Based on the interrupted mating experiment we know the order of markers on the Hfr strain. As a result we are able to determine where the recombination/recombinations have occurred based on the resulting bacterias phenotype. - ones that contained marker A but not B or C had a recombination from a region prior to marker A and between markers A and B (so B and C wasn't included) - A recombinant that contained A and C (no B) had 2 recombinations. One occurred before the A marker and between the A and B marker and the other occurred between the B and the C marker and after the C marker. The distance between the markers can be determined by the frequency of recombinants. Example: If there is a total of 300 recombinants one of the types being A+ B- and C- which had frequency of 25 -The distance between the last recombined marker of A+ to the next marker that wasn't recombined = 25/300 = 8 this recombinant made up 8% of the total recombinants and in turn A is 8 map units away from B on the Hfr insert. - As shown in the image a A+ B+ C- recombinant had a frequency of 75 therefore (75/300) a 25 map unit distance between B and C on the Hfr transfer. It can be said that B and C are 3 times further on the Hfr than A and B are based on these frequencies.

Why is there no phylogenic tree of all viruses?

Because viruses don't have a core genome and therefore it is hard to classify them into groups.

How do you predict whether a variation is significant or neutral within genomes?

Begin by determining if the variation is in a coding region or non-coding region. - if in non-coding is it in a regulatory region or important structural non-coding region. Also begin by determining if it is a point mutation or an indel and is it a SYNONYMOUS or NON-SYNONYMOUS MUTATION??

How can sequencing genome fast help?

Being able sequence genomes quickly helps identify drug resistance.

What are the benefits and disadvantages of plasmids?

Benefits: -Easy to work with - common for expression of proteins - good for cloning small DNA fragments. Disadvantages: - Transformation is a relatively inefficient process - Larger plasmid transformation is even less efficient - Can only go up to about 20kb of insert but usually less than 10kb. *need to use cosmids for larger DNA fragments *Need to use BACS for large genomes.

What are the characteristics of the last 2 boxes of frequently replaced amino acids?

Bottom: F, Y and W amino acids (WYF) are more likely to replace each other within an amino acid sequence as: - they tend to be large, aromatic, hydrophobic amino acids and in turn are able to maintain function of each other 2nd to bottom: M, I, L, V (MILV) amino acids are more likely to replace each other because: - they tend to be large, hydrophobic, aliphatic amino acids.

What conjugation can occur between F+, F- and Hfr strains

Cells containing Hfr or F+ do not mate with any other cells containing F factor. Many recominants - Hfr X F- cells Low levels of recombinants ; F+ X F- cells No recombinants - Hfr X Hfr F- X F- F+ X F+ Hfr X F+

What was the classical view of genetics? - How does this compare to the modern view?

Classical genetics: A heritable change in a gene that results in a change in phenotype. Modern genetics: A heritable change in the sequence of a DNA molecule (mutations may not affect phenotype).

What is cloning? - why do we want to clone?

Cloning produces lots of identical copies of a particular gene or sequence of DNA. - The aim is to obtain isolates and purified copies of specific gene sequences. - can analyse sequence of genes - can purify genes - can look at mutations.

What is the post segregational killing method control of ColE1 plasmids?

ColE1 contains an Ecol1 gene that results in the production and excretion of colicin antibiotic into the environment. However they also contain an imm gene which provides immunity to this antibiotic. - E.coli that don't gain a ColE1 plasmid following cell division will die as they don't contain the imm machinery to combat the colicin within its environment = cell death.

How does comparing one genome to one or more bacteria work?

Comparative genomes is the comparison of the genome structure and function across different biological species and strains. Helpful because: - comparison can aid in identifying differences and similarities (this can allow understanding of the genes that are involved in a particular process) - Can be used to observe changes in genomes (insertions, deletions and inversions)however still are unable tell whether a genomic element was lost by one or gained by the other.

What environmental changes trigger competence in bacteria?

Competence is dependant on stresses induced by antibiotic treatment or the extra-cellular concentration of a peptide. - All bacteria in an environment are secreting this peptide. Therefore as the population increases the peptide accumulates and allows bacteria to sense the population density. Increased population size causes SOME bacteria to produce immunity proteins for themselves as they secrete hydrolytic enzymes into the environment that causes the lysis of neighbouring cells to release DNA to be transformed/recombined into the recipient cell.

How is complementation used to find a gene of interest? (phenotype)

Complementation is the restoration of a wild type phenotype from a mutant by a seperate copy of the gene. 1. Make a library 2. plate on a selective medium 3. Clones that carry the gene should grow 4. Requires an expressed an functional gene. (Leu+ gene can be identified by plating bacteria with various genomic inserts onto a Leu- selective media) - those that grow contain the desired gene of interest.

Define Complementation and Recombination?

Complementation: Production of a wildtype phenotype of a virus when two viral mutant haploid genomes are present within the same cell. - Results from genetic recombination in which a genome that contains all the genes for proper function is created. Each genome provides the other with the gene they are lacking to create a single genome that has both and can function normally. Recombination: A process that creates new gene combinations

What are conditionally essential and essential but redundant genes?

Conditionally essential: Are essential depending on what conditions are used for growth - (if on sucrose media then enzymes for its breakdown and transport will be essential) Essential but redundant: Some genes can do the same essential function therefore deletion of either gene is tolerated but deletion of both is not.

What does conjugation require? - what is the process of this DNA transfer called? - what does it produce?

Conjugation depends on the presence of a plasmid which is considered the F factor (fertility factor) - The donor has a plasmid is F+ (male) - The recipient doesn't have a plasmid and is F- (female) F factor can be transferred from F+ to F- via the ROLLING CIRCLE MECHANISM which involves only a single strand of the plasmid DNA. F- becomes F+ and F+ remains F+ as both are provided with the single plasmid strand that can be synthesised into a double strand in both cells. - This process is strictly heterosexual (F+ doesn't mater with F+).

How was the minimal genome of Caulobacter crescentus determined specifically?

Contains only 517 genes therefore it is a small genome for this method to be used on. - managed to insert 220 transposons within the genome = at every 8bps and only genes with insertion that were non-vital were able to grow. These bacteria bind to surfaces that they come into contact with by forming a stalk and sailfast. - only when they are attached will they divide therefore this allowed for their development to be monitored as old and new cells are in same area.

What are composite transposons?

Contains two IS (insertion) elements that flank the central region which carries other genes such as and antibiotic resistance genes. - The IS elements provide the transposase and recognition signals therefore often ONLY ONE of the IS molecules makes active transposase to make the transposon mobile and make cuts at the insertion sequence. - Transposase is able to recognise IS elements on both transposon and insertion sequence.

What are cosmids?

Cosmids are another type of vector that are plasmids with a lamda cos site but no other features of the lamda phage. - these vectors have a high efficiency of packaging as any DNA with cos sites a suitable distance apart will be packaged into phage heads. - cosmids are used to package DNA (via cos sites) and introduce in high efficiency to E.coli *can package about 50kb (more than the max 20kb of plasmids) and therefore can contain larger gene inserts.

What is the structure of Cro and how does is induce the lytic cycle?

Cro forms a dimer but it doesn't have the N and T domains. - Cro binds operator regions in the order of affinity being: O3 first, then O2 or O1. (they all bind independently as no protein/protein interactions occur) 1. OR3 will be bound to first resulting in silencing of the PRE promotor (no CI) and transcription PR will occur resulting in cro. 2. As cro concentrations increase it will eventually bind to O2 and O1 resuling in its own silencing until it decreases again = lytic cycle. 3. When CI is cleaved it is also removed from the PI site which initiates the transcription of Int and Xer prtoeins = integrase and excisionase promotes the excision of lamda from host genome. 4. The switching off of cro synthesis via Cro binding to lower affinity operators allows planning of the sequence of events that occur within the cell (e.g. DNA replication, machinery, packaging, lysis)

What is Cro, CII and CI?

Cro: A DNA binding protein that represses transcription and promotes the lytic cycle. CII: A DNA binding protein that activates transcription and promotes the lysogenic pathway. CI: A DNA binding protein that can activate or repress transcription (depending on which operators it binds to) - it activates its own expression (Aids RNA polymerase binding to weak PRE) - Represses genes required forlytic cycle - Maintains lysogeny

How can the method of finding a natural small genome be used to determine the minimal genome? - P. ubique

DEFINES THE MINIMAL GENOME TO BE 1.3Mb WHICH IS THE SIZE OF P.ubique The smallest genome of any cell that is known to be able to replicate in nature was isolated being P. UBIQUE. - it has a small genome of 1.3Mbs and only encodes 1354 proteins therefore doesn't contain many extra/useless genes.(compared to M.laprae which has 3.2Mb and encodes 1600 proteins) It grows as free cells in the ocean and utilised dissolved organic carbon in the ocean - it makes uo 25-50% of all bacteria in the ocean

What is transduction? - how does it work?

DNA transfer mediated by a bacteriophage containing previous host bacterium DNA. When viral machinery is synthesised within the host cell and the host DNA is broken down random fragments of bacterial DNA that are similar in size to the viral DNA can be packaged int the head of the virus instead. When the host cell lyses the resulting bacteriophage finds a new host cell to inject its DNA into. - The bacterial DNA is linear and double recombination occurs to incorporate this bacterial DNA fragment into the new host genome.

What has contributed to the evolution of RNA sequences?

Due to the structure and interaction of bases within RNA, when one base is mutated is may support the mutation of another base that it interacts with = co-variation - This can allow for rapid evolution to A-U or C-G to maintain the structural integrity of the RNA therefore functional elements are conserved

How do we know that the P. Unique bacteria genome doesn't contain junk?

Encodes basic functions for growth (such as biosynthesis of al 20 amino acids) and contains almost no non-functional/redundant DNA. - no gene duplications or evidence of horizontal gene transfer. As a result: It has the smallest median intergeneric spaces of only 3bp in comparison to E.coli which has 85bp. - the genes are PACKED VERY TIGHT causing genome to be smaller.

What is the epidemiology of the M. ulcerans disease? - Location - Reservoirs - Modes of transmission

Epidemiology is poorly understood Location: It is thought that the occurrence of Buruli ulcers increases when in closer proximity to slow flowing or stagnant water (water = risk factor) and may be related to deforestation that exposes these water sources. Reservoirs: M. ulcerans is associated with aquatic plants and algae which is thought to be fed on by snails which may act as a transient host to transfer the bacteria to insects. Modes of transmission: Insect vectors thought to be important for the transmission (present in salivary glands) but there is little person to person transmission evidence.

In what ways are we able to alter plastid vectors to get it to do what we want? - to express the cloned gene? - control expression of the gene? - to produce large amounts of cloned gene - purifying the cloned gene product

Expression of the insert gene: To express the cloned gene we would insert a promotor in front of the multiple cloning site (which contains the insert) therefore the gene will be transcribed and expressed within the cell. Controlled expression: The Ptac promotor can be used to control the expression of genes as it can be induced using IPTG when desired. Lots of expression: We can use strong promotor such as the T7 promotor so RNA polymerase binds easily = more mRNA product. Purification of cloned gene: You insert His tags after the promotor and a thrombin cleavage site along with your gene of interest. The the His tag binds to the protein which allows identification of the desired cloned gene. His tag can then be cleaved of by thrombin cleavage at the thrombin cleavage site = purified cloned gene

What are some mechanisms of genome reduction in M. leprae?

Extensive pseudogene formation: M. leprae lacks DNA proof reading in DNA polymerase III therefore more point mutations occur. Genomic reductions: - 26 definitional IS elements (non-mobile) - 4 families of repeats which can be double recombined to delete regions of the genome. - Large scale genomic rearrangements promoted by these repeat families (works much like IS elements).

What are some Plasmid encoded recombination systems that allow avoidance of oligomerisation? - F plasmid - ColE1 plasmid

F plasmid encodes two site specific systems: - ResD: encodes recombinase which acts at the fcr gene near the oriV site of both plasmid genomes to reverse the recombination. - Tn1000: Has is own system of recombination ColE1 uses host-encoded recombination: XerCD is an enzyme that is chromosomal encoded within the cell (not in the plasmid) - The plasmid has a 35bp cer site sequence that XerCD is able to recognise and seperate the plasmids at.

what are F-prime (F') factors and how are they produced?

F" factors are reformed plasmids that sometimes contain chromosomal DNA due to reverse recombination occurring between different IS sequences to the ones it had initially used to recombine into the chromosomal DNA - in this way F' factors can be used as cloning vectors to replicate the chromosomal DNA along with the plasmid DNA each time replication occurs.

How can genomic islands support bacterial cell evolution?

Fitness islands that can adapt a bacterium with core chromosome to specific environmental niches. - pathogenicity - Symbiosis (mycobacterium and plants) - Xenobiotic degradation (allows for breakdown of man made chemicals)

Pangene analysis?

For each gene that you have within a genome you can compare them to other species you are using. - CORE GENES: conserved genes which are found in all or nearly all genomes under consideration. These are usually genes involved in core processes and tend to overlap due to being essential for life. - ACCESSORY GENES: Genes not found in all the genomes under consideration (dispensable and unique) We can identify the highly conserved genes that may be targets for antibiotics and does the presence/absence of accessory genes associated with the traits of interest. - These are generally measured using Venn diagrams however they are hard to scale when more than 2 genomes are involved in the analysis.

How Is the ColE1 plasmid replication controlled?

Formation of persistent RNAII/DNA hybrids that are required for the initiation of replication. Occurs via the rom/rop control methods in which increased plasmid concentrations within the cell increases the concentration of RNA 1 within the cell. - RNA I is transcribed from the top gene and forms a dimer between the RNA II and RNA I = RNA KISSING which inhibits the replication of the plasmid from the OriV site as the RNA II is no longer able to act as a primer for synthesis.

What are Frameshift mutations and how does the Strisinger model explain this? - Strisinger model???? - what can repair these?

Frameshift mutations are as a result of slippage of DNA polymerase during replication - this includes or excludes one or more bases. - this causes all codons and in turn amino acids to be incorrect from this point on. Strisinger model: As shown in the image, DNA polymerase is able to "slip" forward or backward during DNA synthesis. - By doing so base pairs can be excluded or added causing them to loop when the strands come back together. - It is only when DNA synthesis occurs again that a frameshift mutation will be present in one of the cells as DNA polymerase will sequence the entire genome including or excluding the base pairs. *the mismatch repair system can repair frameshift and point mutations.

How did ethylmethylsulphonate (EMS) crease mutations within Jeffery Millers experiment?

GC to AT mutations 1. The ethylmethysulphonate (EMS) reacts with Guanine base's O group resulting in ethylation at this location. 2. The resulting base has no available O group therefore when DNA polymerase comes along it misreads it causing a A to bind instead of a C. 3. This mismatch is called a pre-mutagenic lesion until a second round of replication takes place resulting in a daughter cell with the A-T base pair instead of the original G-C.

What is GWAS good for and was are some important facts for experimental design?

GWAS is a powerful method for linking genotypes and phenotypes. Study is better if: - There is a large sample of cases and controls - diverse samples (not over representing a population) - good phenotyping (easy to identify the desired phenotype)

What did comparative genomics infer about the evolution of M. ulcerans?

Gene acquisition via a plasmids and IS elements has given an adaptive advantage in new niche via an increase in IS, pseudogenes, rearrangements and deletions. M. ulcerans displays: - Mycolactone acquision which resulted in important virulent determinants for proliferation in insect niches and formation of Buruli ulcers. - Removal of genes that are no longer needed by pseudogenisation, deletions and mutations = saves energy.

How were essential operons identified?

Genes are often encoded for within operons therefore if a transposon is inserted at an early point it won't be transcribed and all genes will be determined to be essential even if they aren't. - the outward reading promotor allows definition of the essential genes within operons.... The transposon can insert in the forward or reverse direction the operon won't be transcribed whereas if inserted in the forward direction within the early location of the gene the operon can be synthesised. = can work out which genes are essential based on where transposons are able to insert within it and still grow. E.g. if in-between all genes within it then all are essential and if between and within a gene or two then those genes aren't essential either

What is an essential gene?

Genes hat are required essential for life - if lost no growth can occur. Some genes such as for DNA replication, transcription and translation will always be essential.

How can transduction be used to genetic map?

Genetic mapping using transduction as a technique can be used to determine the distance that markers are from each other as A MAX OF 100KB can be packed into a single viral head and 2 cross overs have to occur for the DNA to be incorporated into the recipient genome (further away they are from each other the more likely they are to be separated during recombination) The frequency at which selected markers are traduced along with another unselected marker can determine how far away they are from each other. 1. Example: Leu+ is transduced along with AriR 50% of the time and with thr+ 3% of the time. This indicates that both AriR and thr+ are within 100kbs of Leu+ and that AriR is closer on the bacterial genome to Leu+ than thr+ is as it is co-transducted at a higher frequency. 2. Thr+ is co-tranduced 3% of the time with Leu+ but 0% of the time with AziR This indicates that Leu+ and Thr+ are within 100kbs of each other by AriZ isn't. *using this information we can determine that the order of markers bacterial fragments is as shown in the image:

What are genomic islands and conjugative elements?

Genomic islands are discrete DNA segments that are absent from closely related strains - when genes come in from different bacteria they have different features. - share features of phages and conjugative plasmids - integrated at specific sites (usually adjacent to tRNA) - Integration similar to lysogenic phages (phage like integrase near one end of island) - MOST GENOMIC ISLANDS HAVE LOST MOBILITY AND ARE STUCK WITHIN THE GENOME

What is the comparison of M. marinum and M. tuberculosis? - Growth rate - Pigment - Niches - Disease

Growth rate: M. marinum - Fast M. tuberculosis - Slow Pigment: M. marinum - Yes M. tuberculosis - No Niches: M. marinum - Diverse aquatic niches, frogs, amoebae, fish M. tuberculosis - None known Disease: M. marinum - Intracelluar infection of fish, frog and human macrophage and skin dermal lesions (like TB) M. tuberculosis - HUMAN TUBERCULOSIS (intracellular) - M. tuberculosis has a narrowed host range and is a specialised pathogen of humans with no environmental niche.

What did the Davis U-tube experiment show?

He used a U shaped tube that contained a fine filter at the bottom. He placed two different strains of bacteria into each end of the tube in a liquid media - The media could pass through th filter but bacteria couldn't. Conclusion: Even after several hours no recombinants were found as PHYSICAL CONTACT IS REQUIRED FOR RECOMBINATION/CONJUGATION to occur.

What did Jeffery Miller find about mutations caused by different Mutagens? - EMS - Aflatoxin - UV light

He used the lacI gene from E coli to determine the effect that the mutagens Ethylmethylsulphonate (EMS), aflatoxin and UV light had on it. He found: 1. Ethylmethylsolphonate (EMS) caused mostly GC to AT substitution mutations and GC to TA mutations to a much lower extent. 2. Aflatoxin caused mostly GC to AT mutations but wasn't as specific to that mutation type as there was a higher frequency of other mutations too. 3. UV light caused mostly GC-TA mutations. *specific mutagens tend to create predominantly one type of mutation.

What conclusions can be made about Hfr strain conjugation based o the Interrupted mating experiment?

Hfr chromosomes are transferred into the host cell in a linear fashion beginning at a specific origin. Markers that are further from this OriT side are mire likely to be interrupted during mating therefore the maximal yield of cells contains a specific donor is lower for donor markers that enter later. - closer to oriT = more recombinants containing this marker than those that are further away as they are less likely to enter the cell.

What is an Hfr strain and how is it formed?

Hfr strains/ High frequency recombinants are bacterial cells that have a fragments or a whole conjugative plasmid (F factor) integrated into its chromosomal DNA. - this allows An Hfr strain can be formed either by a double recombination between identical copies of an insertion sequence within transposons between the F factor (plasmid) and on the chromosome which results in one large chromosome (Hfr strain of bacteria). OR By conjugation of an Hfr strain into an F- cell which can either result in a double recombination to incorporate the plasmid into the F- chromosome = another Hfr cell or the conjugated DNA can degrade if the double recombination doesn't occur.

What is the method segregation within low copy plasmids and high copy plasmids?

High copy plasmids: (generally small plasmids such as ColE1) Generally rely on random positioning of plasmids to incorporate at least a single plasmid within each daughter cell. - if there is at least 15 plasmids within the high copy number cell there is a 1 in a million chance that one daughter cell won't contain a plasmid = death Low copy number: (generally larger plasmids such as F plasmid) Uses specialised machinery such as ParM-like segregation.

What is the genomic comparison Mycobacterium Marinum and Mycobacterium Tuberculosis?

High homology at the PROTEIN level (85%) across 3000 related genes/ differences caused by: - Rearrangements - Inversion - Deletions (2Mb) - Acquisition via Horizontal gene transfer.

Define homologous and analagous traits?

Homologous traits: Any characteristic that is derived from a common ancestor. - E.g. vertebrae forelimbs or all influenza A viruses show common traits. Analagous traits: Similarities between organisms that were not in the last common ancestor. - E.g. wings from birds and bats differ from common ancestor OR ssDNA and dsDNA viruses

How has comparative genomics been used to identify how horizontal gene transfer caused the differentiation between Mycobacterium ulcerans and mycobacterium Marinum?

Horizontal gene transfer resulting in the KEY EVENT IN SPECIALISATION: ALL m. ulcerans acquired a 174kb plasmid which encodes 81 proteins of which most are dedicated toward the synthesis of MYCOLACTONE. - some M. marinum species acquired a plasmid that is resistant to mercury but isn't in all. - Mycolactone synthesis occurs when a 50kb gene (50x larger than normal) if syntheised in modules that are added together.

What are the genomic differences between M. marinum and M. tuberculosis?

Horizontal gene transfer: Has resulted in up to 80 regions being acquired by M. tuberculosis. Reductive evolution: Genomic size: M. Tuberculosis is smaller and less genes. IS elements: M.tuberculosis has more (54 compared to 29 in M. marinum) - has aided reduction of the genome by double recombinations. PE and PPE genes: M. marinum has more Metabolism: Greater metabolic flexibility in M.marinum as it has double the number of transporter class - loss of transporter causes narrow host range for M. tuberculosis.

What can be hypothesised about the P. unique genome streamlining/minimalisation?

Hypothesis: Some niches select for minimalist DNA. replication costs the cell therefore the genomes are streamlined/reduced to conserve energy. - P. ubique grows in nutrient poor sea water therefore most transporters are high affinity in import efficiency and there is NO REGULATION IN THE GENOME. = as a result this bacteria undergoes slow CONSTITUTIVE GROWTH and doesn't respond to changes in increased nutrients (always regulated the same)

How can estimation of the minimal genome be done by mutagenesis?

If you take a small genome and create mutations within it you can determine which genes are essential based on which genes that don't contain a mutation (unable to grow without this gene).

How does recombination differ in bacteria from eukaryotes?

In Eukaryotes recombination occurs between both parental genomes but the remaining recombinants are both viable. In prokaryotes the number of recombinations has to be even in order to incorporate the transferred DNA and produce a circular genome product. - This process also results in one viable recombinant and another linear non-viable fragment that can't be replicated as its not circle and will be degraded.

What were the findings of the Mycoplasma mutagenesis method?

In M. genitalium and M. pneumoniae there were: - 93 disrupted genes in M. genitalum and 150 disrupted genes in M. pneumoniae (NON-ESSENTIAL) - therefore there are 265-350 essential genes that make up the minimal genome.

How is transformation specific?

In some organisms the DNA they uptake is highly-specific to being from their own bacteria species/strains. - they do this by being able to bind to specific short base pair sequences of the donor DNA to bring it into the cell. - Incorporation of this DNA is done by homologous recombination which replaces the original host DNA. - Studies have shown that large amounts of up to 50 kilo bases of donor DNA can be transferred into a recipient bacteria at the same time.

Compare the insert size, features and introduction method into bacteria of plasmids, cosmids and BACs?

Insert size: Plasmids - 10 to 20kb Cosmids - 30 to 50kb BACs - 75 to 300kb Features: Plasmid - can be multicopy Cosmids - Efficient introduction into a host cell and replicated like a plasmid. BACs - Large stable plasmids that have low copy number Introduction to host cell: Plasmids and BACs - Transformation into a host cell (inefficient) via electroporation or treatment with CaCl. Cosmids - Transduction (very efficient)

What is the method of conservative transposition?

Insertion sequence transposons and composite transposons use this method. 1. A double stranded cut is made at the ends of the transposon DNA by transposase enzyme (blunt ends) 2. At the same time a sticky end cut is made in the target insertion location 3. Transposon binds to the 3' OH end of the staggered cuts 4. Repair mechanisms within the cell repair the the missing single strands of DNA crates by the sticky ends = duplicated region on each end.

What are insertion sequences transposons?

Insertion sequences are the simplest form of transposons as they encode: - only the protein for transposition which is called TRANSPOSASE - And has 2 inverted terminal repeats at each end of the gene (IS sequences) are used to move DNA to another location by transposase *The repeats are similar but not identical.

Define conjugation?

Is a process by which one bacterium transfers genetic material to another via direct contact. (known as bacterial sex)

How do you determine whether mutations in a protein coding gene is significant?

Is the mutation synonymous or non-synonymous if it is a point mutation: - Synonymous mutations encode the same amino acid and tend to not be significant as it has no effect on the resulting protein - Non synonymous mutations encode different proteins. These mutations can be (near) neutral if the biochemical properties of the amino acid it encodes is preserved. * Non- synonymous mutations are more likely to cause significant variations but are more significant if amino acid is replaced with amino acids with significantly different properties as it may influence folding and function of the resulting protein. (WYF and MILV are frequent replacements) Indels: Does the insertion or deletion maintain the frame of the sequence? If so it is more likely to be less significant than one that doesn't as it will lead to frame shift. Frame shift mutations: can cause changes in all codons/amino acids downstream of the mutation and tend to be significant as a result. *able to line genomes up to determine were synonymous and non-synonymous mutations have occurs as well as indels/frameshifts.

How can conserved regions relate to the functionality of a coding region/protein?

Is your coding variant in an evolutionarily conserved location? -figure this out by aligning homologous amino acid sequences using sequence logos (larger letter = more conserved) - mutations in highly conserved locations tend to be less likely to be tolerated than mutations in less conserved locations. *non-conserved locations tend to be able to accept lots of different variation and still produce a functional product.

How did the 3 scientists determine that the repressor interacts directly with the operator region?

It was hypothesised that the repressor interacted with the operator which was located near the beginning of the genes it controlled. - they found constitutive mutants in cells that contained two copies of the repressor (unlikely that the repressor mutated) these are known as OPERATOR CONSTITUTIVE MUTANTS as the repressor is unable to bind to the operator as it is mutated. Found that Oc is dominant over lacI+ and lacIs: Found that even when the repressor protein was present within the cell it couldn't bind to the operator to inhibit translation of the operon whereas those that expressed O+ were able to be repressed. - Oc are constitutive as a result. *do questions on lecture 15 at end!!

What was the reason for the genome wide association study based on MRSA by Laabei et al?

Laabei et al (2014) aimed to predictive virulence of the MRSA bacteria from its genome sequence. MRSA = Methicillin-resistance staphylococcus aureus. - the S. aureus bacterium is a natural part of the normal macrobiotic in the upper respiratory tract, skin and gut however it can cause infections in different parts of the body (such as impetigo) - often associated with team athletes, military recruits, prison inmates and children in day care. - hospital acquired MRSA is a real problem in Southern Europe.

How did the 3 scientist determine that inducers are different to substrates?

Lactose (allolactose) is an inducer however lactose is also a substrate. They determined however that other molecules such as the IGTG molecule are not substrates and are able to act as inducers therefore the inducer was not a substrate.

Why is choosing a vector so important?

Libraries can go into various vectors (plasmids, cosmids and BACs) however depending on the size of the genomic library, the type of vector that is used becomes important. E.g. if you want to sequence the human genome it is 3,300,000kb. - you need multiple coverage of the sequence to have a 99% change of cloning the gene of interest within one of the vectors. * if you used plasmids (only contain up to 20kb) you would need 750,00 clones to sequence the whole genome * If you used BACs (75-300kb) you would only need 100,000 therefore if it the better option.

Transmission mapping?

Look at the distribution of species and strains and see where, who and when they came from. Able to determine how wide spread particular diseases are. Example of Mycobacterium leprae which is full of pseudogenes that are non-functional and is the causative agent of Lepracy. They were able to determine how this bacterium spread across the world. - Found that probably originated in east Africa and spread to Asia and Eurasian continent and then to the Americas.

How can screening for recombinant plasmids be done (containing insert)? - pBR322 plasmid

Loss of antibiotic resistance in pBR332: The pBR332 plasmid contains 2 types of antibiotic resistance being Tetracycline resistance and Ampicillin resistance therefore cells that contain the plasmid only are able to grow of plates that contain both of these antibiotics. - If a BamHI digested insert is inserted into this plasmid it will interrupt the Tetracycline resistance gene and therefore insert containing cells can't grow on Tetracycline containing antibiotic plates. You can take colonies and grow them on Ampicillin plates. Those colonies that survive contain the plasmid. You can then transfer specific colonies to Amp and Tet antibiotic plates and those that are able to grow on Amp but not Tet contain the insert.

When is it determined to be a good time for lytic pathway compared to the lysogenic pathway? - when should lytic pathway be educed

Lystic cycle: Good time to make new phages when there is high growth of bacteria and plenty of food/energy within the cell. - lots of ATP and premise which causes degradation of CII and phage will go down the lytic pathway Lysogenic pathway: If the cells are unlikely to have the resources to produce many phages - less ATP and premise which means CII remains stable and phage will go down thelysogenic pathway Induction: If the host cell is damaged and likely to die. - UV shock

What has reductive evolution of M. tuberculosis caused?

M. barium is the closest relative of the M.tuberculosis. M. tuberculosis is undergone extensive genome reduction from the shared ancestor with M. marinum. - M. tuberculosis became a specialised pathogen of humans with no environmental niche. - M. marinum retains genes required for broad life style as a generalist variety of environmental niches.

What is the example of reductive evolution for M. tuberculosis?

M. tuberculosis has half the number of transporters that M. marinum has = less metabolic flexibility and therefore is specific to human hosts. - Acquired genes via horizontal gene transfer (some encode proteins that assists virulence) - Lost many genes through deletion (GENOME REDUCTION) by IS sequence recombination. - Reduced metabolic flexible compared with M. marinum. *M. tuberculosis has a restricted niche (gene loss) and increased pathogenicity.

What is mycobacterium tuberculosis?

M. tuberculosis is thought to have evolved from an ancestor similar to M. marinum. - it has infected 1/3 of the worlds population and kills about 2 million people pre year - common form of infection is pulmonary which infects the Jung macrophages (intracellular). - it is spread via coughing and is most infection in its latent (dormancy) 10% of latent infections become active of which 50% fatal if untreated. Dangerous synergy with HIV/AIDS Long antibiotic course of 6 to 12 months.

How has IS sequences caused reductive evolution? - deletions - inverstions - Pseudogenes

M. ulcerans contains more IS sequences (304) than M. Marinum (29). This has lead to rearrangement within the genome as well as pseudogene formation (gene inactivation). - these genes are able to jump into the mycolactone genes but this causes cell death so no cells seem to contain IS sequences within this gene. Deletions: IS sequences that are directly orientated can double recombine to exclude the genomic information between the two IS sequences. - Inversions: IS repeats that are in inverted orientation double recombination to result in the inversion of the sequence in-between them. - generally well expressed genes point in the same direction as DNA replication (maintains function) Pseudogenes: Are inactive relatives of known genes that have lost their ability to be expressed. Can be caused by point mutations, insertions of IS sequences, deletions or substitution which encode premature stop codons.

What are some comparative genomics of M. leprae to M marinum?

M.leprae = 3.27Mb and M.marinum = 6.6Mb 29% of M. leprae is pseudogenes 1,100 which is many more than M. marinum - caused by mostly point mutations as it contains many less IS sequences. Only 49.5% of its genomic content is coding genes. 23.5% are unrecognisable genes. - M. leprae is very inefficient as a result as half its genes are useful, a quarter are pseudogenes and the other quarter is unknown.

When was MRSA discovered and when how did it acquire its resistance?

MRSA was discovered in 1961 and was named MRSA due to its development of multiple drug resistance to BETA LACTAM ANTIBIOTICS (includes penicillin derivatives such as methicillin and oxacillin). This resistance is likely to have been acquired through horizontal gene transfer and extensive NATURAL SELECTION thanks to wide spread antibiotic use. - mecA is a biomarker gene responsible for resistance to methicillin and other Beta-lactam antibiotics. - MecA encodes a penicillin binding protein that is required for peptidoglycan synthesis and maintaining the bacterial cell walls (the MecA active site does not bind to methicillin or other beta-lactam antibiotics which is what causes MRSA's resistance).

What are some uncultivable containing clades in the tree of life?

Many bacteria such as the: - microgenomates, candidate and pyhla radiation and cyanobacteria clades contain uncultivable organisms. Many archea are uncultivable too.

How can the Ames test be made better?

Many chemicals that are not mutagenic to bacteria are mutagenic to mammals this is because these chemicals are converted into an active form by enzymes in the liver after ingestion. Solution: Prepare liver extracts and incorporated them into the medium that are used in the Ames test. - Mutations within the genome can increase the effectiveness of the mutagens in creating mutations due to their effect on the cell.

What are transposons? - E.g. of flower

Mobile elements of DNA that are able to jump between chromosomal locations and replicate themselves. - A transposon was discovered due to the effect it had on the pigmentation of a flower. Transposons had inserted themselves in and out of pigment genes within the chromosome of a flower which altered its phenotypic colouring.

Why are error prone DNA polymerases used in apurinic sites?

Most DNA polymerases have a proof reading activity to synthesise DNA. - These DNA polymerases can't synthesise damaged DNA such as apurinic sites so error prone polymerases take over at this point onward. These DNA polymerases however are more likely to incorporate the wrong base even in locations of DNA that isn't damaged.

What are the modes of transmission and treatment of M. leprae?

Most prevalent in the equatorial regions. Transmission: - Not well understood but due to person-person contact via the respiratory route. - Not very contagious. Treatment: - Multidrug therapy (6- 12 months) - some protection by BCG vaccine

What are mycobacteria? - variability of mycobacteria?

Myobacterium is a genus of actinobacteria which contains 40 species including: - mycobacterium tuberculosis - mycobacterium ulcerin - mycobacterium marinum - mycobacterium leprae *they are categorised by thick cells that are hydrophobic and waxy. Varriablity: They are generally aerobic, non-motile rods which are free living an parasitic (0.2-0.6 um in width and 1.0-10um in length) - They can be fast growing (colonies within 7 days) or slow growing with colonies after 7 days) and some can be very difficult to culture so a lot of information about them is learned by genome sequencing and comparison.

What is the NDM-1 plasmid and what are some features of it?

NDM-1 plasmid is responsible for the synthesis of proteins that are able chop up antibiotics that contain beta lactate. - very successful plasmid type that has spread globally. - 88803bp - Found in dangerous E.coli and other virulent bacteria. Grey area: Contains the backbone DNA - tra (allows conjugation) - OriV - parA/B (segregation of plasmids) - toxin/antitoxin system Red area: - multiple antibiotic resistant genes (resistant to Bete Kaftans, aminoglycosides and macrolides) - Flanking IS26 (insertions) which suggest a large part of this region is as a result of transposons.

How does CI maintain lysogeny via negative and positive control?

Negative control - CI bound at OR1 turns off the cro gene by preventing RNA polymerase from binding to the cro promotor (blocks/repressor protein) - will also bind to the PI (integrase promotor) to suppress the transcription of integrase and exisonase enzymes so the prophage remains stable within the bacterial genome. Positive control - CI binds at OR2 heps the RNA polymerase to begin transcription of self. The CI helps RNA polymerase bind to the wear PRM promotor by enabling the formation of more stable contact with the DNA (Protein-protein interaction) = a 10 fold increase in expression for the PRM promotor to increase CI production and reinforce circuit of maintenance. * the stable structure formed between CI bound to OR1 and OR2 can be inherited by daughter cells (epigenetics)

What is positive and negative regulation?

Negative regulation: When gene expression is turned off by a repressor protein that binds to the operator region to inhibit transcription and must be removed by an inducer - inactive repressor = CONSITUTIVE EXPRESSION Positive regulation: Gene expresión is turned on by a regulator protein called an activator. - NO EXPRESSION

What is the method of replicative transposition?

Non-composite transposons use this method of transposing. 1. The transposase enzyme makes two staggered double stranded cuts. One in the transposon and the other in the insertion location. - sticky ends same length to allow binding to each other. 2. The single strands within the transposon bind to each of the single strand sticky ends of the insert location. 3. DNA repair mechanisms repair the single strand structures that result = 2 double strand copies of the same gene which appears like a large plasmid structure. 4. Resolvase enzyme catalyses the site specific recombination reaction to seperate the 2 DNA molecules - 1 in original location - 1 in new location.

How is chromosomal DNA sometimes transferred in the process of conjugation?

Normally, chromosomal and plasmid DNA remains seperate within bacterial cells. However, in some F+ cells the plasmid is recombined/integrated into the chromosomal DNA which makes it appear to be a giant plasmid. As a result when Relaxase cleaves the OriT site this will allow the rolling circle mechanism to transfer plasmid AND chromosomal DNA into the recipient cell. - due to the weak cross bridge the whole chromosome is rarely transferred. - The transferred DNA must be incorporated into the recipient DNA or it is lost.

How can you clone using cDNA?

Once you have created a cDNA copy of mRNA you can insert it within a plasmid with blunt ends (less efficient) OR More efficient to ligate on sticky end adaptors and then clone into a vector of choice that has complementary sticky ends.

What are the PE and PPE gene families and how are they reduced in M. leprae

PE and PPE are surface and secreted proteins with a role in virulence and host evasion. - they are important for intracellular growth OR increased host range 167 in M. tuberculosis (8% of genome), 280 in M. marinum and only 9 in M. leprae. - This reduction in M. leprae is due to them being unneeded due to long term stable parasitism with humans during evolution. most reduction in: - catabolism genes - energy metabolism genes - transport gens - PE and PPE genes.

How is the Photodymer mutation caused by UV light repaired?

PHOTORESTORATION: Photolyase enzyme uses energy from white light to convert the photodymers (double bonded adjacent thymines) back to pyrinidines. - They then use EXCISION REPAIR which involves the AP endonuclease enzymes detecting the apurinic sites, cutting the DNA here and excursion endonuclease proteins removing this location as well as adjacent bases. - DNA polymerase the uses the other stand as a template as it remains undamaged to replace the removed bases.

How does ParM segregation occur within low copy number cells?

ParM like segregation is used during the partitioning of low copy number plasmids at cell division. 1. ParM is is an actin-like protein that is encoded by the par gene within low copy number cells. 2. Part binds to plasmids at each end and filementates to extend within the cell. 3. This filamemntation provides a force of movement for the plasmids causing them to be pushed to each pole of the cell. 4. Once poles are reached the filament depolymerises and cell division can occur with a plasmid in each daughter cell.

What is pathogenicity?

Pathogenicity is the ability of a bacterium to cause disease due to temperate phages encoding violent factors. - virulence is the degree of pathogenicity strains.

How does biological interpretation of genome content occur? - 3 ways

People are required for this process and it is limited by our current knowledge of biology. - genes that are present within a bacteria can be useful to determine its biology and the niche it inhabits. (e.g. Bacillis subtilas has over 200 genes for amino acid biosynthesis and is free living in soil whereas Treponma palladium has no known amino acid biosynthesis genes as it gets these from its host = paracite) 1. Functional grouping of genes: Grouping of genes into vague functional purposed allows us to determine trend in the gene content and function. 2. Metabolic modelling: Sets of metabolic genes can be analysed and used to determine what metabolism pathways the organism has (limited by what we know) - can be useful to culture new bacterium as we can provide all it needs in the culture. 3. The accessory genome: transposons, IS sequences, Genomic elements (pathogenicity), prophages and plasmids can be active or inactive and are highly variable between bacteria - drives evolution.

why are CI and CII named how they are?

Phage is lysogenic form produces repressor protein which silences any new phages that enter before they can do anything = therefore immune to lamda virus. - When cells of E.coli lawn are infected shows turbid colouration rather than clear plaques which is due to infection leading to lysongenic pathway and in turn immunity to other incoming phages. If you get turbid plaque it is likely that you've got a temperate phage. - Scientists found mutants that only form lysogenic plaques (CII stimulates lysogeic, CI maintains and allows lysogeny) = clear plaque genes.

What are some examples of Plasmid-encoded phenotypes in: - Plasmid F - Plasmid RK2 - R plasmids

Plasmid F: Contains the phenotype that allows in to undergo conjugation horizontal gene transfer with same and similar cell types. - large size of 100kb - Found in E.coli Plasmid RK2: Contains the phenotype that allows it to undergo conjugation with many different cell types (such as gram +ve to -ve) - Found in P. aeruginosa and smaller in size with 60kb. R plasmids: Encodes enzymes that modify or degrade antibiotics. - Often on transposons too - significant medically.

What are plasmid free cells and why does it need to be controlled?

Plasmid free cells are plasmids that have lost plasmids following binary fission. - this is a problem as metabolic stresses from plasmids (more energy used) may slow bacterial growth compared to those that don't contain plasmids therefore they may be able to out-grow plasmid bearing cells. Solution: Kill plasmid free cells using POST SEGREGATIONAL KILLING SYSTEMS.

What are plasmid multimers? How is this issue resolved?

Plasmids are double stranded circular structures therefore double recombinations can occur between them creating multimers and resulting in a larger chance of plasmid loss during cell division. Solution: Plasmid encoded site specific recombination systems?

What are plasmids of the accessory genome and how are they contributes to bacterial evolution through gene acquisition?

Plasmids are extrachromosomal genetic elements that are: - usually supercoiled within the cytoplasm - Capable of autonomous replication - Not essential to cell (not required for survival) They contribute to bacterial evolution and genetic plasticity/change. - Very important in recombinant DNA technology - Can encode important phenotypes.

How do point mutations occur spontaneously?

Point mutations generally occur spontaneously (without any input) Occurs during DNA replication when DNA polymerase makes a mistake. - This results in mismatching/Tautomerisation of bases within the genome (substitution of a base)

What is the purpose of High resolution mapping? - how does this relate to conjugation?

Purpose is to determine the distance between markers on an Hfr genome. - interrupted mating can determine the order of these markers and the FREQUENCY at which each marker is incorporated into the viable recombinant can be compared to determine the distance between them on the genome.

Why is RNA more interesting than mRNA? - RNA world hypothesis

RNA may have been part of the RNA world as they are able to act as ribozymes and are able to carry genetic information (catabolic and genetic potential). RNA world hypothesis: RNA fulfils all essential biochemical functions of life such as Information storage and replication. Enzymatic activity and regulation. - As a result, it is likely that at some point RNA acted as the first genetic material as it was able to replicate, splice and peptide bond itself within a cell. - It can also act as a regulator to support or inhibit processes such as transcription and translation.

What are recombinations?

Recombinations are new combinations of alleles or of genes due to crossing over. - involves some kind of breakage and rejoining of DNA

How do these regulatory proteins work?

Regulatory proteins have allosteric sites that substrate effectors can bine to to alter its configuration to allow binding to an operator. - can allow activation or repression of the gene.

Who is Robin Holliday and what is the Holliday model?

Robin Holliday is a Scientist that studied recombination of DNA using fungi that expressed mutations. Holliday model: Was an early model for recombination. 1. Breakage occurs in single DNA strands of each chromosome. 2. Holliday junction forms between the 2 strand breaks (fused together) 3. Branch migration occurs when Holliday junction slides along the DNA by twisting the DNA giving HETERODUPLEX (hybrid) which are each derived from different parent molecules. 4. RESOLUTION occurs either by horizontal or vertical cuts to the Holliday junctions. 4. Results in 2 DNA molecules with new combinations of alleles: - Hetroduplexes and recombinants OR - Heteroduplexes and No recombinants.

How was transposition between Enterococcus and S.aureus dangerous?

S. aureus is resistant to majority of antibiotics therefore treatment was very limited as it was. When transposition occured between S. Aureus and Enterococcus faecalis it transferred a Vansomysin resistance gene which was the only antibiotic left that was known to be successful in killing this type of infection. - frequency of transposons on conjugative plasmids enables the rapid acquisition of multiple antibiotic resistance genes.

What does the Salvador Luria and Max Delbruck study show about how mutations arise? - 2 response types

Salvador Luria and Max Delbruck study: They infected a large amount of E. coli with TI using bacteriophage (bacteria virus). Most of them died but a few survived and from which colonies formed. - All the new colonies were immune to the virus too. Theories explaining results - 1. Amarkian response: Mutation was induced by the virus, It was suggested that mutation was an adaptive response to changes in the environment. These changes are induced for the purpose of survival. 2. Darwinian evolution theory: Resistant mutants randomly evolve and pre-exist in the population and were selected for when bacteriophage was present. How they determined which theory was correct: They used the fluctuation test by which they exposed different cultures to the TI bacteriophage. - For the adaptive/Amarkian response they can expect to find approximately the same number of resistant mutants in each culture. - If a random mutation occurs/Darwinian evolution theory is correct you will find variation in the number of mutants in each culture. Conclusion - They found variation in the number of microbes that survived in each of the cultures which supports the idea that mutations arise spontaneously and they can be selected for by environmental conditions. = Darwinian evolution theory is correct.

How can screening for recombinant plasmids be done (containing insert)? - pUC18/19

Screening by colour change in pUC18/19: The pUC18/19 plasmid contains the LacZ alpha gene which encodes the Beta-galactosidase alpha chain. The rest of the LacZ gene is encoded for within the bacterial genome. - The alpha chain is required for the successful activity of beta-galactosidase to cleave colourless x-gal into a blue insoluble product. As a result if an insert is present within a plasmid the colonies will appear white and the alpha chain is not present and therefore x-gal remains intact.

What did Seymour Benzers experiment do to determine the location of genes in the T4 genome? - wildtype features - mutant features - method

Seymour Benzer - Used mutant bacteriophage T4 to determine the structure (base ordering) of genes within the T4 virus. Seymour knew that there was genetic diversity in the E.coli bacteria which has resulted in different strains including stain B and strain K. - He found that wildtype (contains no mutation) T4 viruses had the rII genes that are responsible for the infection of the E.coli virus which allowed it to infect both strand B and K. - He found that the rII mutant phage formed large bacteriophage plaques on E.coli strain B but cant form any plaques on stain K. He discovered about 2400 rII mutants which all showed the same phenotype (was discovered that these mutations occurred in 2 genes within the genome which created the same phenotype). Method: 1. Benzer infected the E.coli B bacteria with both of the RII T4 mutants. 2. Inside the cells genetic recombination will occur by which DNA is exchanged between the two viruses. 3. In E.coli that contain 2 mutants that have mutations on the same gene no complementation will occur causing no lysis of the E.coli host cell. In E.coli that contain 2 mutants on different genes complementation can occur as a double mutant and a wild-type will result. The wildtype will contain both the rII genes required for cell lysis to release the progeny phage. 4. These viruses were all inserted into strain K bacteria which selected for the progeny wildtypes which were able to grow on this strain. 5. The amount of plaques indicated the frequency of recombination events and this corresponded to the distance between the mutations in the DNA (more plaques = mutations further away in the viral genome and less plaques = closer on the genome). Conclusions: - Complementation would occur in pairs of mutants that have mutations of different genes as after genetic recombination they could provide each other with their missing rII function. - No complementation would occur in some pairs as they did not give rise to a wildtype as neither phage could provide the other with the rII function they were missing. - The location of the two mutation sites could be determined based on the number of plaques that were present of the strain K bacteria. More plaques indicate the mutations are further away from each other on the genome than if there are less plaques due to the theory of recombination. - Genetic maps are linear and genes themselves are likely to be linear. - Most mutations are changes to only one mutable site and some sites are particularly prone to mutations (called hotspots)

How significant is Transformation in pathogen virulence evolution? - flesh eating bacteria

Single transformation can have large scale effects: There was a single transformation of a 36 kilo base section of DNA from a donor bacteria to the recipient bacteria that contained 2 virulence factors. - This resulting in a flesh eating function in the bacteria once recombined into the genome. - resulted in >600 million cases.

How can Streptococcus Pneumoniae avoid treatment/vaccine by INTERSPECIES transformation?

Some bacteria are able to incorporate DNA from other bacteria types into their DNA. This bacteria has many capsular types (over 90) which are each attacked differently by the immune system of the body. - Transformation between species can allow the bacteria to switch capsule type which gives it the ability to evade the immune response to the pathogen or become resistant to antibiotics that can't penetrate this capsule type. - Interspecies transformation is responsible for the appearance of penicillin resistance isolates of the bacteria as it reduced the affinity of it to penicillin.

What are some virulence plasmid encoded phenotypes?

Staph aureus - coagulase and haemolysin Pathogenic E. coli - Enterotoxin Salmonella, Yersinia - host cell invasion Agrobacterium - Plant tuberculosis (able to invade plant cells to get them to produce nutrients for the bacterial cell)

How was transduction discovered using the U-tube method by Leaderberg and Zinder?

Strain A which lacked 2 genes and had 3 and Strain B that lacked 3 genes but had 2 were used. - When they were crossed over phototrophic colonies resulted which are the wilhype (can produce all 5 of the proteins as they have all 5 genes) In the U-tube the colonies were separated by a fine filter so no recombination would be expected - however.... a filterable agent (bacteriophage) was able to transduce Strain A genetic material into the strain B colonies which caused wildtype strains when the B stain was plated = TRANSDUCTION of bacterial DNA.

What are synonymous and non-synonymous mutations?

Synonymous mutations: Are mutations in a genome that cause the same amino acid to be encoded for (redundant mutation). - usually these mutations are in the 3rd position of the codon. Non-synonymous mutations: Are mutations that lead to a different amino acid being encoded for. - these mutations are 2x as likely to occur randomly as they generally occur in the first 2 positions of an amino acid.

What is the Ames test? - who developed it? - How does it work with example of Aflatoxin mutagen?

The Ames test was developed by Bruce Ames to determine the mutagenic activity chemicals by observing their ability to cause mutation in microorganisms. He used auxotrophic strains which where mutated in their ability to synthesise histidine that is required for their growth. - They are then spread on an agar place that doesn't contain histidine and any bacterial growth are revertants and have gone back to being able to synthesise the protein. - If the test compound increases the number of revertants then it is a mutagen E.g. of test compound Aflatoxin: Expose the strains of auxotrophic salmonella to different concentrations of aflatoxin. Found that increased concentrations of Aflatoxin does increase the frequency of mutations back to wldtype and in turn is a mutagen.

How is decision made to do into the lysogenic cycle?

The CII protein is still the deciding protein. 1. Cells that are starved contain less ATP and in turn protease enzymes. 2. CII remains stable as it isn't degraded. 3. CII presence causes the E.coli to go down the lysogenic pathway as CII acts as an activator at the Promotor sites of PRE (synthesises CI repressor) and Int (Intergrase and exisonase) - int which results in the integration of lamda into the host genome - CI also activates its own expression and binds to the OL and OR sites to repress all other phage genes. 4. Integrase works by causing a site specific recombination between attP and attB sites. This results in the lamda genome being linearised within the bacterial chromosome. 5. The lysogenic state is maintained by CI repressor protein which represses all phages but itself. It works to keep the prophage genome silent until induction occurs.

How is a decision made to go into the lytic cycle?

The CII protein is the deciding protein. 1. Within healthy cells there is an abundance of ATP and in turn protease within healthy cells. 2. Proteases degrade the CII causing the (right) Cro pathway to win. 3. Replication from Promotor R occurs toward Q which is an anti terminatior that enables the late lytic genes to be transcribed in preparation for lysis. 4.Cro then represses the expression of CI as well as all early genes, itself and replication genes 5. Assembly of the phage occurs and host is lysed to relate the phage. *default pathway

What are the post segregational killing methods of the F plasmid?

The F plasmid contains 2 methods: - hok/sok method - ccdAB another TA system Hok/Sok method: Hok is a host killing gene that is transcribed into a very STABLE mRNA. Sok is a suppressor of host killing which forms an unstable antisense mRNA (half life 5 minutes) that is able to form an RNA/RNA duplex which stimulates the degradation of both mRNA types = no toxicity to cell. - If No plasmid is incorporated within a cell following cell division the stable Hok mRNA will remain within the cell cytoplasm however there will be no synthesis of the unstable Sok mRNA as the plasmid is absent. As a result, the Hok mRNA becomes a toxic protein which results in the death of the plasmid free cell.

What did they find about the genes associated with virulence due to changed in the toxicity of individual strains?

The GWAS identified 121 genetic loci that were significantly associated with the toxicity of individual isolated of MRSA. - some genes had known roles in virulence but many were novel

How has pseudogenes caused and resulted from the evolution of M. ulcerans?

The M. ulcerans genome is smaller (5.6kb) and contains more pseudogenes (771) than the M. marinum bacteria (6.6kb and 65) - this may be due to some genes becoming un-needed which allows less sensitivity to mutation in these genes as well as increased dominancy as less energy is expended on producing gene products that are no longer of use. Example of ESX secretion systems: M. tuberculosis has ESX secretion systems that are important for virulence, intracellular spread and immunogenicity. - M. ulcerans' ESX systems have been deleted which may be due to or have caused its extracellular pathology.

How did they present the data collected from the diffusion disk method on a Manhattan graph and what did they find?

The Thailand and USA data sets were analysed separately. - They identified 51 loci that had high numbers of SNPs * they identified 3 loci strongly associated with resistance in BOTH of the countries strains and no indels associated with the beta-lactam resistance.

What is genomics?

The discipline involving the mapping, sequencing and analysis of genomes.

What are bacteriophage?

The most abundant biological entity on earth which is known to infect all domains of life. - were used to discover what genetic information was by Hershey and Chase. * can under go 2 life cycles: - lytic - lysogenic

How do the results of the mRNA comparison between S. Tympani and P. aeruginosa reflect negative selection?

The results: - there are 6 synonymous mutations - only 4 non-synonymous mutations. * The expected number of non-symonymous is twice that of the synonymous mutations (2:1 ratio) as they generally occur in the first 2 positions whereas the synonymous ones generally occur in only the last position of the codon. - this is evidence for negative selection as it is more likely that the amino acid would be replaced with any of the other negatively scoring amino acids (as there are more of them) on the BLOSUM62 matrix therefore they have different chemical properties and may not survive. * the non-synonymous mutations that did occur were generally M-V. S-T and L-M which are known to be biochemically similar. *also indels that conserve the frame are more likely to survive * It is unlikely to get 3 positively scoring amino acids within a codon by chance therefore this points to negative selection as the protein is conserved over time.

How has reductive evolution occurred in M leprae?

The size of the genome is 3.27 Mb therefore you would expect there to be about 3000 genes - however only contains 1,100 therefore almost 2,000 genes have beed lost. = GENOME DOWNSIZING - HGT may enable a pathogen to proliferate more effectively due to having a more specific niche. Almost half the genome is non-coding: Genome reduction is common in parasites as they gain nutrients from their host (as other niches are redundant) -Genes/regions might be in process of being eliminated - Genes not required in specialised niche become deactivated - Mechanisms of genome rearrangement enables deletion of genomic regions no longer required. (unlike M ulcerans the genomic reduction is due to point mutations rather than IS sequences).

how does ligation of the DNA work?

The sticky ends are able to base pair to each other and DNA ligase enzyme is used to stick them together by forming phosphdiester bonds between them.

How did the 3 highly variable loci lead to increased resistance?

They all encoded Penicillin binding proteins. Normally the function of these proteins is inhibited by penicillin which causes cell death however the mutations in these locations disabled the ability of penicillin to bind to its active site therefore the strains because beta-lactam resistance. Penicillin binding proteins are involved in the synthesis of peptidoglycan cross-linking therefore disruption of their function leads to cell death. *penicillin binding proteins seem to be important for bacterial pathogens.

What are covarying sites?

They are bases at the secondary RNA structural contact points that covary across evolution - It means that is one base changes the base it interacts changes too therefore selection is favouring the reversion or compensatory mutation at the contact site.

How was mutagenesis used to determine the minimal genome in M. genitalium and M. pneumoniae?

They are both intracellular parasites that are able to grow as free living cells too. - They have very small genomes of 580kb and 816kb and lack cell walls. The Mycoplasma mutagenesis method: Performed TRANSPOSON MUTAGENESIS on M.genitalium an M. pneumonaiae which meant that only mutations that did not interrupt an essential gene could grow. - IS sequences in survivors were determined by DNA sequencing and non-essential genes were identifies. - this study had high mutational coverage to guarantee that all genes could have been mutated.

How did they validate the mutations that lead to increased toxicity?

They attempted to validate significant results (virulent SNPs and indels) with Tn5 mutagenesis. - takes the toxic mutants an expose them to Tn5 mutagenesis to see if interference in these genes leads to reduced toxicity of the microbe. - they expected an inverse relationship between SNPs that were related to virulence and mutations that lead to reduced toxicity. * as a result they are able to see if identified mutations (SNPs and Indels) have a causative role in the virulence of the strain OR if it was a false +ve.

What are non-composite transposons?

They don't rely on Is elements for mobility and therefore encode genes for their own transposition and have inverted terminal repeats. - These transposons require both TRANSPOSASE AND REVOLVASE enzymes and may contain antibiotic resistance genes.

How did the 3 scientists determine that the LacI gene was a repressor rather than an inducer? - PaJaMo

They found lacI- mutants where were unable to synthesis repressor protein for the lac operon therefore it was CONSTITUTIVE (always on) for the production of the enzymes. PaJaMo experiment: Initially thought that the LacI- mutant makes an internal inducer (as this lac operon is always on) therefore thought that I- would be dominant over I+ when conjugation between a lacI+ and lacI- cell occurs. - however found that I+ was dominant as transcription of beta glucasidase the lac operon decreased when I+ was conjugated over *DISCOVERED THAT I+ ENCODES A REPRESSOR PROTEIN THAT IS DIFFUSIBLE WITHIN THE CYTOPLASM. confirmed with F prime factor study in which a heterozygote of I+ and I- where both repressed therefore repressor must be diffusible.

What did they find about the toxicity phenotype of various MRSA strains and how did they validate this?

They found that there were strains of MRSA with low, medium and high levels of toxicity. They validated this toxicity measurement using a toxicity assay using high and low doses of MRSA. - lower doses of mouse inoculation with MRSA that was deemed as more toxic from the vesicle-mediated lysis experiment resulted in less survival than the same dosage with MRSA with a lower toxicity reading. (40% of death with highly toxic and only 10% of death with lowly toxic)

What data was collected for the GWAS study of beta-lactam resistance in S. Pneumoniae?

They gained 3,701 sample isolates of the bacteria mostly from Thailand but also from the USA. They measured to phenotype of Beta- lactic susceptibility (found that 50% of strains were susceptible and other 50% were not). GWAS found that there were about 600,000 SNPs associated with the beta-lactam resistance and no indels.

How was the MRSA Genome wide association study (GWAS) designed?

They isolated 90 MRSA isolates (mostly from Turkish hospitals and from a range of sources: blood, woulds, catheter) They measured the phenotype of the TOXICITY AND ADHESIVENESS of the bacteria. They did this by breaking up the genome and sequencing each fragment many times, they then pieced them back together which allowed for: - single nucleotide polymorpisims called SNPs (point mutations) and indwells from whole genome sequence to be identified.

How can conjugation be used for genetic mapping? - Wollman-Jacob interrupted mating experiment findings

They isolated HfrH strains of bacteria that donated The and Leu at high frequencies compared to other markers. - They allowed conjugation between cells (HfrH which contained functional markers and the other cell that we auxotrophic for those markers) but interrupted the act at different time intervals and analysed the exconjugants (conjugation pairs) for the presence of unselected markers. They found: 1. Each marker first appeared at a particular time (which indicates that transfer of markers is in a linear fashion which is why markers further from the oriT took longer to get into the cell) 2. Order of markers is always in a particular sequence with more of the markers closer to the OriT appearing in shorter time intervals. Interpretation of the graph - The Hfr strain transfers aiR then tonR then lac+ then gal+

How did the 3 scientists discover that the inducers regulated the new synthesis of beta gal?

They originally thought that inducers led to the increase in the activity of beta gal that was ALREADY present within the cell HOWEVER: They added radioactive amino acids before and after induction therefore radioactivity accumulated in the beta gal enzyme. * After removing the inducer the synthesis of B gal stoped NOW KNOW THAT AN INDUCER IS RESPONSIBLE FOR THE INDUCTION OF SYNTHESIS RATHER THAN JUST FUNCTION.

How was the concept of conjugation discovered by Leaderberg and Tatum's experiment?

They took two mutant bacteria which lacked more than one gene each. The genes that each of the mutants lacked were different to the other. 1. When grown separately the mutants didn't grow as they didn't have all the genes required to do so. 2. The 2 mutant strains were mixed together which resulted in growth on the mixed media. 3. The growth on the media of wild-type bacteria that was able to synthesise all proteins to grow indicated that there was some gene transfer between the 2 strains.

Why was GWAS used in the study of Streptococcus Pneumoniae by Chewapreecha et al?

They used GWAS for comprehensive identification of SNPs associated beta-lactam resistance within Streptacoccus Pneumonae bacteria. - this bacteria colonies healthy individuals in an asymptomatic was in their respiratory tract, sinuses and nasal cavity. * however can lead to pneumonia and meningitis.

What was George Beadle and Edward Tatums finding based on their experiment: - one-gene-one-enzyme hypothesis

They used Neurospora fungus (common bread mould) and isolated auxotrophs that could only grow when provided with arginine. - They used genetic mapping to locate genes in which mutations caused arginine auxotrophy. 3 loci were identified of 3 different chromosomes. - They then determined whether the mutants would grow with any compound that was similar to arginine - from which they found 3 different classes of mutants. They found that all Arg-1 mutants had the same locus and Arg-2 and 3 had different ones. Arg-1 would grow with all 3 amino acids that were tested. Arg-2 would only grow with 2 and Arg-3 would only grow with Arginine. Conclusion - Each of the 3 genes gives rise to a different enzyme that catylises a different chemical reaction. Basis of ONE-GENE-ONE-ENZYME hypothesis. Explains how if one enzyme isn't present the metabolic pathway to convert the other amino acids into arginine is inhibited. - Gave insight into the idea that genes (DNA) is the basis of genetic material that codes for proteins.

How did they measure the susceptibility of S. Pneumonae strains to beta-lactam antibiotics?

They used the disk diffusion method (Kirby-Bauer method) on various lawns of specific bacterial stains. - then dropped antibiotic infused paper disks with various beta-lactam antibiotics into them *repeated this for all of the isolated strains. * measured the size of clear zones around the disks to determine the overall susceptibility of the bacteria to particular antibiotics.

How was the case study of Haemophilus influenzae sequenced?

This bacteria causes meningitis and ear infections and was the first bacterial genome to be sequenced (1995) - 1.8Mb in size Was sequenced using by TIGR using the whole genome shotgunning methods. This genome was assembled from 24,000 shorter sequences that were about 500 bases in size. - The sequences were aligned to define its place in the genome (assembly) - Assemble sequences are called contigs. The COVERAGE WAS UNEVEN between contigs (less coverage in some locations than others.) = increased risk of error in sequence to 1/10 genes. The gaps in the sequence were filled using weither Lamda lytic clones or PCR followed by sequencing.

What is DNA proof-reading by DNA polymerase?

This is a method by which mutations are prevented within the genome. DNA polymerase proof-reads the new strand to ensure the correct base has been added. (exonuclease activity in palm domain) - if mistake is found then base can be removed and correct one added for DNA synthesis to resume - 99% effective in removing mutations.

How does recombination play a role in repair of damaged DNA?

This is another method of DNA repair as recombinations that exclude a mutation and create new DNA using the invading DNA as a template. - pre-mutagenic lesion is lost and mutation doesn't hold.

What is a merozygote?

This is the name for a cell state in which it contains an endogenote (complete genome) from the F- recipient cell and an Exogenote (part of the DNA from the Hfr cell) following conjugation between an Hfr cell with a F- cell. = partial diploid cell

What is Gateway cloning?

This method of cloning allows DNA fragments to be efficiently transferred between plasmids using a set of recombination sequences (att sites) - based on the lambda recombination system - no restriction enzymes used - no ligation - cloned into an entry vector first and then recombined into many other plasmid types

What is the Mismatch repair system? - How does it work?

This method repairs mismatches AND INDELS that have gotten passed the proof-reading process. - There are a group of proteins that scan DNA and detect mismatches and are then able to remove that chunk of DNA for it to be replaced. How it works: The proteins that make up the mismatch repair protein complex are MutS, MutH and MutL. 1. MutS scans the DNA and locates the mismatch. 2. MutH is then able to determine which base is the correct one as it is able to detect the methylation of the template strand and creates a cut in the daughter stand at this point. 3. MutL then connects the MutH and MutS by removing the chunk of DNA between the methylated location in the DNA and the cleaved DNA mismatch location. 4. DNA polymerase then replicates this location again but correctly to rid the point mutation.

How can (ddNTP) Dye labeled terminator sequencing be used to sequence a library?

This method uses 4 different dideoxynucleotides (one for each base) which don't have the 3' OH group for DNA polymerase to extend from. There is one for each base and each have a different florescent dye. 1. The DNA is heated to 96˚C for double strands to seperate. 2. 50˚C primers are added to the edge of the plasmid so DNA polymerase can extend the unknown insert sequence. 3. Normal dNTPs and ddNTPS are added and DNA polymerase binds them to the template strand randomly 4. Once a ddNTP is added the sequence can't be extended any further. (repeated many times = many different lengths of sequences) 5. A capillary tube containing gel allows the electrophoresis of DNA fragments toward a +ve electrode 6. At the end of the tube there is a laser that is able to detect the colour of the ddNTPs as they reach the end. (smaller fragments move faster) 7. Coloured peaks on a computer can be used to translate the ddNTPs into a sequence of the gene. *limited to about 1000bp - difficult to distinguish size and less likely for a ddNTP to incorporate at every base position.

what are integrative and conjugative elements?

Those genomic islands that have retained mobility are known as INTEGRATIVE AND CONJUGATIVE ELEMENTS. - Integrative and conjugative elements transfer by conjugation after excising from the chromosome. A copy of ICE remains in the donor and reintegrates. 1. The integrative conjugative element excises from the bacterial genome using exisonase enzyme. 2. transfers to neighbouring cell via the rolling circle mechamism 3. Replication occurs within both cell to create double stranded structure. 4. Integrase enzyme allows integration into the bacterial genome again by crossing over at the attB and attP sites.

What is Ordered clone sequencing?

Thought that this was the safest way of sequencing DNA fragments. - BACs are used as they can contain large gene inserts. 1. They used restriction enzymes that made cuts in the DNA rarely therefore there were many DNA fragments of different sizes and compositions. 2. Organised the different inserts into overlapping sequences to create a physical map. 3. They selected genes with minimal overlap 4. Divided the clones into many sub clones and sequenced them 5. Assembled the sub clones to create the genome sequence

How does in vitro packaging occur?

To infect bacteria phage particles must be produced. Phage outer coat proteins will spontaneously form from phage particles and a "stuffing" enzyme will automatically pack DNA of a certain length into those particles if it detects the COS sequences.

Why do we use genomic libraries?

To isolate genes for bitechnology To identify genes in an organism The obtain GENOME SEQUENCE and gene function.

Which NON-CODING genes did they find to be essential within Caulobacter crescentus ?

Total of 130 non-coding elements essential: - 29 tRNAs are essential not all tRNAsare as there are duplicates of them and required high resolution mapping as they are only about 70bp in length on the genome. - 8 essential small non-coding RNAs - The origin of replication is essential. without it there can be no replication of the circular genome and no growth. - 91 unknown non-coding functional genes may be regulatory region where proteins need to bind to turn on or off genes.

How can DNA alignment prove the conservation within RNA?

Trp operon example: (Green = co-varying sties) - There are conserved blocks of RNA information which tend to be involved in structural base pairing. - There tends to be more variation in bases that are within the looped region as they are not involved in structure. - The terminal regions tend to be well conserved *Negative selection tends to conserve RNA sequences involved in structure therefore regions that interact to create structure tend to be conserved and co-varrying sites tend to be in these locations.

Why were bacteriophage a good model for Benzer's analysis?

Used bacteriophage T4 to determine the structure (base ordering) of genes. - He used bacteriophage because viruses easily infect bacteria and are rapidly grown. - They are also genetically simple organisms that have similar genetic mechanism to the host cells they infect. - We are able to analyse billions of them very easily and in a small amount of space - The way the experiment was set up meant that the K strain of E.coli selected for the wild-type recombinants and therefore didn't have to be identified among many non-recombinants.

How did the 3 scientists determine that the genes within the lac operon were controlled together?

Used mutated strains of the gene to determine that the genes were seperate but also genetic mapping showed that the lacZ, Y and A genes were closely linked.

How can estimation of the minimal genome be done using genome comparison? - H. influenza/M. genitalium

Uses biotechnology to determine which genes are conserved between genomes to identify which ones are essential. When H. influenzae and M. genitalium were compared it showed that 256 genes were conserved between them which were perceived as essential.

If you were to create a phylogenic tree for viruses how would you do it?

Using the (David) Baltimore classification system of viruses: Able to place viruses into 1 of 7 groups based on: - Type of nuclear acid (RNA or DNA) - strandedness (ds or ss) - linear or circular - method of replication - number of segments and size of genome - nucleotide sequence comparisons.

How does bidirectional transcription prepare the cell for a decision?

When the circularised phage forms within the cytoplasm there are 2 promotor regions which create bidirectional transciption - Promotor L allows transcription of an N protein which stimulates anti-terminator proteins (promotes the lysogenic pathway) which enables transcription past the early gene expression regions as terminator signals in both transcription directions can be ignored (produces machinery for both pathways. - anti terminator proteins bind to the terminator sites causing RNA polymerase to pause and allows the formation of an anti terminator loop. - Promotor R allows transcription of the Cro protein which represses further transcription and promotes the lytic cycle. Also results in the transcription of a DNA binding protein called CII which promotes the lysogenic cycle.

Phylogenetic trees?

Which strains/species are closely related and how does theis relate to the genography of phenotypes. You can map species that are more closely related closely within the phylogenetic tree and diversified ones further away. - Can identify adaptations to pathogenicity or phenotypes. E.g. Salmonella can be commensal within the gut and cause no illness or can be pathogenic by causing typhoid fever and colonise different locations within the body.

Genome wide association studies (GWAS):

Widely used study methods which tries to identify all the variation within the genome such as SNIPS (Single ucleotide polymorphisms) which are point mutations or indels for various traits. - Key advantage is that it is unbiased therefore identifies ALL variation rather than in just one gene which means that even if there are less allele frequencies they will still be identified.

How can conserved regions relate to the functionality of non-coding regions?

With non-coding regions you can also use sequence logos but do it with nucleotide sequences rather than amino acid sequences that are used in coding variant comparisons. Conserved regions tend to be less tolerant of variation than on-conserved regions. - E.g. start codons and the shine dalgarno sequence within prokaryotes tend to be conserved as variation if these locations tend to result in negative selection as the cell cans survive.

Recombination hotspots?

With whole genome comparisons you can determine where more mutations occur than what you would expect by chance. These changes in the genome are likely to be due to horizontal gene transfer such as transformation (direct DNA uptake), Transduction (DNA introduced by a virus), Conjugation (cell to cell gene transfer) - Hots spots are often associated with surface structures and membrane structures that allow immune evasion. The diversification can be complex therefore we tend to focus on point mutations and mobile genetic events.

Where can genome variation occur?

Within population variation can be: -SNPs (point mutations) - Indels (insertions or deletions) There can also be between species variation Coding variation can be: - synonymous or non-synonymous - can result loss or gain (indels). Non-coding variation can be: - intergenic (between genes), intronic (between exon of a gene) or in ncRNA There can be gain or loss of genes due to structural variation, duplication. Need to determine if variation was vertically or horizontally inherited.

How does cloning of PCR products work?

You can add extra sequences to the RNA primers used in PCR that can be recognised by a restriction enzyme - allows removal of PCR products. Taq polymerase adds an Adenine at the 3' end of the PCR product and the vector contains a Thymine overhang that it can base pair with = easy insertion of PCR product *due to overhang the plasmid is unable to relegate onto itself therefore all colonies will be white (not intact to produce blue colonies)

How is the issue of introns within eukaryotic DNA solved?

You can create cDNA libraries(complementary DNA libraries): - cDNA is a DNA copy of synthesised mRNA using the viral reverse transcriptase enzyme. RT generates a double stranded DNA fragments which lacks the introns and in turn can be translated within bacteria. = smaller fragment sizes = restriction enzymes aren't required. 1. An oligoT primer complementary base pairs with the polyAtail. 2. Reverse transcriptase is able to synthesise a DNA strand from this primer in the 5' to 3' direction = RNA/DNA hybrid 3. RNaseH degrades RNA in the RNA/DNA hybrid and the reverse transcriptase enzyme is able to loop around and synthesise a double stranded DNA structure. 4. Enzyme cuts the loop = double stranded cDNA

How can you find a gene of interest using antibodies? (protein)

You can use antibodies that bind to the gene product of interest. - A cDNA library is cloned to express the cloned genes in the bacteria. clones expressing proteins are detected by the antibody. This allows for identification of clones that encode the protein of interest. 1. Overlay a membrane to which the proteins will bind to. 2.Incubate the membrane with primary antibody (binds to the protein of interest) 3. Wash the membrane and incubate with the secondary antibody (radiolabeled and binds to the primary antibody) 4. Can autoradiograph using an x-ray film to determine which clones on the master plate contained the gene of interest.

What is a GFP fusion vector and when would you use it?

You would use a GFP fusion vector when you want to measure the expression of a gene. A GFP fusion vector is a green fluorescent protein vector. It is a vector that has an insert that is fused with GFP. As a result the GFP allows visual perception of how much cloned DNA is present within a cell using a computer. - used localise the gene product.

What is lysogenic conversion?

the incorporation of phages into host genome. - this process is efficient as phages are specialised to recognise host cell receptors. - only requires the 2 enzymes (integrase and excision enzymes) as well as homology dependent incorporation at the attP/B sites. *can survive in harsh conditions that eliminate bacteria.