GENE221

Isolation of Hfr strains

Can isolate and purify strains with F integrated, all cells will have F integrated at the same location. Every cell in the population can then transfers chromosomal genes, giving a high frequency of recombination (Hfr strains)

F plasmid IS

As F can integrate at more than 18 sites in the chromosome, at least 18 Hfr strains, each with a characteristic origin (depends on where the IS was) and direction of transfer (depends on orientation of IS on chromosome). Gene a will transfer first, while gene b transfers last

Pre mutagenic lesion to mutation

At least one round of DNA replication is needed to turn a pre-mutagenic lesion (a change to DNA that may lead to a mutation) to a mutation. DNA repair takes place at the pre-mutagenic lesion, but once the mutation is established, it's too late. The DNA repair systems repair the damaged DNA (pre-mutagenic lesions) before it's replicated

Avery-MacLeod-McCarty experiment

Avery-MacLeod-McCarty (1944) recognized the genetic determinant. They subfractionated the smooth cell lysate into macromolecular components (polysaccharides, lipids, RNA, proteins and DNA) and tested these against R strains. The DNA fraction converted a small portion of the R strain to the S strain (virulent). Seen as clear evidence for DNA, but criticized DNA as polymer and not the genetic material

Ames test - improvements

Adding features to the bacterial test strains to create mutations that increase their sensitivity to mutagens. This can be done through a mutation in a gene (rfa) that affects the cell envelope, making the bacteria more permeable to some chemicals. Causing defect in a gene (uvrB) encoding a protein that repairs damaged DNA, therefore increasing freq. of mutations. Another example is containing a plasmid pKM101, which enhances the effectiveness of some mutagens. All these increase the effectiveness of mutagens in causing mutations

Activator and repressor mechanisms

Activator has DNA binding site and allosteric site (effector binding site), binding of effector molecules changes conformation and allows DNA binding site (well positioned) to bind to DNA and activate gene expression A repressor binds to DNA, but in presence of effector molecule changes conformation and unable to bind DNA

M. leprae

Affects myelin Small genome of 3000 genes Half gene is non-coding (reduction) Not many IS elements Lacks DNA proofreading in DNA polymerase III Less PE and PPE Large-scale genomic rearrangements, promoted by repetitive sequences in genome (some found inside pseudogenes, suggesting mobility), which can promote recombination much like IS elements

Aflatoxin mutation system

Aflatoxin reacts to guanine, changing the electron distribution of the structure, resulting in depurination loss of purine base from DNA). The reaction caused an destabilization in the single bond joining guanine to the DNA backbone, resulting in an apurinic site (site without purine) where guanine should be. During DNA synthesis, a base (often adenine) can be inserted opposite the blank, causing changes in base pair sequence. This results in GC to TA mutation

Maintaining high copy number

Copy number maintained at 15 has less than 1 in a million chance of not inheriting plasmid, can rely on this random partition. Low copy number cells rely on specialized molecular machinery e.g. ParM system

Core and accessory genome

Core genome includes chromosomes, essential genes acquired by vertical transmission, carrying out macromolecular synthesis, transcription, growth etc. Accessory genome includes plasmids, transposons, ICEs, lysogenic phages, non-essential genes, adaptive genes acquired by horizontal transmission. Accessory genome contributes to microbial diversity and evolution through gene acquisition. Plasmids, transposons, bacteriophage and integrative and conjugative elements (ICEs) are acquired by HGT and contributes to bacterial evolution (mutation and recombination also contributes but not as much as HGT)

Davis U-tube experiment

Davis U-tube experiment (1951) showed physical contact is required for genetic recombination (not transformation) U shaped tube with fine filter separating the two arms, the pore of the filter didn't allow bacteria to pass, but allowed passage of fluid medium and any dissolved substances. Strain A on one side, strain B on other side and applied pressure/suction to mix the two. Mix for several hours, then plated samples from each side of U-tube. Showed no recombinants from the plated strains

Ames test

Developed by Bruce Ames and others. Ames test uses strains of Salmonella typhimurium bacterium that have a mutation in a gene that is required for the synthesis of histidine. The bacteria is spread on a growth medium, which does not contain histidine. This means any bacteria that can grow are revertants, if the test compound increases the number of revertants, it is likely to be a mutagen

Sanger sequencing

Dideoxyribonucleotide chain termination sequencing sequences DNA up to 1000 base pairs in length. The DNA fragment sequenced is denatured into single strands and incubated in a test tube with the necessary ingredients for DNA synthesis. This includes the DNA template strand, A primer designed for the base-pair with the known 3' end of the template strand, DNA polymerase, four dNTPs and four ddNTPs (dideoxyribonucleotide fluorescent tags) The synthesis of each new strand starts at the 3' end of the primer and continues until a ddNTP happens to be inserted instead of the equivalent dNTP. The incorporated ddNTP prevents further elongation of the strand. Eventually, a set of labeled strands of every possible length is generated, with the color of the tag representing the last nucleotide in the sequence The labeled strands in the mixture are separated by a passage through a gel that allows shorter strands to move through quicker than the longer stands. For DNA sequencing, the gel is in a capillary tube, the small diameter allowing the fluorescent detector to sense the color of each fluorescent tag as the strands come through. The strands differ in length by as little as one nucleotide can be distinguished from each other. The results can be printed out as a spectrogram and the sequence, which is complementary to the template strand, can then be read from bottom to top (shortest to longest)

Diversity of E. coli

Diversity of E. coli due to horizontal acquisition of mobile genetic elements, contributing to evolution shows genomic fluidity (e.g. enterotoxins). Plasmids that provides enterotoxins can convert commensal E. coli into an enterotoxigenic E. coli

Initiation of recombination in eukaryotes

Double strand breaks of one chromatid may be the normal way of initiating recombination in eukaryotes. Recombination generates new combinations of DNA

EMS mutation mechanism

EMS transfers an ethyl base from EMS to the oxygen molecule on guanine, while the rest (methylsulphonate) is released. The oxygen in guanine is important in forming hydrogen bonds for its base pairing with cytosine. Two remaining groups on guanine will bind to thymine. EMS affects thymine the same way, ethyl group binds oxygen, causing thymine to bind to guanine resulting in a TA to CG mutation (less common). DNA polymerase uses the mutated template, reading thymine and pairing it with adenine, causing the AT mutation

Normal mutations - parent to offspring

Each person has around 40 heritable sequence changes (mutations) compared to their parents (parents did not pass this down, was mutated within said person)

S. aureus

Emergence of strain of Staphylococcus aureus resistant to most antibiotics in clinical use, with last resort antibiotic used (vancomycin, used in multiresistant bugs). Isolate of S. aureus found with high-level resistance to vancomycin, with multiresistance conjugative plasmid (with Tn1546 (vanA gene)). Also had other transposons for other antibiotic resistance S. aureus isolated from diabetic patient's foot ulcer. Also isolated was E. faecalis that contained Tn1546 that encoded vancomycin resistance. Found S. aureus strain with multi drug resistance plasmid. Showed transposon transferred via conjugative transposition from E. faecalis to S. aureus, but could not replicate, but jumped into plasmid forming S. aureus strain resistant to all antibiotics

Purpose of historic genetic experiments

Historic experiments shows where science comes from, the experiments have clear examples of how the experiments where thought of and carried out. Fast moving science, giving timeframe to genetics

Merozygote genetics and recombination freqeuncy

In eukaryotes, zygote has copies of each gene from each parent, whereas merozygote genetics in bacteria (due to gradient of transfer/time of entry) recipient strain will receive closer markers rather than the further apart one. Recombination frequency will depend on distance between markers and distance from origin of transfer

Library screening via sequence hybridization

Hybridization screens a known sequence to identify a clone of interest. The known sequence could be a target gene from a related organism, an oligonucleotide designed to a short sequence or a short fragment of the gene. This sequence is labeled (usually radioactively) and used as a probe to identify a clone with a similar DNA sequence Hybridization is easy, cheap, and the gene doesn't need to be expressed, but it needs some sequence information of the gene we are trying to isolate Take library (e.g. cosmid library), transferring bacteria to absorbent membrane, membrane will pick up DNA that is in each clone, treated to obtain ssDNA. Then mixed with radioactive probe, probe designed for sequence, and binds to DNA. Film highlights any clones that has sequence in them. On original plate, select out desired clone, then can amplify desired gene

Transformation HGT

In transformation, the cell ends up lysing, releasing DNA fragments that can be uptaken by recipient cells, the fragments have no way for replicating by themselves (no origin). To inherit the gene, must be recombined into a chromosome, recombination requires a double cross-over, leading to a replacement of the gene that was present in the recipient of the incoming gene (gene replacement), changing the recipient cell permanently (by recombination)

Very early stage

In very early stages, two small parts of the genome is expressed. The pathway is shared up until the decision, with same patterns of very early and early gene expression. Lytic pathway expresses genes for head and tail production, lysogenic pathway has only two specific genes expressed 1. In very early gene expression, host RNA polymerase transcribes the PL that produces N protein and PR that produces Cro (Cro encodes the cro protein, helps the lytic pathway). N encodes an anti-terminator protein that enables transcription path 2 terminators (binding to nut sites), resulting in early gene expression (relates to timing). The protein products for both possible pathways are produced early, transcription goes past termination point after N (to int gene), and past termination point after Cro (to Q gene) Cro is a DNA binding protein that represses transcription (promoting the lytic cycle). CII is a DNA binding protein that activates transcription (promoting lysogenic cycle). CI is DNA binding protein that can activate or repress transcription, activating it's own expression, repressing genes required for lytic cycle and maintaining lysogeny

Regulation of plasmids

Incompatible plasmids share the same regulatory mechanism and are subject to each other's inhibitor, meaning these are unable to co-exist. One will eventually gain the upper hand and eliminate the other

HIV history

It is now widely accepted that the transmission from chimp to human occurred in South Cameroon Changes in Africa, specifically urbanisation and some aspects of colonial rule explain why this zoonosis occured now

Loss of plasmid - metabolic stress

Metabolic stress (plasmids require proteins etc.) imposed by plasmid can slow growth of host so plasmid free cells outgrow plasmid bearing cells. This will result in the lost of the plasmid from population

Purpose of mutations

Mutations provide a way for organisms to evolve, generate diversity, giving insight to how genes and their products work. For a practical purpose, mutations can lead to cancer and other diseases, can result in resistant to antibiotics in microbes, can give animals, plants, microbes improved commercial properties

Mycobacterium

Mycobacterium is a genus of actinobacteria with own family (mycobacteriaceae). The genus includes many important pathogens and over 40 species including M. tuberculosis (TB), M. leprae (leprosy) and M. ulcerans (Buruli ulcer). They have a characteristic thick cell that is hydrophobic and waxy (hard ot antibiotics to penetrate) Mycobacteria are typically aerobic and nonmotile, free-living or parasites and have rods between 0.2 - 0.6 μm wide by 1.0 - 10 μm long. They can be fast growing (colonies within 7 days) or slow growing (colonies after 7 days), and some are very hard to culture. This means genetic manipulation is poor, therefore a lot to be learnt from genome sequencing (providing good insight)

Ordered clone sequencing

Ordered sequencing order a physical map, create a BAC library (BAC was developed for this purpose) with overlap and selecting clones with minimal overlap. Digested using restriction enzyme, analyze different patterns and found overlapping. Sequencing ones with minimal overlap, dividing into subclones to sequence as assemble to create the genome sequence

Insertion of F depends on IS - important (restatement)

Orientation that F inserts into the chromosome depends on orientation of the IS in the chromosome that it recombines with. As F can integrate at more than 17 sites in the chromosome, several Hfr strains, each with a characteristic origin and direction of transfer, are available

Prophage induction

Prophage induction requires CI repressor, Cro components (regulatory proteins), the operator sites on the phage DNA and their promoters, and RNA polymerase (essential for transcription). The switch has two positions: lysogeny (CI ON and Cro OFF) for maintenance and lytic (CI OFF and Cro ON) for induction

Bacteriophage lifecycle

Protein shell containing nucleic acid, protein tube to insert DNA into bacteria. Bacteria cell is infected by bacteriophage, injecting its genetic material into the bacteria cell. The phage genes are expressed inside bacteria to make more phage proteins and genetic material, assembling progeny phages (genetically identical). Lysis of host cell for phages to infect other bacteria cells

RNAP binding

RNAP transcribes DNA into RNA and is from the bacterial host. RNAP will bind to either PR or PRM, never to both. PR activity by RNAP for expression of Cro (strong promoter, doesn't require regulatory protein), PRM activity for expression of CI (weak promoter, needs CI as activator)

Transposons facilitate in (acting as a portable region of homology

Replicon fusions: example, fusion of F with chromosome to form Hfr strains Deletions: homologous recombination between two copies of transposon present in direct orientation. DNA can be lost for can form plasmid (if it has an ori) Inversion: homologous recombination between two copies of transposon present in inverted orientation. Occurs often in bacterial genome Insertions: by transposition or homologous recombination These are the major generators of chromosome level genetic variation

DNA digestion for Genomic library

Restriction digestion generates a wide range of fragments sizes, needing different vectors for the different size fragments. The genes are always on the same fragment. Difficult to obtain complete libraries for large genomes, this is when partial restriction digestion can be used (limiting time or enzyme, generating different size fragments, selecting size by cloning vector). Can also fragment genomic DNA using nebulizer for random shearing of DNA. It is sequence independent shearing that can be calibrated for the desired size. It is fast, simple and reproducible. The DNA is passed through the tube, solution speeds up as forces through constriction and the acceleration stretches DNA and snaps it. The flow rate and constriction determines the size of the sheared DNA

Robin Holiday - proposed

Robin Holliday did a lot of work on fungi (diploid), and used genetic crosses to develop a theoretical model of what is happening at the DNA level. He proposed the Holliday model (1960s) to account for available genetic information.

M. ulcerans

Slow grower Produces mycolactone No pigments Extracellular disease Acquired 174 kb plasmid (pMUM001) with 81 proteins for mycolactone production. Speciation event IS elements acquired through HGT, used as diagnostic markers Reductive evolution, lost 1 Mb Many pseudogenes (carotenoid pigments, ESX secretion systems), caused by point mutations and insertion events Mycolactone is a cytotoxin, immunosuppressant and an injection is sufficient to cause skin lesions via apoptosis (critical virulence factor)

M. tuberculosis

Slow growing No pigments Intracellular disease in humans Acquired 80 regions via HGT More IS elements Less PE and PPE genes Restricted niche and increased pathogenicity Half number of transporters EXS secretion systems, important for virulence for intracellular spear and immunogenicity

Temperate phage

Temperate bacteriophage can enter lysogenic or lytic style, including bacteriophage λ. Differs from lytic bacteriophage (can choose lysogenic lifestyle), instead of replicating and destroying bacteria cell, the phage genome integrates into the bacteria chromosome (usually at a specific site) to become part of the bacteria chromosome (lytic genes repressed). Phage can make choice to excise from chromosome and resume the lytic state. If remaining lysogenic, phage can become a fixed part of bacteria chromosome through mutations. In the lysogenic phase, the phage carries genome and also carry other genes, which create marked effect on the bacteria phenotype

Ames test - Aflatoxin experiment

Test compound aflatoxin used, exposing His strains of Salmonella to different concentrations of aflatoxin. Results were aflatoxin does increase the frequency of mutations in TA100 strain (GC to TA), however aflatoxin had no effect on strains TA1538 and TA1535 (these two required different types of mutations to grow)

Capsule importance

The capsule is important for virulence but is the thing our immune cells recognize. The capsular types can be switched by intraspecifes transformation, with 90 types known, human vaccines use the 5 most common capsules. The switch aids in immune evasion to attack our bodies better, generating vaccine escape strains and antibiotic resistant strains observed in vivo. Interspecies transformation is responsible for the appearance, in penicillin-resistant isolates, of mosaic penicillin binding protein (pbp) genes that encode proteins with reduced affinity for penicillin

Persistent RNAII/DNA hybrid

The formation of a persistent RNA II/DNA hybrid at origin of replication is required for initiation of replication. RNA binds persistently to DNA due to RNA II having an unique secondary structure that helps binding. RNA II/DNA hybrid is cleaved by RNaseH at a point that forms a primer required for initiation of replication

Different frequency of recombination classes

The frequency of different recombinant classes will depend on the distance between each pair of genes

Transformation competence causes

The induction of competence triggers (certain cell population) the release of a fratricide (murein hydrolase) that break down cell walls causing lysis and DNA release from a subfraction of the cell population (5-20%). Some cells dies and aid genetic recombination/assortment in recipient cells. Early in the competence phase the cells make an immunity protein, while later on releases hydrolytic enzyme for a source of DNA

Minimal genome

The minimal genome is the minimal number of genes required to support cellular life independently of a host (or the minimal set of essential cellular genes). This excludes viruses and obligate intracellular parasites/endosymbionts that cannot be cultured outside of their hosts

Benzer - complementation

The production of a wild-type phenotype when two different mutations are combined in a diploid or a heterokaryon. This means two mutants compensate for each other, and together, combine to give a wild-type outcome (functional phage)

Repressor protein

The repressor protein controls the lac operon, lac operon only transcribed in the presence of lactose. Operon is a series in genes, transcribed as a single mRNA and translated into multiple protein products (promoters controlling multiple genes). Lac operon contains lacZ (enzyme breaking down lactose), lacY (permease that brings lactose into cell) and lacA (transacetylase, enchance these processes). LacI is the repressor gene, made as single mRNA translated into lacI protein (repress lac operon, until an inducer (e.g. lactose) is present)

Transduction HGT

Transduction: Bacteria virus infects cell, destroying chromosomes and making more viruses. The viruses incorporate genome into head, transducing bacterium have a head full mechanism (their own DNA and other), with bacteriophages with own DNA and some with DNA fragment from host cell. The bacteria DNA can be incorporated via recombination to replace preexisting genes

Jacques Monod and Francois Jacob findings

The synthesis of enzymes in bacteria follows a double genetic control. Regulator and operator genes (functionally specialized genetic determinants) control the rate of protein synthesis through the intermediacy of cytoplasmic components or repressors. The repressors can be inactivated (induction) or activated (repression) by certain specific metabolites. This system of regulation appears to operate directly at the level of the synthesis by the gene of a short lived intermediate (mRNA) which associates with ribosomes (protein synthesis)

Prokaryotic genome dynamics

There is a dynamic prokaryotic genome, with a core gene pool (chromosomes containing ribosomes, cell envelope, key metabolic pathways, DNA replication, nucleotid turnover), that can overlap with a flexible gene pool (genomic islands, genomic islets, phages, plasmids, integrons, transposons). Best adapted will survive, adapted by gaining variations of properties via HGT

3 Types of HGT

Transformation is the uptake of naked DNA into a competent recipient cell (not all cells can be transformed), conjugation is the DNA transfer through cell-to-cell contact mediated by a plasmid or transposon (accessory genetic elements) and transduction is the DNA transfer mediated by a bacteriophage (accessory element). All these methods can be used to transfer chromosomal DNA

Transformation occurance

Transformation occurs naturally in some bacteria (e.g. Strep. Pneumonia, Haemophilus influenza, Bacillus subtilis, Neisseria spp. And Acinetobacter, these are pathogens) but not in others (e.g. E. coli). Its dependent on specialized cell state called competence, which occurs in a small proportion of the culture at a specific growth stage for short period

Griffith's experiment - transformation discovery

Transformation was discovered by Griffith's (1928), streptococcus pneumoniae caused disease, needed to be a smooth (S) strain (with a polysaccharide capsule). Mutant rough (R) strain (lacking polysaccharide capsule) would not cause disease. But heat killed S strain mixed with viable R strain would cause disease due to transformation, could recover S strain from dead mouse. Griffith's hypothesized a transforming principle that was transformed from the lysate (S strain) to the R strain

Transduced markers

Two crossovers are required to incorporate transduced DNA into recipient. Two markers co-transduced means they are relatively close together, the closer together the less likely to be separated by recombination. The further apart the two co-transduced markers are the more likely they are to be separated by recombination

Chance of recombination

Two markers on the same chromosome, long distance apart, greater chances of recombination between the two compared to two markers relatively close. Recombination occurs at random along the chromosome

Repair of UV light damage

UV light damage often causes two adjacent thymines to become covalently bonded (cross linked), causing problems in base pairing properly during DNA replication. The error-prone DNA polymerase can replicate past these but can incorporate the wrong base. To repair the damage, there are multiple mechanisms such as excision repair and the use of photolyase enzyme. This enzyme uses energy from white light to convert photodimers back to pyrimidines

Watson and Crick model

Watson and crick discovered structure of DNA and how the genetic code could be incorporated in DNA

Conjugative transfer F

When F is integrated into chromosome, essentially converts chromosome into giant plasmid, able to undergo conjugative transfer. RCR initiated by nick at OriT, replication is unidirectional with one side of OriT starts transfer and other side is last to be transferred, with chromosome inbetween. Will have a population where F is integrated at a number of different sites, each able to transfer chromosomal genes. Mating bridge is usually stable enough for whole F plasmid to transfer, but chromosome takes longer, usually get a fragment of DNA transferred (fragment will only have part of F, recipient does not become F+)

When cloning a gene

When cloning a gene, need to decide what vector to use, based on the ease of use and the size of DNA fragments (e.g. plasmids, fosmids and artificial chromosomes). Also dictated by what hosts are use, which are based on ease of use and correct production of protein products (e.g. bacteria like E. coli, yeast like S. cerevisiase and mammalian cells)

Discovery of transduction

Transduction discovered by Lederberg and Zinder (1951), using Salmonella typhimurium. Zinder did an U tube experiment, even with filter, had recombinants transferred from strain A to strain B. This showed transfer didn't require cell to cell contact, postulated filterable agent was bacteriophage P22

Accuracy of DNA polymerase and mismatch system

Inherent accuracy of DNA polymerase is ~1 in 10^5, proofreading corrects ~99%, mismatch repair corrects ~99% of remainder. The overall spontaneous mistakes is ~1 per 10^9 basepairs

Genetic transfer HGT

It is common for genetic exchange mechanisms to be unidirectional, from the donor (male) to a recipient cell (mother), only part of the donor genome is transferred to the recipient, changing recipient cell genome. In most cases, transferred DNA consists of linear fragments that can't be replicated autonomously. Consequently, for transferred genes to be stably inherited by recipient cells, they must be recombined into the recipient chromosome, or it gets lost

pBR322

pBR322 (E. coli cloning vector) plasmid lost cer (didn't understand importance at that time) causing these cloning vectors to be unstable (due to no cer)

Time of entry

"Time of Entry" of each marker can be used to construct a genetic map, using "minutes" as the measure of distance. Genetic map of E. coli, expressed in units of minutes (100 units long, F plasmid takes 100 minutes to transfer the entire chromosome Time of entry mapping is crude, gives estimation, hard to determine order between two close genes and hard to determine actual distance between the two genes

Comparative genomics

1. Determining genome sequences of bacterium: via sanger sequencing and next generation sequencing. Sequencing followed Moore's law, sequencing getting cheaper and cheaper, until new methods were made (BOOM SO CHEAP). Think illumina sequencing 2. Genome annotation: Take input DNA sequence, apply computer tools to find protein coding regions (ATG until stop codon). Use comparative search (e.g. RAST) to find similar genes in databases. If none found, labeled a hypothetical protein, but can be used to predict protein domains. Its like copying, hope data is done well. RefSeq is more trustable BASys (bacterial annotation system) is a good start but human annotations yield higher quality data 3. Genome data interpretation: People are needed to interpret the data and compare this to the biology of the organism. Genes present in genome can determine the biology of that organism, conversely the biology or niche inhabited by a bacterium can determine its genome content. E.g. Bacillus subtilis has over 200 genes for amino acid biosynthesis and is free-living in soil. Treponema pallidum (syphilis) has no known amino acid biosynthesis genes as it gets these from host (obligate parasite) Can use metabolic modeling, sets of metabolic genes can be analyzed and used to determine what metabolism a bacterium has (still just a prediction, but a good starting point). E.g. whipplei causes GI infection, associated with host cells, computer modeling of genome sequence data used to define media to grow previously unculturable bacteria (had to be grown in hosts) The accessory genome can be active/inactive and are a highly flexible part of a bacterial genome that drives rapid evolution 4. Comparative genomics is the comparison of genome structure and function across different biological species or strains. Comparison can aid in identifying differences. The regions of difference can help identify genes involved in particular process (e.g. find pathogenicity) Closer comparison to spot smaller changes. Distant comparison can see larger changes that happened over millions of years Useful for slow-growing or unculturable bacteria, due to no need to grow, only comparing Genes can switch around in order, still conserved but different orders, changes in genome

Variations of vectors

1. Expression vectors express the cloned gene produce in the cell, vector includes a promoter in front of multiple cloning site. The expression of gene can be controlled by Ptac promoter (commercial hybrid promoter), which is induced by IPTG. TO produce large amounts of the cloned gene, use strong promoters like T7 promoter 2. Expressed protein can be purified via added tags. Produced protein can pass through a column, the tag binds (reversibly) to column to isolate the protein. The tag might interfere with protein function, can be cleaved via addition of cleavage site (e.g. thrombin cleavage site) to cleave out the tag after purification 3. pGEX has Amp resistance, lac repressor, polylinker (DNA insertion), Ptac promoter and GST gene. Ptac can be induced by IPTG. GST (tag) binds to a GSH column, allowing purification. Prescission protease can be used to purify protein with out GST tag 4. GFP fusion vector, expressing protein along with GFP expression to visualize where protein is being made 5. Clone a promoter of Gene X (in front of a reporter gene) to see if promoter is activated (activated will produce reporter gene) 6. Shuttle vector has mammalian promoter (to make proteins in mammalian cell), SV40 origin (mammalian virus, allows replication within mammalian cells), and selection markers. It is a vector constructed to propagate in two different host species (google) 7. Gateway cloning is based on λrecombination system. Cloned into an entry vector first and then easily sub cloned into different clones for different purposes. There are no restriction enzymes or ligation used 8. Polymerase chain reaction (in vitro) amplifies specific regions of DNA. Adding sequences to the ends of PCR primers can make 'sticky' ends of PCR product sequences, ends correspond to restriction enzyme sites 9. TA cloning: Taq DNA polymerase (PCR) adds on an adenine at the 3' end of the PCR product. The plasmid vector has a 3' thymine overhand to insert the PCR product (and can't relegate) 10. Gibson assembly developed by Dr. Daniel Gibson (2009), rapidly assemble multiple DNA fragments in one reaction (cloning lots of DNA sequences in one plasmid). Design PCR products with specific sequences on the ends, with exonuclease to chew back the 5' ends (allows DNA to base pair) allowing DNA polymerase to extend and DNA ligase seals 11. If sequence is known, can buy synthetic DNA to insert into plasmid

PaJaMo experiment

1. Inducers thought to lead to an increased in β-gal activated pre-existing enzyme. Radioactive labeled amino acids added before or after induction, radioactivity accumulated in newly synthesized β-gal enzymes (fast, up to 3 mins). This showed induction when lactose is added leads to new synthesis, not to activation of pre-existing enzymes. When the inducer is removed, synthesis stops (repressed) 2. Lactose broken by β-gal, IPTG (different but similar to lactose) could act as an effector and induce enzyme synthesis. There are molecules broken down by enzymes, with a different set of molecules inducing enzyme synthesis. This shows the enzyme doesn't directly sense the inducer, another molecule (lacI) that senses the repressor Inducers differ from substrates, the inducer is distinct from the enzyme. Lactose is an inducer (allolactose) and a substrate. Other molecules that are not substrates act as inducers (IPTG 3. Induction of permease leads to co-induction of β-gal (coordinately controlled). Mutations in these genes (in mapping experiments) showed that they were separate genes, and showed lacZ, lacY and lacA are closely linked. This is concept of operon and mRNA (later verified experimentally) 4. Did mutagenesis, selecting for mutants that were on for β-gal activity. A lacI mutant (lacI-) could no longer repress, causing lacZ to be constitutively on for the production of enzymes, found to be close to structural genes (close to lacZYA) 5. PaJaMo wanted to generate other ways of putting two copies of different alleles/mutations into the same strain. In this way, they can see if a mutation was dominant over another. But bacteria have one chromosome, they wanted a way to generate diploid cells, and did it by F' plasmids where plasmid has picked up a piece of the E. coli chromosome and carry it (old school way of cloning). Chromosome has genes, F' plasmid has different genes (with different mutations) and how do these different genes work together. Found that lacI+ (wildtype) worked on both copies to repress expression, and still be induced with presence of inducer, showed mutation was an inactive gene. LacI could diffuse in cells and work on two copies of the operon (trans-acting), had a diffusible signalling factor 6. Found lacIs mutant (super repressor) did not express lacZ, lacY or lacA, even in the presence of inducers. LacIs was mapped to the lacI gene, showed mutations in repressor that lead to repressions all the time (mutated sites that inducer binds to, lost ability to sense signal). LacIs is trans-dominant over lacI+ and lacI-, even with lacI+ and lacI- in the cell, because lacIs will go and repress. LacI protein was able to sense effector directly 6. Found lacIs mutant (super repressor) did not express lacZ, lacY or lacA, even in the presence of inducers. LacIs was mapped to the lacI gene, showed mutations in repressor that lead to repressions all the time (mutated sites that inducer binds to, lost ability to sense signal). LacIs is trans-dominant over lacI+ and lacI-, even with lacI+ and lacI- in the cell, because lacIs will go and repress. LacI protein was able to sense effector directly 7. It was hypothesized that the repressor interacted with the operator located near beginning of genes it controlled. They searched for constitutive mutants in cell containing two copies of repressor (i.e. hard for repressor to mutate) They had two copies of the lacI gene in the cell when they do the mutagenesis. Even if mutation is in one, there is still another copy, hard to have mutants in lacI gene. Now they can start to find these rarer mutations, found OC mutants (operator constitutive). OC mutants constitutively switch on the lac operon (genes always being expressed). Repressor can bind to O+ but can't bind to OC (even in absence of inducer). OC is cis-dominant (upstream of genes its controlling) over lacI+ and lacIs, mapped between lacI and lacZ

Examples of capsule (and changes)

1. Looked at 240 isolates of PMEN1 multidrug resistant lineage of Streptococcus pneumonia, could distinguish large polymorphisms that arose through HGT. Detected 700 transformation events where genes encoding major antigens frequently affected, including 10 capsule switching events leading to a vaccine escape strain. This strain emerged in USA and caused widespread disease. Also detected multiple drug resistances (fluoroquinolones, rifampicin, macrolides) on multiple occasions. Single lineage of resistant bacteria evolved new codes through transformation over a short period 2. Streptococcus flesh eating bacteria had virulence acquisition and HGT of region encoding two virulence factors. Occurred in a single cell in 1983, leading to 600 million cases of pharyngitis and necrotizing fasciitis (flesh eating disease). Shows power of one HGT event, nasty. 3. Streptococcus pneumonia transformation allows maximum plasticity potential, making it able to continually adapt to environment, giving advantage in constant struggle with us and them

Steps for making plasmid clone in E. coli

1. Restriction: Plasmid vector and DNA fragment of interest must be digested with restriction enzyme to produce sticky ends. Restriction enzyme recognizes a specific site and cleaves within that site to create sticky ends, cutting DNA that will be inserted and plasmid vector (same enzyme for these sites) Restriction enzymes are bought commercially, named after bacteria they come from, all with different recognition sequences 2. Ligation: DNA fragment of interest must be ligated into plasmid vector, DNA ligase enzyme covalently joins DNA (completes phosphodiester bond) to produce recombinant plasmid 3. Transformation: Ligated plasmid vector must be transformed into a bacterial host (usually E. coli) to make many copies of the gene. In transformation, the cells are made competent to take up DNA by treating with ice cold CaCl2 (neutralize negative membrane charge) then heat-shocked at 42 degrees (causes pores in membrane, driving DNA into cell). This method is relatively inefficient (~10%). In electroporation the cells are made competent to take up DNA by treatment with an electric field (often 2500 V), electric pulse causes DNA uptake The ligation mix will have uncut vector, unligated insert, cut vector and vector plus insert. Transformation will generate uncut only (grows on Amp plates, blue), insert only (can't grow), cut only (can't grow), correct with insert in vector (Grows on Amp, white) or empty (can't grow) populations of E. coli cells 4. Selection: Bacterial colonies carry plasmid with DNA fragment of interest must be selected from those that do not Select via antibiotic resistance inactivation. pBR322 has beta lactamase gene (cleaves ampicillin) and Tet gene (pumps tetracycline antibiotic out of cell for resistance). Insertion in BamHI (in Tet sequence) will disrupt Tet resistance but retain Amp resistance. Put vectors in cell and plate cells onto Amp plates to select for bacteria with plasmids (with Amp resistance). From the Amp plate, take colonies and plate on Amp AND Tet plates to identify tetracycline sensitive colonies. The colonies are derived from a single bacterium containing plasmid and insert, meaning it is cloned gene Select via color change. pUC18/19 vector has Amp resistance with a multiple cloning site (polylinker) on the lacZα (encodes α -peptide, not whole otherwise vector is too large). α-peptide is required to make functional β-gal, now able to cleave X-gal (artificial substrate of β-gal) to form blue cells. If no α-peptide is present, β-gal not functional, cannot cleave X-gal and remains white. This is α complementation, rest of β-gal is produced in the cell, just needs α-peptide from vector

Early stage

2. In early stage, Cro and CII protein has both been produced, CII is the deciding protein (sensing). Host proteases degrade CII, healthy cells produce high levels of protease (actively growing cells, CII gets degraded), meaning Cro protein causes lytic cycle

Lytic pathway mechanism

3a. In the late lytic pathway, CII is degraded, Cro represses expression of CI and all early genes (not needed). O and P genes allow replication of phage genome, Q is an anti-terminator allowing expression of late lytic genes (head, tail and lysis genes), transcription and translation of all viral particles

Lysogenic pathway mechanism

3b. In late lysogenic pathway (starved cells produce less protease, CII intact and stable). CII turns on promoters for int (Pint) and CI (PRE) genes, int results in integration of λ into host chromosome, CI 'repressor' represses all other phage genes (silence entire virus genome) and activates it's own expression (to make sure virus genome is silenced). PRE) transcribes Cro gene but antisense, which can help reduce Cro levels in cell (binding between mRNA) Lysogeny is maintained as a prophage by CI, keeping phage genome silent in bacterial chromosome until induction. This means viral genome is stably inherited

Conjugative plasmid components

A general conjugative plasmid contains four gene modules: replication (related to vertical transfer), stability (ensuring stable propagation), adaptation (provide adaptive traits like antibiotic resistance), propagation (related to HGT, conjugation)

Summary L30

A WGD event occurred in the Saccharomyces lineage after the divergence from K. waltii and S. cerevisiae is therefore derived from a tetraploid. WGD may have occurred by endo-duplication (auto-polyploidy) or fusion of two close relatives (allo-polyploidy) Following WGD duplication in most cases one duplicate copy of each gene is lost. Occasionally both copies of a gene are retained as paralogs. In some cases the two gene copies diverge, one gene copy remains conservative while the other paralog is innovative, evolving faster (ohno's idea) In the Saccharomycete yeasts a large number of rearrangement events have shuffled the duplicated chromosomes after the WGD

Clone, Cloning vector, restriction enzyme, DNA ligase

A clone is a large number of identical cells or molecules with a single ancestral cell or molecule. DNA cloning vector is a carrier DNA molecules that allows attached DNA to be replicated in a cell. Restriction enzyme is an enzyme that recognizes and cleaves DNA at specific sequences. DNA ligase is an enzyme that covalently link DNA molecules

Genome

A genome is the total complement of genetic information of a cell or a virus. Genomics is the discipline involving the mapping, sequencing and analysis of genomes Genome is dsDNA, usually circular (some are linear e.g. Streptomyces sp.), usually one chromosome (but can be multiple) with sizes ranging from 0.112 Mb to 14.7 Mb. Genomes can contain plasmids

Robin Holiday - intermediate structure

A key intermediate Holliday structure is proposed to arise during the recombination process after strand exchange and branch migration. Two chromosomes covalently joined together by a junction has been observed experimentally using electron microscopy.

Library screening

A library can contain thousands of different clones/sequences. To find something, needs information about gene/protein. Information includes phenotype (function), DNA (sequence) and protein (what it encodes)

Mismatch repair system

A protein detects non-matched pairs of bases in DNA molecules and determines which base is wrong and another protein removes the wrong base. This system distinguishes the original (correct) and new (incorrect) strands of DNA molecule. DNA inside E. coli is chemically modified by methylation, a chemical modification of bases (adenines) in the DNA, it does not affect pairing of bases with their partner thymine but methylated strand shows the original strand

Phage virus

A virus is an infectious agent that must grow or reproduce inside a host cell, considered obligate intracellular parasites. They are composed of nucleic acid genetic material and protein coat (some also contain lipids in their coats)

AIDS is caused by HIV

AIDS is a newly emerged distinct disease (acquired immunodeficiency). It therefore requires a new cause (such as a new virus) The symptoms of AIDS respond to antiretroviral therapy (suggesting it is a retrovirus). HIV (a retrovirus) is present in AIDS patients (the antigen and antibodies can be detected). The disease is transmitted between sexually active adults suggesting an infectious organism. It is also transmitted in blood (haemophiliacs and intravenous drug users). Treatment failure is associated with sequence changes in the HIV that make the virus resistant to the antiviral. There is an animal reservoir (wild chimps) that have a related virus (SIV) and that live in the area where AIDS first arose and from whom the virus could easily have been transmitted (bushmeat) Calculation of the most recent common ancestor (MRCA) of the M group HIV (the pandemic strain) fit well with the idea that the virus was transmitted from chimps (SIV) to man (HIV) in the early 20th century giving rise to a newly emerged disease (AIDS). From the perspective of our lectures we have emphasised that comparative genetics and genomics is critical for this reasoning. The error prone replication system of HIV and the persistence of HIV have resulted in the rapid evolution of HIV

Aim of gene cloning

Aim of gene cloning is to obtain isolated and purified copies of specific gene sequences (via in vitro/test tube in vivo/in cell) to study the gene, sequence it or express it. Cloned genes allows determination of sequences of gene and protein (if expressed), obtain leads into functions of the gene, and manipulate the gene (mutate, insert gene into cells, make large amounts of protein). E.g. making large amounts of insulin for medical use for diabetics, produce human growth hormone, producing EPO (increases RBC numbers) for athletes, gene resistance for plants, genes used to alter bacteria for cleaning up toxic waste

Amount transduced

Amount of DNA that can be transduced limited by how much DNA can be packed in a phage head. E.g. in E. coli, phage P1 can carry ~90 kb of DNA (~4700 kb in the whole E. coli genome) Phage host specificity determined by cell-surface receptors (phage requires specific receptors to bind to) to transduce genes into same species, means host range is limited

Mutagen

An agent capable of increasing mutation rate (from the natural rate). Can cause mutations in organisms including people. Mutations are associated with hereditary diseases and cancer

Essential gene

An essential gene is essential for bacterial growth or survival, therefore a mutation of an essential gene results in no growth. Some genes will always be essential (DNA replication, transcription, translation etc.)

BACs

BACs (bacterial artificial chromosomes) have low copy number origin of replication (1 - 2 per cell), contains genes to stabilize plasmid, antibiotic resistance marker, and can carry large DNA inserts (75 - 300kb). BACs enable packaging of much larger DNA inserts from large genomes such as humans BACs are based upon F plasmid of E. coli, containing parA,B,C genes to stabilize plasmid. Its oriS and rep E is used for replication/autoregulation. BACs contain selective marker (e.g. chloramphenicol) that may include color selection (e.g. lacZα). BACs were developed for genomic cloning (human genome project). BACs are transformed by transformation and electroporation (like plasmid)

Resistant bacteria

Bacteria being resistant has the cost where it is less able to take up iron (an essential nutrient), impairing its growth. Bacteria that can uptake iron (therefore can be infected by bacteriophages) grow faster than the resistant mutant bacteria

Bacteria and induction

Bacteria is exposed to different environments/nutrients, (selection leading to evolution of efficient systems) with different genes able to bring in different carbon/nutrient sources for metabolism. In enzyme induction, bacteria only produce enzymes required for growth on a particular substrate in the presence of this molecule/inducer (inefficient if producing all enzymes for all substrates, even if not in environment) Bacteria induction is controlled by negative and positive regulation (transcriptional regulation, controlling mRNA produced to control amount of protein/enzymes present). These are found through mutants. Repressors and activators need to sense effector molecules (e.g. lactose) to alter expression accordingly (of enzymes)

λ phage att

Bacteria phage λ integrate into attachment site (short region (15 - 20 bps) of homology within the chromosome), via homologous recombination creating hybrid attachment sites (attL - attachment site on the left, attR - attachment site on the right). Recombination between these attachment sites (at a particular site in the middle) is catalyzed by λ integrase (site specific recombination enzyme), leaving a structure where λ is part of the bacteria chromosome. The phage like genes are shut down and phage propagates along (lysogenic) This process can be reversed using λ integrase and excisionase, pushing recombination direction towards excision, instead of integration

Bacterial recombination

Bacteria recombination and genetics differ as it results in one genome (endogenote, DNA from F= recipient, this is the complete genome) with a part of another genome (exogenote, part of DNA from Hfr donor recipient). This is a merozygote, this transient state is partially diploid, but has a single copy for the whole. One recombination event of circular and linear DNA forms a nonviable linear strand (unable to replicate in bacteria, needs to be circular to replicate). Two recombination of circular (one cross over either side of the linear gene to be incorporated) and linear DNA forms a viable plasmid and a nonviable fragment (reciprocal product of recombination). Only one product of recombination survives, requires an even number of cross overs to incorporated transferred DNA, odd numbers end up with linear unviable product

Bacteria basics

Bacteria replicate clonally without the need for mating/missing of gametes (although some can have mixing of DNA from different individuals. Most bacteria have one chromosome (circular) per cell, with few exceptions having more than one chromosome and/or with linear chromosomes

Bacterial transposons

Bacterial transposons (transposable genetic elements) include insertions sequences, composite transposons, non-composite transposons and conjugative transposons

Bacteriophage transduction

Bacteriophage acts as passive carrier of bacterial DNA, which is injected into recipient cell and incorporated by recombination. Any bacterial gene can be transduced at low frequency called generalized transduction. Amount of DNA that can be transferred limited by size of phage head, ~90 kb for P1 (used for transduction in E. coli). Generalized transduction very useful for high resolution mapping by analyzing frequency of co-transduction. Transducing mutations is very useful for strain construction

Phage lysogenic conversion

Bacteriophage lysogenic conversion is efficient, no cell to cell contact required, and incorporation is not homology dependent. Bacteriophage can act at population level and survive harsh conditions that eliminate bacteria

Benzer - analysis

Bacteriophage plaques are holes (clear patches) in a lawn of bacteria where the bacteria have been killed by the phage Benzer analyzed the rII genes (two specific genes) of bacteriophage T4 (has ~150 genes). The rII mutant phage, unlike wild-type T4, cannot form plaques (grow) on E. coli strain K12 (λ) but forms larger plaques on E. coli strain B. Benzer isolated over 2400 rII mutants, all with the same phenotype. He wanted to know how many genes were mutated in the rII mutants to cause this phenotype

Bacteriophages and eukaryotic viruses

Bacteriophages and eukaryotic viruses are genetically simple (10 - 100 genes), with single or double stranded DNA/RNA genomes. They usually range from structurally simple to complex, relying on the host cell to provide nutrients and chemicals needed to replicate. They can kill the host cell (lytic) or integrate into the host genome (lysogenic)

Use of bacteriophage in Benzer's experiments

Bacteriophages were suitable for this analysis because Benzer was able to screen a huge number of T4 plaques to identify a large number of mutants. He was able to carry out a huge number of genetic crosses simple in test-tubes in the complementation and recombination methods Benzer had a powerful screening system allowing him to select recombinants rather than trying to identify by eye one recombinant in the background of a million non-recombinant phages

Basic plasmid vector

Basic plasmid vector has origin of replication, selectable marker and multiple cloning site

Transposon mutagenesis to determine essential genes and minimal genome

Beat Christen used Caulobacter crescentus, found insertions every 8 bp. C. crescentus is swarmer cell and permanently attaches to surfaces, dividing in stalked state, budding off. Christen used transposon Tn5 (jumps into DNA at random) to disrupt sequence, but designed Tn5 with promoter reading out from the edge Transposon mutagenesis, introduce transposon on bacteriophage. Plate selection and select for marker. Carry out a random primed PCR to amplify Tn5 junctions, resulting in a pool of products flanking where the Tn5 inserts and sequence those. Christen did one illumine sequencing run generating 120 million DNA clusters with 12 giga bp sequenced Christen mapped 428,735 unique Tn5 insertions in Caulobacter crescentus. 80% of genome showed no gap that was greater than 50 bp, average gap size 7.65 bp (shows fine resolution mapping) Longer the gene, more insertions Can have some mutations that cause growth defect He found 29 tRNA that are essential for translation

Benzer - conclusion

Benzer concluded from the rII analysis that genetic maps are linear, hence the genes themselves are likely to be linear structures. Most mutations are changes in only one mutable site, some mutations caused the deletion of one or more mutable site (one base pair mutation), some sites were particularly prone to mutations (hotspot sites). He could relate these findings to his rIIa and rIIb classes of mutants

Benzer - complementation system

Benzer took pairs of rII mutants to infect E. coli K12 (λ), which individual mutants could not infect/grow. With mutations in different locations, he thought they could compensate for the other pairs defects. Possible for the pair to have mutations in the same gene (no complementation, no cell lysis, no release of progeny phage), or the mutations in the pair are in different genes (complementation, cell lysis and release of progeny phage)

Minimal genome - synthesis

Bottom up approach Synthesize the genome, transplant the genome (into cells devoid of DNA) then optimize and minimize the genome. This estimates the minimal genome of 473 genes

DNA extracted from E. coli

Broth of bacteria is resuspend cells in lysozyme (breaks open cells), sodium dodecyl sulphate is added to form complexes with protein and then the sodium hydroxide causes DNA to become ssDNA. Neutralization buffer precipitates genomic DNA, protein and other cellular components, leaving the plasmid DNA in solution. The supernatant is added and spun through column with a silica surface to bind DNA. This is washed with water to remove DNA from silica surface

CI

CI is dimeric via C-terminal interactions in ~95% of lysogenic cells. CI dimers use their N-terminal domain to bind DNA (operator sites). Each OR site can bind one CI dimer along one site of the DNA helix CI dimers lean towards each other (co-orperativity). CI binding at OR2 can still bind RNAP since RNAP interacts with N-terminus of dimer Examples: In negative control, CI at OR1 turns off Cro gene by preventing RNAP from binding to Cro promoter (exclusion). In positive control, CI at OR2 helps RNAP bind and begin transcription of CI gene (10x upregulation) CI increases RNAP affinity for PRM by providing protein-protein interactions in addition to the protein-DNA interactions CI affinity: OR1/OR2>OR3 CI bound to OR1 causes no CI activation (not close enough) and Cro off due to exclusion CI bound to OR3 causes CI off due to RNAP exclusion and Cro on (translation along that direction). Never find this situation, due to affinities

Chromosome recombination systems

Chromosomes has xerC site specific recombination system because chromosomes during replications are identical DNA molecules, can undergo recombination forming one big molecule. xerC avoids this, recombinant chromosome can cause problems during division

Chytrids

Chytrids are any various usually aquatic and often parasitic or saprophytic fungi in the division Chytridiomycota having flagellated gametes A particular chytrid, Batrachochytrium dendrobatidis, is causing a massive pandemic, resulting in the extinction of many species of amphibians This disease, chytridiomycosis, is another example of a newly emerged disease Analysis of the complete genome sequence of the chytrid shows that: Bd is diploid (there are heterozygous sites revealed by clone sequencing or PCR sequencing That the same heterozygous sites occur in strains from all over the world, the Bd pandemic is due to a clone That Bd is asexual (otherwise the heterozygosities would segregate into homozygosities at each meiosis) It is possible that the chytrid is a hybrid between two related species/strains. This would explain the sudden appearance, the asexual (sterile) state

ColE1 killing plasmid free cells

ColE1 excretes colicin, the cells that don't produce an immunity protein will die (own cells produce immunity protein, plasmid free cells don't)

ColE1 plasmid replication

ColE1 has less coding capacity, needs to be gene efficient, has oriT and mob gene to hitch a ride through tra pore. Plasma replication controlled by oriV (replication inside the cell) and rom (RNA involved). Initiation of replication in ColE1 requires RNA II (relatively large primer, 600 nts) to bind to ColE1 oriV. RNA II is made by transcript upstream of the origin. RNA I is transcribed off the same region of DNA as RNA II but transcribed in the opposite direction (anti-sense RNA). Rom protein present facilitates the binding of RNA I (relatively smaller, 108 nts) to RNA II (complementary and can pair), which prevents RNA II binding to oriV (therefore replication stops). The concentration of RNA I and rom protein are critical for replication. Low concentration of plasmid means low concentration of RNA I and rom, leads to plasmid replication. Higher concentration of plasmid means accumulation of RNA I and rom, preventing replication. Rom protein is the inhibitor, preventing RNA II binding to oriV for replication Normal transcription RNA and DNA come off readily (as a comparison)

ColE1 plasmid

ColE1 is smaller plasmid than F plasmid, used to make cloning vector, with a high copy number. ColE1 has oriV (origin of vegetative plasma replication, copy number maintained at 15), imm (immunity to Colicin E1), ColE1 (synthesis of Colicin E1, a bacteriocin), mob (nuclease required for bolisation acting at oriT, makes the nick), rom/rop (protein for copy number control), oriT (origin of conjugative transfer, site of nick) and cer (sequence for site-specific recombination to resolve multimers

ColE1 recombination systems

ColE1 uses host-encoded recombination systems, doesn't encode its own, using chromosomal recombinase enzyme (xerC). xerC acts on cer (35 bp sequence) specific DNA sequences just recognized by the site-specific recombinase enzyme. Contrasted with recA that recognized most sequences with lower affinity, site-specific recognizes specific sequences with high affinity. xerC moves DNA around until cer sites are next to each other, promoting recombination at high frequency to make two monomers from a dimer (cis acting/on own DNA molecule). Trans acting is on two other DNA molecules by diffusing freely. These are essential plasmid adaptations

Transformation competence phase

Competence occurs in stationary phase, when running out of nutrients, at a high cell density. Streptococcus pneumonia is competence dependent on extracellular concentration of a secreted small peptide, which each cell releases, high concentration means more cells in certain space (quorum sensing). Reaching a certain quorum (concentration of peptide), sensed by cell to activate gene for DNA transport. Competence can be induced by stresses including antibiotic treatment. It allows sensing of population density to know when more potential donor cells are available

Library screening via complementation

Complementation is the restoration of a mutant phenotype by a separate copy of the gene. Making a plasmid library, transform plasmid DNA into a lacking phenotype E. coli, plate on selective medium and the clones carrying the gene (phenotype) should grow. This requires an expressed and functional gene

Complementation

Complementation lets you tell how many genes there are and if mutations were in the same or different gene while recombination allows the mapping of relative locations of mutations

Composite transposons

Composite transposons (e.g. Tn5 kanR, Tn10 tetR) consists of two identical IS elements on each side of a central region carrying other genes (e.g. antibiotic resistance genes). IS elements supply transposase and ITR recognition signals Often only one IS module makes active transposase

Conditionally essential gene

Conditionally essential gene is essential depending on what conditions are used for growth Essential but redundant genes are some genes that can do the same essential function but deletion of either gene is tolerated

Conjugation in organisms

Conjugation is demonstrated between diverse bacteria (gram + and -), between F and yeast, between agrobacterium (can spread plant disease) and plants, fungi, yeast and HeLa cells (system also used for GM of plants), and between pathogen Bartonella hensalae and human cells (Mating pore VirB/VirD4 can mediate conjugative DNA transfer and transfer proteins into eukaryotic hosts, occurring naturally in infections, could possible use system to deliver vaccines (knock out virulence first))

Conjugation, recombination for linear DNA fragment

Conjugation is fragile, the whole chromosome is rarely transferred, mating bridge often disrupted due to motion/stress in culture. Linear fragment is unable to be replicated, can only be incorporated by homologous recombination or it is lost (replaces gene). Several different sites F can integrate (different oriT)

Conjugation HGT

Conjugation requires F factor plasmid in E. coli, F factor has the ability to integrate into the chromosome to transfer the chromosome as a single strand into the recipient. Usually a part of the genome is transferred, has to be incorporated to replicate. Recipient cell is permanently changed. The recipient is permanently altered by the acquisition of the gene, where the incoming gene is inserted or by recombination and thus replacing old gene in recipient

Conjugative transposition

Conjugative transposition (like conjugative plasmid) is able to excise (recombination event) from the genome, replicate (RCR) and transpose from one cell to another via conjugative intermediate. It inserts randomly into recipient and reinserts into the donor genome. This is important in dissemination (distribution) of antibiotic resistance and virulence, especially in gram positive bacteria

pUC19

Contains: ORI (allows replication), Amp resistance gene, lacZ gene containing alpha complementing portion Target DNA cloned into lacZ gene at polylinker cloning site, disrupting lacZ gene (prevents complementation) Using indicator media, we can find recombinant plasmids where lacZ gene is inactivated (lacZ-), and non-recombinant are lacZ+ DNA to be cloned is part of pNRE1 plasmid, carrying gene contained within 1.3kb BamHI fragment

Cosmid

Cosmid is a plasmid with a λcos site. Any DNA with cos sites a suitable distance apart will be packaged into the phage particles Cosmids have origin of replication, antibiotic resistance gene, restriction enzyme sites and cos site for λDNA packaging. λused to package DNA (via cos sites) and introduce in high efficiency into E. coli cell, packing up to ~50 kb Restriction enzyme cuts plasmid and DNA inserts, DNA and plasmid is ligated. In vitro packaging recognizes two cos sites that are 35 - 45 kb apart, introduced into the E. coli (via transduction) and becomes circularized inside E. coli (replicates as plasmid)

Cro dimers - drives lytic cycle

Cro dimers can bind to each OR site in the absence of CI (same binding sites but opposite effects). Cro binds on one side of the DNA, binds to operator sites independently (no co-operativity). Almost all Cro in cell is dimeric, only a negative regulator Cro affinity: OR3, OR2/OR1 Cro binds to OR3 (overlaps promoter for lysogenic growth) and stops CI expression. Expression from PR enables phage lytic gene expression to enter lytic cycle. Later Cro shuts off its own transcription and that of early phage lytic genes

Next generation sequencing

DNA fragments of up to 1000 base pairs can be sequenced, sequencing parallel fragments of up to 900 million nucleotide sequenced in 10 hours The genomic DNA is fragmented and the fragments of 400 - 1000 base pairs are selected. Each fragment is isolated with a bead in a droplet of aqueous solution. The fragment is copied over and over via PCR technique. All the 5' ends of one strand are specifically captured by the bead, eventually with 106 identical copies of the same single strand attached to the bead. This will be used as a template strand The beads are placed into a small well along with DNA polymerase and primers that hybridize to the 3' end of the template strand. The well is one of 2 million of a multiwell plate, each containing a different DNA fragment to be sequenced. A solution of one of the four nucleotides is added to all wells and then washed off, done sequentially for all for nucleotides. dATP, aTTP, dGTP and then dCTP, repeating this process over and over In each well, if the next base on the template strand, is complementary to the added nucleotide, the nucleotide is joined to the growing strand, releasing PPi causing a flash of light, which is recorded. The nucleotide is washed off and a different nucleotide is added. If the nucleotide is not complementary to the next template base, it is not joined and there will be no flash. The process of adding and washing off the four nucleotides is repeated until every fragment has a complete complementary strand. The pattern of the flashes will reveal the sequence of the original fragment in each well The results are shown in a flow gram, analyzed using computer software, which will stitch together the fragments into a whole sequence (an entire genome). If the template strand has two or more identical nucleotides in a row, their complementary nucleotides will be added one after the other in the same flow step

DNA polymerase in mutations

DNA polymerase checks each new base-pair, correcting mistakes and removing the incorrect base (99% effective), then DNA synthesis resumes. DNA proof reading reduces frequency of spontaneous mutation in bacteria. In mice, the lack of DNA proofreading causes increased mutations in mitochondrial DNA and premature ageing of mice and prone to tumors

Similar repair systems

DNA sequencing shows that molecular nature of mutations in humans is the same as bacteria. Higher eukaryotes have repair systems that are equivalent to those that have been identified in bacteria, working in the very similar way

Variations of vectors summary

Drive gene expression (expression vectors), product protein that can be purified (pGEX), allow visualization of gene product (GFP fusion), study when a gene is expressed (clone a promoter), replicate and express genes different system (shuttle vectors or gateway systems), easily clone PCR products (TA cloning), clone multiple fragments in one step (Gibson assembly). Can also design and buy synthesized DNA in a vector

E. coli chromosome

E. coli chromosome (100 mins long, min 0 and min 100 is Thr gene). Genes with similar function are clustered together, transcribed off a single RNA molecule (opern) E. coli strain K12 has 4,638,858 base pairs, sequenced in 1997, with around 4200 genes. Two-thirds have experimentally determined functions and 48 transposable elements. 'Clean' E. coli has no transposable elements for protein production (industrially). Good correlation with maps from genetic experiments

E. coli recombination

E. coli found to be first bacterium that sexual recombination was discovered in and able to isolate sexual recombinants, meaning genetics was possible. Able to carry out genetic crosses, analyze genetic properties

Use of E. coli

E. coli used as the foundation for molecular biology studies E. coli chosen to study in 1940s because its non-pathogenic, has rapid growth rate and simple nutritional requirements. It supports the growth of a range of bacterial viruses, chosen by a group of physicists and biologists, in the phage group, to study the problem of replication. The phage group wanted find to understand how organisms replicate, started with tobacco mosaic virus but took too long so they went with E. coli

Packaging of cosmid

Empty phage particles must be produced, phage outer coat proteins will spontaneously form phage particles and a stuffing enzyme will automatically pack DNA of a certain length into those particles if it detects the correct sequences Bacteria that have been infected with λmutants are used to make packaging λ DNA. The first strain has a mutation in gene E, encoding head protein and the bacterial cells will accumulate tail proteins. The second strain has mutation in gene D (or A) causing immature heads to accumulate. THe protein extracts are prepared from these bacteria, added with cosmid DNA (containing λ cos sites) and causes it to packaged into infectious bacteriophage particles

Transformation - process

Environmental signals trigger the onset of natural competence in a pneumococcal cell. Once competent, cells can capture foreign dsDNA via a long transformation pilus. Transforming dsDNA is captured at midcell (red foci), suggesting the pilus is localized here during transformation, some bacteria can recognize sequences using the pilus. After capture, dsDNA is passed to the DNA receptor apparatus ComEA, also preferentially at midcell, before transfer to the EndA nuclease. EndA degrades one strand of the captured dsDNA to use as nutrients, and the other ssDNA is transferred across the membrane via the ComEC pore. After internalization, ssDNA is coated with single strand binding protein (SsbB), which protects it from nucleases and creates a reservoir of ssDNA for homologous recombination. The transformation-dedicated recombinase loader DprA (replaces SsbB) can also bind transforming ssDNA, and promote binding of the recombinase RecA onto the ssDNA. RecA can then polymerize on the ssDNA, a process that may be facilitated by SsbB, and promotes homology search and strand exchange (highly recombinogenic with RecA bound). When inside the cell, incorporation is efficient due to protection from SsbB then RecA binding. This process is adapted for transformation, highly specific, only lasting short periods

Repair of apurinic sites

Enzyme AP endonuclease recognizes an apurinic site and breaks the DNA backbone to cut out that strand of DNA containing the apurinic site. The defective DNA and some adjacent DNA is then removed by excision exonucleases. The gap is filled in by DNA synthesis. This is an example of excision repair, which can repair many damaged DNA

Making cell transformable

Escherichia coli is not naturally transformable but can be made competent to take up circular DNA or plasmids by treatment with calcium chloride in the cold, permeabilizing its membrane. Other methods used to introduce naked DNA molecules into cells include electroporation (creating transient pores in the membrane using an electric pulse) and biolistic transformation (gene gun). Most organisms can be transformed by one of these methods

Repair of EMS damage

Ethylmethylsulphonate reacts with guanine to form ethylguanine. Repair enzymes (alkyltransferases) reverse the reaction, taking the ethyl group off to give back a guanine. The protein can only do this once, must be degraded after. This means no mutation will occur

Eukaryotic recombination

Eukaryotes have two genomes fusing and recombination and segregation but results in two separate genomes

Evidence for Robin Holiday and modern model

Evidences that support these models of recombination include genetic information data from eukaryotic systems (fungi), visualization of DNA in Holliday-type structures, the phenotypes of bacteria mutants lack the relevant proteins/enzymes have predictable phenotypes, and the biochemical reactions catalyzed by relevant proteins done in lab conditions still function as predicted based on the recombination models

Molecular parasites - IS and transposons

F plasmid and chromosome harbor molecular parasites (insertion sequences (IS) or transposons, able to integrate into plasmid or chromosome and remain stable within), often occurring in multiple copies in the genome (identical sequences). These same DNA elements makes DNA homologous, able to undergo recombination

Transduction class example

Experiment 1 shows Leu+, Thr+ and AziR are within 90kb of one another as they can all be co-transduced. Also suggests AziR is closer to Leu+ than Thr+, as AziR is co-transduced with Leu+ at a higher frequency than Thr, but don't know what order they are in Experiment 2, shows no AziR meaning Thr is opposite of AziR, Thr+ and AziR cannot be co transduced, therefore more than 90 kb apart. Thr+ and Leu+ can be co-transduced at low frequency and therefore lie within 90kb of each other Experiment 3 had used up 90kb along the way. Selected for Leu+ and Thr+ means AziR is never co-transduced, confirms the distance between Thr+ and AziR is greater than 90kb

Fluctuation test

Exposing different cultures (batches) of bacteria to T1 bacteriophage. For an adaptive response, will have approximately the same number of resistant mutants in each culture. For random mutation selection, there will be variation in the numbers of mutants

Extraction of mRNA for cDNA library

Extraction of mRNA is mostly ribosomal with a poly(A) tail for the stability of the mRNA, use poly(A) tail to isolate mRNA using immobilized oligo-dT that will base pair to poly(A) tail. Remaining RNA is removed by washing

F plasmid site specific recombinations systems

F has encodes two site-specific recombination systems, resD and Tn1000, for plasma maintenance functions. resD is site-specific recombinase, acting at fcr (next to oriV), to resolve multimers. Tn100 is a transposon that encodes its own site-specific system

Control of F plasmid replication

F plasmid control of replication is mediated by a protein binding to repeated sequences (iterons). At low concentrations RepA binds to oriV (high affinity site, AT rich region) and initiates replication, RepA required for replication. F plasmid replication is tightly controlled (low copy number) At high concentration of plasmid, there are more RepA, RepA also binds a lower affinity sites (iterated sequences) which "hand-cuffs" plasmids together making a plasmid dimer. This prevents replication (ori blocked) until plasmids are separated at cell division (ParM system)

P plasmid TA systems

F plasmid encodes two toxin antitoxin systems: hok/sok and ccdA/ccdB Hok encodes killer peptide of 52 amino acids (makes holes in membrane), translated from stable messenger DNA (half life 20 mins). Sok (suppressor) is unstable antisense RNA (half life of 5 mins), binding to hok mRNA and preventing its translation, made at a rapid rate (but rapid degredation) ccdA is stable killer protein (inhibiting DNA gyrase) while ccdB is unstable blocking protein TA systems include restriction systems, often plasmid encoded, restriction enzyme (toxin) and methylation (modification/antitoxin)

F plasmid

F plasmid structure is 100kb, 1/3 involved are tra genes (involved in making mating pore and DNA mobilization functions). RepF1A determines vegetative replication and incompatibility properties. RepF1A has oriV, par, res/fcr and ccdA/ccdB regions. OriV ensures plasmid only divides 1 to 2 times per cell, keeping copy number low. Par is a partitioning loci. Res/fcr functions for a site-specific recombination system that resolves dimers. ccdA/ccdB involved in host-killing system. F plasmid also includes regions for hok/sok (another host-killing system), pif genes (inhibit phage T7 from infecting) and Tn1000, IS2, IS3 (transposable elements, facilitate interactions between F and other DNA molecules)

IS in F and E. coli

F plasmids have 4 copies of molecular parasites, IS2, two copies of IS3 and transposon. IS2 and IS3 are important in making Hfr strains, because they are successful molecular parasites The E. coli K12 chromosome has 12 copies of IS2 and 6 copies of IS3 that provide regions of homology with the F plasmid, meaning there are 18 locations on the chromosome where F can integrate into by homologous recombination

F' plasmid

F-prime (F') factors carry chromosomal DNA, which is an 'in vivo' cloning vector. This is useful for doing complementation tests

M. marinum

Fast grower No mycolactone Has pigments Intracellular disease Opportunistic pathogen Mercury resistance plasmid 29 IS elements (low) Lots of PE and PPE genes

Minimal genome - conserved genes across genomes

First two genomes H. influenzae (1740) and Mycoplasma genitalium (480) compared. There are conserved genes that were identified and predicted to encode essential products. This defines the minimal genome of 256 genes. Similar studies have now used more genomes to refine this number Caveats (warnings) include that some proteins share no sequence or structural homology but can perform the same function, these are omitted from these analyses, and more than one different metabolism/nutrient can support life

Wollman-Jacob - results

Found that 10 minutes after mating, no recombinants gained any other markers, after 10 minutes, Azi marker started to show plateauing at 90%. 15 minutes in, some got Ton marker in recipient, 20 minutes in lac+ started to appear, and Gal+ appeared at around 30 minutes in Data showed each donor allele first appears in recipients at a specific time after mating begins. Donor alleles appear in a specific sequence and the maximal yield of cells containing a specific donor marker is lower for the donor markers that enter later

Frameshift mutations

Frameshift (indels of one or more base-pairs) mutations thought to arise through an error during DNA replication, explained by the Streisinger model. DNA slippage results in loss or incorporation of a new base-pair into the DNA, this model fits the experimental data

Mutation rates

Frequency of 1 per 10^9 mutations represents a balance between occurrence of mismatch and frameshift lesions and their repair by one of the repair systems. In some situations, bacteria have higher mutation rate (mutator strains). Strains have also been isolated in the lab where frequency of mutation is lower than usual, put in a lot of energy into repair systems

Generalized transduction

Generalized transduction (any gene on chromosome can be transferred), bacteriophage injects DNA into host (donor bacterium), breaks up bacterial chromosomes and replicates phage components. Self assembles within host, phages able to transduce have a head full mechanism (carrying DNA in head until full, often also incorporating host DNA as it doesn't recognize its own DNA), released from cell via lysis able to re-infect. Re-infection requires phage particle to inject DNA (linear) into recipient cell, doesn't require any genetic function. Fragment of DNA from original donor and can reincorporate via recombination, as it has no origin therefore cannot replicate

Genetic diversity

Genetic diversity in eukaryotes mainly comes from recombination through meiosis. Bacteria have asexual reproduction via cell division, efficient but not genetically diverse. Bacteria can gain genetic diversity from mutations and horizontal gene transfer. Vertical gene transfer is from mother to daughter

Genomic island

Genomic islands is generic term for acquired large number of elements (DNA regions in chromosome), many of which have lost mobility, while ICEs have retained ability to transfer between bacteria Genomic islands have different content of GC bases compared to AT bases than the normal bacterial chromosome (characteristic content). Often inserted into chromosome adjacent to a tRNA gene, directly flanked by repeat sequence (17 bp). By tRNA, there is also an intergrase gene (same type as Φλ) along with IS. Genetic islands also encode cargo genes, which define the type of genetic island it is. E.g. Pathogenicity islands encode virulence cargo genes Genomic islands are key players in bacterial HGT and evolution. Genomic islands are in essence discrete DNA segments that may be absent from closely related bacterial strains and which usually show some evidence (integrase, conjugation genes) of past or present mobility. Most genomic islands have lost mobility functions and have become fixed in the genome Genomic islands in bacterial evolution serve as fitness islands that adapt a bacterium with a core chromosome to specific environmental niches (e.g. pathogenicity, symbiosis and xenobiotic degradation)

ICEs

Genomic islands that have retained mobility are known as Integrative and Conjugative Elements (ICEs). ICEs share features of phage, conjugative plasmids and transposons. ICEs integrate at specific sites in chromosome, usually adjacent to a tRNA gene flanked by direct repeat of the 3-prime end of the tRNA gene (attachment site). Mechanism of integration (like lysogenic phage) mediated by integrase (encoded within one end of island), can excise from chromosome to enter conjugative state (instead of lytic state), conjugating single strand into recipient bacteria (copy remains in donor and new copy in recipient that can reintegrate) An ICE is integrated into one site in the host chromosome and is bounded by specific sequences on the right (attR) and left (attL). Excision yields a covalently closed circular molecule as a result of recombination between attL and attR to yield attP (in the ICE) and attB (in the host chromosome). An ICE-free cell can serve as a potential recipient. During conjugation, the donor and recipient are brought in close contact, and a single DNA strand is transferred to the new host through the action of rolling circle replication. Following transfer, DNA polymerase in the recipient synthesizes the complementary strand to regenerate the double-stranded, circular form. A recombination event between attP and attB results in integration into the host chromosome Conjugative transposon insert randomly, ICE inserts at a particular site

Genomic library

Genomic library is a collection of all the DNA fragments of a given species that have been taken from one organisms and inserted into a vector for transport (cloning) into a host, using BACs, cosmids and for smaller genomes can use plasmids Genomic libraries are used to isolate genes for biotechnology, identify the genes in an organism, and to obtain the genome sequence and gene function. E.g. finding particular gene that is little known, bacterium isolated that produces unknown anticancer compound, isolate and identify the genes responsible Genomic library is made from genomic DNA from eukaryotic genome (human), prokaryotic genome (bacterial) and viral genomes

George Beadles and Edward Tatum

George Beadles and Edward Tatum (early 1940s) used microbes to understand how genes work. They used Neurospora crassa (eukaryotic fungus, also common bread mold), which has nucleus with chromosomes (haploid for much of life cycle, meaning one gene copy per cell) They had a test tube, with a gel that provides basic nutrients at the bottom, and grew the fungus on the gel. Wanted to look at genetic mutants of neurospora properties, and isolated mutant (defective) Neurospora that couldn't grow, called auxotrophs. These auxotrophs could only grow when provided in a gel with arginine amino acid. This means the natural Neurospora can make their own arginine, therefore doesn't need it to grow. They found that the offspring of the mutant Neuorspora could not grow in the absence of arginine, meaning the mutation was heritable

H. influenzae

H. influenzae (1.8 Mb) causes meningitis and ear infections, it is the first bacterial genome to be sequenced (1995). It was sequenced by TIGR using whole genome shotgun sequencing methods Each of these sequences were ~ 500 bases, sequences were aligned to define its place in the genome, assembled sequences are called contigs The coverage of the genome was uneven, with an average of 6.3 coverage but some regions only represented once, thus the risk of error rate in the sequence was estimated a 1 in 5,000 - 10,000 (average gene ~1000, 1 in 10 might have errors) After assembly there were still gaps in the sequence due to sequences that didn't clone. Gaps were filled in using lambda lytic clones or PCR followed by sequencing

Hershey-Chase experiment

Hershey-chase experiment (1952) helped confirm DNA is genetic material. Experiment showed bacteriophage, made of DNA and protein, only injected DNA into bacterial cells while the proteins remained outside

Hfr strain formation

Hfr strains are formed by homologous recombination between identical copies of an insertion sequence or transposon present on F and on the chromosome. This indicates that the F plasmid and chromosome share regions of identical DNA sequence (homology)

High resolution mapping by analysis of recombination frequency

High resolution mapping by analysis of recombination frequency determines the distance between markers, the interrupted mating will determine the order of markers. The problem in bacteria is there will be more recombination with markers closer to the origin. The key is to select for the latest (last) marker to enter (using time of entry) then screen for earlier markers, because then it must have receive all the other markers too

Robin Holiday - Modern model

Holliday model doesn't quite match all data, so subsequent development proposed DNA degradation and resynthesis. In this (modern) model, after a double-strand break on one chromosome, some DNA is degraded (erosion) by enzymes and results in a region of single stranded DNA. This is not a desirable state, so base-pair with DNA in a partner chromosome (homologous chromosome) occurs, in a process called invasion, where the single stranded DNA displaced the homologous DNA to base pair. Polymerization generates complete DNA strands, by using the single strand as a template. The heteroduplex region is produced by the strand invasion process. DNA synthesis and ligation can lead to a Holliday junction, which are resolved by cutting Holliday junctions. Resolution sites where taking the Holliday junctions and cutting DNA and rejoining it so we get back to having two individual chromosomes (post recombination). This process can occur by accident due to radiation or can be a programmed event

Recombination and crossing over

Homologous chromosomes cross over during meiosis at the chiasmata (the physical appearance of crossing over) Due to the exchange of genetic material between chromosomes, involves breaking and rejoining DNA. The molecular mechanisms are best understood in microorganisms, also applies to eukaryotes

HGT

Horizontal/lateral gene transfer (bacteria sex) in bacteria occurs when the organism acquires genes directly from another cell, incorporating them into its genome. This is responsible for the acquisition and spread of fitness-enhancing traits, including antibiotic resistance (we are on the edge of the antibiotic resistance era). It provides an awesome mechanism for ongoing adaptive evolution (mutations are slow), helps with survival in a particular environment. E.g. Transferring symbiosis genes required for nodulation, nitrogen fixation to other bacteria in the soil, the recipient bacteria in the soil can now associate with eukaryotic organisms (e.g. plant) and produce nodules and fix nitrogen

Experimentally described ICEs (specific notable phenotypes)

ICEMlSym (symbiosis with Lotus plane, forms root nodules and fixes nitrogen) lives in host Mesorhizohium loti (acquired symbiosis island). ICEMlSym is 500 kb (10% of genome), residing in chromosome next to tRNAPHENYALANINE gene, under circumstances can excise and enter non symbiotic bacteria in soil that belong to the Mesorhizohium genome (gaining 10% of genome, genetic leap)

IPTG induces lacZ

IPTG must be added to induce lacZ, binding the lacI repressor, acting like allolactose, however it's not broken down by β-gal (because IPTG is artificial) pUC must be used in a compatible E. coli strain e.g. DH5-alpha

IS and composite transposon transposition

IS and composite transposons transpose by a conservative, cut and past mechanism. Transposon is recognized by transposase enzyme via inverted repeats making a ds cut, excising IS/transposon from donor DNA. Transposases pastes IS/transposon to target site, cutting target site stagger (up to 5 - 9 bp) and ligated, the staggered gaps filled by DNA polymerase. It results in small direct repeats of target DNA either side of transposons. The non-replicative donor DNA is lost or repaired

IS reformation

IS1 and IS2 are actually identical, ignore the picture connotations. Here, F integrates into IS1. Homologous recombination can actually be reversed (at a lower frequency usually), would just lead to the reformation of F and its excision from the chromosome. Occasionally, F excises from the chromosome by homologous recombination, but not by the initial points it inserted. Picture shows recombination between IS1 and IS2 (further along down the chromosome). Results in excision of plasmid and along with a fragment of the chromosome DNA (in this case included the lac+ gene). Plasmid is now smaller (F') carrying lactose gene, transferring (along with lac gene) to recipients with high frequency. Mutant recipients (lac- gene) provides lac+ genes in trans, restoring ability to utilize lactose

Blue white selection

In a non-recombinant, an intact pUC vector can make α peptide, complements rest of β-gal, cleaves X-gal and produces blue colonies In recombinants (plasmid with insert), will disrupt lacZα gene, cannot make α peptide therefore no X-gal is cleave and produces white colonies (carrying single insert, and are the clones of interest) Plate on Amp to select for pUC vector inserted into cell, then plate on X-gal plates to select for plasmids with an insert and those that do not (after the white colonies with the insert)

F+ male population

In a small proportion of cells in a F+ (male) population, F exists not as a plasmid but integrated into the bacterial chromosome, recombine with chromosome at a single cross over point. When F is integrated into the chromosome, it can still transfer chromosomal DNA to the recipient cell, instead of just transferring F, transfers whole chromosome starting at oriT. The few cells with F integrated can transfer chromosomal genes at a low but detectable level (Lederberg and Tatum observations)

Mutation definition

In classical genetics: A heritable change in a gene that resulted in a change of phenotype (see change in organism) Modern molecular terms: A heritable change to the sequence of a DNA molecule (may or may not affect phenotype)

Induction of prophage

In induction, prophages excise from host chromosome and starts lytic growth. UV induction can cause prophage to enter lytic cycle. Induction via UV results in DNA damage that is sensed by RecA, resulting in DNA becoming a co-protease. Activated RecA assists CI cleavage into two domains (non-functional) and therefore clears the CI operator sites CI cleavage causes no activation of PRM is lost, loses repression of Pint (making Int and Xis, promoting excision of λ from host). RNAP binds to PR (strong promoter) and transcribes Cro, Cro now determines the course of events

Transformation DNA uptake

In some organisms, DNA uptake is highly-specific. E.g. H. influenza binds at specific 9-bp sequence (AAGTGCGGT) that occurs 1471 times in its genome, and N. meningitides has 1891 copies of 10-bp sequence, these two have evolved mechanisms to only uptake their own DNA by recognizing the sequence, not bacteriophage or other environmental DNA. Incorporation of DNA is by homologous recombination replacing original host DNA. Recent studies have shown multiple regions up to ~50 kilobases can be transferred simultaneously

Gene loss after WGD

In the Saccharomycete yeasts a WGD event doubled the number of chromosomes in the lineage, but subsequent gene-loss events led to the current S. cerevisiae genome, which is only 13% larger than K. waltii and contains only 10% more genes. The ploidy of the genome (tetraploidy) returned to functionally normal ploidy (diploidy), by a large number of deletion events. Meiosis may have been impossible or error prone in the tetraploid and in the yeasts that were reducing the number of duplications

Phage lysogenic pathway

In the lysogenic pathway, the circular viral genome in cell integrates into host by recombination event. The prophage needs to be maintained in the daughter cells in a stable state, for the prophage to be passed to daughter cells Integration occurs between two sites, attP (on phage) and attB (on bacteria), sequences similar but not identical. Sites can only undergo recombination in presence of integrate enzyme, causing λ DNA to linearize in host chromosome (site specific recombination)

Phage lytic pathway

In the lytic pathway, transcription and translation of viral genes produce viral proteins that encode the heads and tails (head and tail proteins are synthesized). DNA recognized by heads and packaged into heads, then the tails are added. The host is lysed and releases a new phage

Lederberg and Tatum - Double marked strains

Initially Lederberg started with singly marked strains, plated out colonies to get single autotroph control, and got some revertant colonies (mutant reverted to wild type). The mixture plates had colonies but unsure if revertants or recombinants. Lead to the use of doubly marked strains (e.g. a strain that is met- and bio-), colonies would most likely be recombinants, reducing chance of revertants

IS in recombination

Insertion sequence can appear in forwards or backwards orientation. Homologous recombination goes from chromosome in IS to the IS on the F plasmid, end up with hybrid IS (identical so no change), goes along F plasmid to oriT (pointing towards IS, when integrated into chromosome would still point towards IS). Continues around plasmid to IS, finish off crossover (another hybrid IS) then integrate into chromosome. Recombination of these two form a larger circle. This is now a Hfr strain, with F plasmid integrated into chromosome, able to transfer chromosomal genes. In this case, gene D+ will be transferred first, while gene C+ will be transferred last

IS elements

Insertions sequence (IS) elements are simplest type of transposon (ultimate selfish DNA), only encoding protein needed from own transposition/transposase and their markers (on each end). There are several IS present in multiple copies on E. coli chromosome (IS1 has 5 - 19 copies depending on strain). The ends of all known IS elements show inverted terminal repeats (ITRs) of 20 - 50 base pairs, one end has one sequence, other end has inverted sequence. Inverted end repeat recognized by transposase enzyme to facilitate transposition

Genetic transfer eukaryotic system, compared to HGT

Interesting to compare to eukaryotic systems, parents mates and both contribute full genome into zygote (haploid genomes) and undergo recombination to result in a diploid genome

Wollman-Jacob - interpretation

Interpreted that a specific strain started transfer at a specific origin in a specific order. This explains time of entry. Chances of mating bridge breaking at random increases as time lengthens, no more transfer when bridge breaks. Shows greater transfer for those closer to the origin of transfer This showed Hfr chromosome is transferred in linear fashion, beginning at a specific origin. The further from O, the later a gene will be transferred and the more likely the transfer process will be interrupted before the gene is transferred

ESTs

Isolate mRNA from cells or tissue, copy mRNA into ds cDNA by reverse transcription and ligate into plasmid vector to make library. Sequence many clones randomly from one end. A group of single pass sequences to give representation of expressed genes (only sequencing bit next to vector) By sequencing random clones from cDNA library genes were identified that were expressed in the cells, tissues at the time the library was made. An EST database of gene expression data was created, ESTs were most commonly used for eukaryotic species. Now we would use RNA-seq (next generation sequencing of cDNA)

Jeffrey Millar

Jeffrey Millar (1970s/80s) found that different mutagens cause different effects on DNA. He looked at three different known mutagens (EMS, UV light and aflatoxin), using a screen to identify mutations and the nature of the mutation (base pair changes) in the lacI gene in E. coli. EMS caused many GC to AT base pair changes, UV light causes broader range of mutations and aflatoxin causes predominantly GC to TA mutations

Lederberg and Tatum - experiment

Lederberg and Tatum's experiment (1946) used autotrophic mutants (e.g. met- requires met to grow) that Tatum developed. They mixed tube A (met- bio- thr+ leu+ thi+) and tube B (met+ bio+ thr- leu- thi-) together, and plated a million cells for tube A, B and mixture on minimum medium plate (contains glucose and ammonium, without the needs of the strains like met or bio). No colonies formed for tube A and B plates. They found the mixture plate grew colonies, found prototrophs (able to grow without additional growth supplements). The mixture colonies was thought to have arose from cross-feeding, that each strain leaked out the required supplements to allow the deficient strains to grow (e.g. met+ leaked met to allow met- to grow). But single colony purifications and careful control, showed no cross-feeding. Concluded that bacteria have sex, prototrophs arising through recombination

Lederberg - first conditional mutants

Lederbergs approach represented the first use of conditional mutants (e.g. autotrophs) to select against the parental type. The mutants were double mutants so reversion artefacts were avoided (due to low reversion frequencies). The prototrophic recovery technique had enormous sensitivity

Lambda phage lifecycle

Lifecycle decision depends on state of the host bacterium (and other factors), taking default pathway (lytic) or integrate into host chromosome and become prophage and replicate with the host (lysogenic). Lysogenic state is stable, but can be induced to release more phages λ phage is a temperate phage, able to undergo lytic or lysogenic cycles, virulent phages are only able to undergo lytic cycles. Temperate phage latent form that remains within the host is a prophage, and the host with the prophage is called a lysogen Lytic pathway used in high growth of bacteria with plenty of food/energy. Lysogenic pathway taken if cell is unlikely to have resources to produce many phages. Prophage induction (leave lysogenic and enter lytic) when host cell is damaged and likely to die UV irradiation inactivates CI, and Cro is synthesized for lytic growth

Salvador Luria and Max Delbruck - Limitations and conclusion

Limitations: Bacteria rapidly killed, no time for them to adapt. It could take time (several generations) for resistant mutations to give rise to resistance phenotypes Concluded their data fit best with the random mutation model, mutations arising spontaneously and randomly in bacteria, able to be selected by environmental conditions

Seymour Benzer - used bacteriophages as fundamental tool to understand genetics

Logic built on previously done experiments. Bacteria are grown easily, so bacteriophages (viruses that infect bacteria) are grown easily and rapidly (generation time around 15 minutes). Phages are genetically the simplest organisms, with similar genetic mechanisms to the host cells (probes of genetic mechanisms). Able to analyze many millions easily, able to identify rare genetic events

Salvador Luria and Max Delbruck - method and results

Luria and Delbruck infected a large number (~ 109) of E.coli with T1, causing most E.coli to die with a few (resistant) ones that survived. The progeny of the survivors were also resistant to T1 (shows resistance is inheritable genetic trait) Hypothesis: The resistant mutations were induced by exposure to the bacteriophage in an adaptive response OR natural mutation occurred at a low rate (resistant mutants pre-existing) and were selected for by the addition of the bacteriophage (Darwinian evolution) To test the hypotheses, Luria and Delbruck developed the fluctuation test to distinguish between the two theories

Minimal genome - mutation analysis

M. genitalium and M. pneumonia have very small genomes. They are intracellular parasites of humans, lacking cell walls, causing urogenital and respiratory tract infections respectively. These are ideal candidates to growth in vitro to asses essential genes and minimal genome Performed transposon mutagenesis on M. genitalium and M. pneumonia where only mutants that could survive would grow (mutations in essential genes would result in no growth). The insertion sites of transposons in the survivors were determined by DNA sequencing and non-essential genes identified. There was a high mutational coverage to try to guarantee that all genes could have been mutated in the screen No transposon insertions identified in genes involved in DNA metabolism, transcription and translation. This is a good proof-of-principle that it has worked, many predicted essential genes have no known function (microbial processes are not fully understood). This estimates the minimal genome of 265-350 genes Functional redundancy means may underestimate essential genes by mutational studies

Maintenance of lysogeny

Maintenance and regarding the switch, only concerned with portion between CI and Cro. Two diverging promoters, PRE drives expression of Cro and PRM for repressor maintenance. The switch is either in one direction (maintenance) or other direction (lytic). Switch region (80bp) is between genes containing the operator (binds CI and Cro) and promoter sites (binds RNAP). There are 3 operator sites for CI and Cro, and 2 promoters for RNAP (PRM and PR), these two don't overlap (only one promoter working at each time). Operator sites either overlap 1 (OR1 & OR3) or both promoters (OR2). Operator sites (17 bp) are not identical, but similar, so CI and Cro can distinguish between them (different affinities, allows control order of binding). Protein binding to DNA is reversible

P element in Drosophila

Many DNA transposons in eukaryotes have similar structure to IS elements. P elements are key tools for genetic analysis of Drosophila, similar to transposons in bacteria. P element is IS, has inverted repeats with transposase gene between and introns in transposase gene. Tc1/mariner family elements (e.g. Minos) show low insertional bias (often inserting in single TA base pair sites) and transposes with high frequency in vertebrates and invertebrates. Mariner Tns also adapted to be used in bacteria. Ac and Ds transposons insert in maize. Retrotransposons can be modified to use in gene therapy

Causes of mutations

Many mutations occur spontaneously, with no evident external cause. Mutations require DNA synthesis, which can involve mistakes by DNA polymerase (DNA synthesis enzyme inside cells). This can arise through tautomerisation of bases (transient flip of base into different isomer that has different base pairing properties)

DNA polymerase mutation mechanisms

Many other chemical mutagens also alter bases in DNA and affect base pairing, EMS is typical in this aspect. Other mutagens also cause the absence of pairable base, in the case of aflatoxin and UV light. Some mutagens mimic normal bases and become incorporated into the DNA, later paired with the wrong bases. All these mutation mechanisms involve DNA polymerase

Mating pore, encoded by tra genes

Mating pores are promiscuous, made by Tra genes encoded by plasmid, making connections between wide range of cells. Plasmids can also adapt to replicate in a wide range of cells

Multimeric plasmids

Monomeric plasmids (with identical regions) can form multimeric plasmids/oligomer (one big plasmid with multiple ori) via recombination (RecA system). This means bigger chance of losing plasmid during cell division, as cell still recognizes multimeric plasmid as the "number of plasmids that joined together" (not recognized as just one plasmid). Multimers must be resolved back into monomers (single plasmids), by forcing recombination in the opposite direction using plasmid-encoded site-specific recombination system (working at high efficiency)

DNA polymerase and error prone version

Most DNA in cells is made by DNA polymerase that is very accurate with proof reading activity to synthesize DNA. Most DNA polymerase don't synthesize past damaged DNA (e.g. apurinic sites), instead special error prone DNA polymerase (trans-lesion DNA polymerase) synthesize, most likely incorporating the wrong base (even when DNA isn't damaged)

Testing for mutagens - animals

Most direct approach is using laboratory animals. They are mammals, meaning a good laboratory model for humans but there are issues with practicality/cost (need to screen large number), problems with sensitivity (how would you detect mutations) and ethics

Mutagens and their mutation effects

Most mutagens increase the frequency of substitution mutations though some result in indels (frameshift mutations). Mutagens work by a variety of mechanisms e.g. ethylmethylsulphonate (EMS) and aflatoxin. Mutagens can be chemicals or radiation

Negative and positive regulation

Negative regulation of gene expression turned off by a regulatory protein called the repressor. RNA polymerase binds to promoter region, repressors bind to operator sites blocking ability of RNA polymerase to bind or move along the DNA. Inactivated repressor (due to mutation) results in constant expression Positive regulation is when the gene expression is turned on by a regulatory protein termed an activator. Some promoter regions are weak and need help to turn on, activators bind to activator binding sites (normally upstream of promoter region) helping RNA polymerase recognize and bind on promoter for transcription to occur. Inactivated activator (due to mutation) results in no expression

George Beadles and Edward Tatum - results

Neurospora has 6 chromosomes, mutation found in 3 different loci (sites) on 3 different chromosomes. Each class has same genetic mutation site, class 1 has different site to class 2, and different to class 3. If given citrulline or ornithine (chemically similar), the class one mutants are able to grow. If given ornithine, class two mutants are not able to grow, but still grows in citrulline. Class three mutants cannot grow in citrulline or ornithine. This found the different classes to be defective at different steps (e.g. lacking enzymes) of the converting pathway (compound X to ornithine to citrulline to arginine, normal Neurospora has compound X therefore didn't need arginine to grow).

Non-composite transposons

Non-composite transposons are simpler than composite, not relying on IS modules for transposition (e.g. Tn3 ampR, Tn1000). They encode genes for own transposition and accessory functions. They have inverted terminal repeats, transposase and resolvase genes. Transposition requires transposae and resolvase gene products

Ohno hypothesis

Ohno has hypothesized that after gene duplication, one paralog copy would preserve the original function and the other copy would be free to diverge and adopt new properties of adaptive value. Have a resource able to tweak In Saccharomyces 76 of the 457 retained paralogous gene pairs (17%) show accelerated protein evolution relative to K. waltii, defined as instances in which the amino acid substitution rate along one of the S. cerevisiae branches was at least 50% faster than the rate along the K. waltii branch This suggests that one of the Saccharomyces paralogs (duplicates) retained an ancestral function while the other was free to evolve more rapidly

cDNA synthesis

Oligo-dT primer base pairs to poly(A) tail, of mRNA (introns spliced out). This allows reverse transcriptase to add to the primer and make cDNA. Hybrid strand of mRNA and cDNA has RNaseH added to degrade the DNA (into chunks), and DNA polymerase uses RNA bits (as primers) to add on DNA to make ds cDNA cDNA can be cloned as blunt fragments, but more efficient to ligate on adaptors (on sticky ends) then clone into vector of choice

PaJaMo hypothesis

PaJaMo experiment initially hypothesized the I- alleles makes an internal inducer (always ON) so therefore I- will be dominant over I+ (normal allele). Tested using Hfr I+ Z+ strain, conjugating with F- I- Z-, looking at how two alleles act together. Killed donors, and β-gal levels increasing, but then β-gal reduced due to lacI synthesis (could bind and switch off synthesis of β-gal). This showed lacI+ was dominant over lacI-, if lacI- was dominant then the induction would have continued (β-gal increasing without decreasing). But if an inducer was added, then β-gal production kept increasing, shows lacI was repressor and in presence of inducer it could not repress gene expression Showed a diffusible repressor initially absent from the cytoplasm of the recipient cell

Pathogen diversity

Pathogenic isolates differ from benign isolates by presence of plasmids and clusters of horizontally acquired virulence genes (pathogenicity islands) on their chromosomes

Minimal genome - looking in nature

Pelaginbacter ubique has the smallest genome of any cell to replicate in nature. It grows as free cells in the oceans, utilizing dissolved organic carbon in the ocean. This group of bacteria accounts for 25 - 50% of all bacteria in the oceans. Its genome is 1,308,759 bp, encoding 1254 proteins (compared to M. leprae 3,268,203 bp encoding 1604 proteins). P. ubique encodes basic functions for growth, containing almost no non-functional/redundant DNA with no gene duplications and no evidence of HGT. It has the smallest median intergenic spaces of 3 bp (in comparision, E. coli has 85 bp between genes for gene regulation), meaning genes are packed very tightly in the genome P. ubique is hypothesized to have some niches select for minimized DNA replication costs for streamlined genomes (save energy during replication). It grows in nutrient poor niches (can synthesize all 20 amino acids), most transporters has high affinity and import efficiently. There is almost no regulation in genome, and has constitutive slow growth that doesn't respond to increased nutrients. This defines a minimal free-living genome of 1354 genes. Might not be the smallest genome

Phage infection

Phage infects cell and injects dsDNA, genome is linear in phage head but following injection, the λ genome circularizes by its cohesive end sites (cos sites, ssDNA that are complementary). λ genome is organized into modular domains, with similar functions

Plasmid replication via RCR

Pilus, encoded by the F+ plasmid, contacts recipient cell (no pilus), pilus retracts, bringing donor and recipient into close proximity. DNA relaxase (mobilization protein) makes a single stranded nick at oriT and covalently binds to 5' on a tryptophan residue. Rolling circle DNA replication initiates at 3' OH (3' acting as primer) and proceeds 5' to 3' while outer strand 'peels' off. Tra proteins form a pore complex that spans the cell membranes (mating pore). Membranes close in proximity form a mating bridge (type IV secretion system). DNA relaxase interacts with membrane Tra pore complex, guiding and pushing outer ssDNA through pore, DNA replication pushes the ssDNA into the recipient cell. Lagging strand DNA replication in recipient cell converts ssDNA plasmid to dsDNA plasmid again. DNA replication in recipient cell occurs through okazaki fragments. Upon complete replication of plasmid, DNA relaxase nicks to separate the old and new plasmids. The ds plasmid DNA in the donor (old strand inner) and recipient (old strand outer) are religated. The mating complex collapses and the cells separate, yielding two new donor cells

TA system - removal of plasmid free cells

Plasmid free cells can be killed through toxin-antitoxin (TA) systems (genetic addiction systems/post-segregational killing systems (PSK)), killing host after segregation. Based on toxin and antitoxins produced by plasmid, without antitoxin the toxin is left in cytoplasm and kills cell, antitoxins less stable than toxin will break down and toxin also kills cell. Needs plasmid to produce antitoxin. Without plasmid, stable toxin remains longer than unstable antitoxin

Maintaining low copy number

Plasmid in cell containing ParR (DNA binding adaptor protein) that binds to ParS (centromere-like region). ParM (actin-like protein that polymerises to form filaments) keeps polymerizing until it locks onto ParR sites, bridging the two plasmids with a filament. The filament is relatively stable Replicated plasmids are paired by ParR bound to parS, thereby forming a partitioning complex. The partitioning complex forms a nucleation point for ParM filamentation (polymerizing via ATP hydrolysis). Continuous addition of ParM to the filament poles provides the force for active movement of plasmids to opposite cell poles (elongation), whereupon they are stably inherited on cell division. Upon the plasmids reaching the poles, the filament depolymerizes. This ParM system is used in partitioning of low copy plasmids (should be at least 2 copies in cell)

Plasmid can replicate

Plasmid is accessory genetic element, ds circular DNA that can replicate and exists separately from the chromosome. Has an oriC (ori of replication) for normal vegetative replication and an oriT (ori of transfer) where the conjugate of transfer initiates

Plasmid replication

Plasmid replication during cell cycle determines the plasmid copy number and incompatibility (if two plasmids can co-exist in same cell). The plasmid DNA replication is controlled by plasmid encoded inhibitory that acts at the oriV (frequency at which replication is initiated). As cell size increases, inhibitory concentration decreases (if above threshold will inhibit initiation of replication) and plasmid replication is initiated. The replication results in further copies of inhibitor gene and more inhibitor (increase inhibitory concentration), which limits plasmid replication again

Plasmids, ideal cloning vectors

Plasmids are dsDNA molecules that replicate in cells independently of the host chromosome. They are inherited by daughter cells and are non-essential for growth Plasmids are ideal cloning vectors because they are well characterized (sequence and function of genes are known), small, easy to purify and manipulate Plasmids have their sequences known, has an origin of replication, selectable marker (select for plasmid), has unique restriction enzyme cleavage sites (polylinker, multiple cloning site, contains restriction enzyme recognition sites), easy methods to screen for recombinants and are able to have high copy number (vary by purpose) A plasmid vector must contain an origin of replication, selectable marker, and an unique restriction enzyme cleavage sites. Want plasmid to only open once, and where the DNA is being inserted into

Plasmid functions

Plasmids are essentially molecular parasites as they are often of no use to their host. They must replicate, segregate, keep host happy, keep host under control and spread. Must segregate, ensuring each daughter cell receives at least one copy upon division. Must keep host happy by constraining (trying to minimize) metabolic load (regulating copy number), large plasmids typically have 1-5 copies/cell (low copy number), while small plasmids have ~10-50 copies/cell (high copy number). Must keep host under control via genetic eviction systems (killing off cells that remove the plasmids, using a toxin). Must spread via conjugation, the non-conjugative plasmids are often able to be mobilized. Small plasmids are often non-conjugative (can't self spread), but can often hitch a ride (mobilisable)

Plasmid accessory elements

Plasmids are extrachromosomal (separate from chromosome) genetic elements, capable of autonomous replication, called replicons. They are not essential, not required for day to day survival, but has ability to integrate into chromosome. Antibiotic resistance plasmid has no use when no antibodies are present, but when present is essential for survival of cell. Plasmids contribute to bacterial evolution and genetic plasticity (genetic diversity), highly important in recombinant DNA technology. They can encode important phenotypes like pathogenicity and antibiotic resistance

Lost plasmids

Plasmids may be lost through failures in replication, partitioning or multimer resolution.

RNA II structure

RNA II is able to fold on itself, RNA II has crucial loop of guanines that interact with cytosines on DNA to make very stable interaction. Above threshold concentration, rom protein is able to dimerize (monomer to dimer) and dimer is capable of binding RNA I and RNA II together, winding them together (RNA kissing) destroying RNA II structure (that makes interactions)

Recombination in E. coli

Recombination at a molecular level is best understood in E. coli (bacteria) and involves a number of enzymes, with enzymes identified that catalyze all the steps in the recombination model. RecBCD protein nicks and unwinds the DNA to generate single stranded DNA. The RecA protein coats the single-stranded DNA to keep it inert, then catalyzes base-pairing of the DNA with the target double stranded molecule (e.g. strand invasion). Proteins (RuvA & RuvB) cause branch migration. RuvC protein resolves Holliday junctions by DNA cleavage

Recombination as a repair mechanism

Recombination can be a further system for repair of damaged DNA (important), providing a correct double stranded DNA template for damaged DNA repair. In E. coli, its catalyzed by a RecA enzyme. Recombination can also allow repair of double stranded DNA breaks in eukaryotic cells. Bacteria that lack enzymes for recombination are more sensitive to UV light and X rays, showing recombination as important to repairing DNA

Recombination frequency giving map units

Recombination frequency aims to work out proportional distance between markers and the correct order of genes. Map units are arbitrary, worked out using percentage of recombinants divided by total recombinants (e.g. 75/300 recombinants with cross over between b+ and c+ gives 25 mu (25% of 300) between b+ and c+)

Benzer - recombination analysis

Recombination is a process that generates new gene combinations, his analysis aimed to map relative positions of mutations. Benzer infected E. coli strain B with pairs of mutants, all rII mutants could grow in this strain. Infected the progeny phage onto E. coli strain K12 (λ), where the rII mutants couldn't grow, plaques indicated that the wild-type (rII+) phage were formed by recombination between mutant chromosomes inside the E. coli strain B cells. The number of plaques indicated the frequency of recombination events (random events). This corresponded to the distance between the mutations in the DNA, mutations close together have lower frequency of recombination (less chance)

Recombination discovery

Recombination was first identified in eukaryotic organisms (diploid), seen as a new combination of alleles of genes. Recombination also occurred in bacteria (haploid) as shown in Benzer's experiments, crossing over doesn't occur often in bacterial because they are haploid. When two bacteriophages insert their DNA into the host bacteria cell, results in having two homologous chromosomes where crossing over can occur

Salmonella and E. coli

Salmonella and E. coli are similar bacteria, both containing the same core with different pathogenic lifestyles (especially K12). Able to trace evolution of salmonella by studying acquisition of genes. Salmonella pathgenocity island (SPI) blocks give salmonella its pathogenic characteristic (different to other S. enterica strands and E. coli), SPI encodes a type III secretion system (secreting effector proteins from bacterial cell direct into cytoplasm of eukaryotic host cell). Salmonella also contains a number of lysogenic bacteriophage, able to produce effector proteins that influence host cell, contributing to virulence

Salvador Luria and Max Delbruck

Salvador Luria and Max Delbruck (early 1940s) wanted to study spontaneous mutations. They analyzed a bacteriophage (bacterial virus) called T1 that infects and kills Escherichia coli.

Library screening via antibodies

Screen using antibodies to find protein of interest. cDNA library is cloned to express cloned genes in bacteria, clones expressing proteins are detected by the antibody. This allows identification of clone that encodes protein of interest. Can use cross reacting antibodies that can detect the same gene from quite distant species Membrane is overlaid on plate, also keeping a master plate. Membrane has proteins are expressed from expression library. Incubate membrane with primary antibody, wash membrane and incubate membrane with radiolabeled secondary antibody (labeled fluorescently or radioactively). Overlaid with X-ray film and find protein we are after Primary antibodies can be produced in an animal (rabbit) and purified, secondary antibody is bought (goat anti-rabbit) that is labeled Screening using antibodies will detect genes that are translated into proteins, but needs antibodies specific to protein of interest (often non available)

Double marked strains

Sensitivity is dependent on frequency of reversion. Use doubly marked strains is a powerful selection method to detect genetic recombination, if single reversion is 10^-6 then double reversion would have 10^-12 reversion rate

Phage influences on phenotype

Some phages have importance influences on bacteria phenotype. Vibrio cholerae, usually benign bacterium, acquires virulence due to lysogenic bacteriophage (cholera toxin phage). E. coli O157:H7 (hamburger bug, causes diarrhea) contains shiga toxin phage, which is largely lysogenic but can excise. C. diphtheria acquiring genes from diphtheria toxin phage. Staph. aureus can contain enterotoxin A phage

Types of plasmids

Some properties/phenotypes that plasmids can encode include conjugation, antibiotic resistance, antibiotic and bacteriocin synthesis and resistance, virulence and symbiotic nitrogen fixation. Conjugation includes F plasmid, do not know any other adaptive markers that F might encode, only replicates in E. coli and closely related species while other plasmids have a broad host range (e.g. gram +, gram -). Antibiotic resistance plasmids (R plasmids) can encode enzymes that modify or degrade antibiotics, often on transposons, which are highly significant medically. Antibiotic and bacteriocin (antibiotics that only kill closely related strains) synthesis plasmids include ColE1 (6.5kb) and SCP1 (350kb linear plasmids acting on Streptomyces) to kill off competing bugs, but also resistance to the antibiotic it makes. Virulence plasmids include Staph aureus (coagulase, haemolysin), E. coli (enterotoxin), Yersinia (host cell invasion) and Agrobacterium (plant tumourigenesis). Symbiotic nitrogen fixation plasmid (250 - 1500kb) to form root nodules, white clover forms symbiotic relationship with Rhizobium, that is able to infect white clover roots and induce the plant to form root nodules to convert nitrogen into ammonia (NH3) that the plant can use as an energy source

Class Time of entry example

Strain 1 and 2 have similar orders, except strain 2 has pro first, shows different site of initiation but in same direction for strain 1 and 2 This concluded that E. coli and bacteria have circular chromosomes Shows evidence of rolling circle replication

Ames test - gene practical

Suspension of bacterial cells, suspension spread on petri plate with agar gel. Single cells develop into visible colonies (each colony corresponds to a single cell)

Ames test - mutagenicity on animals and microorganisms

The Ames test uses many chemicals that are not mutagenic to bacteria, which are mutagenic in mammals. These chemicals are converted into an active form in mammals by enzymes in the liver, after ingestion. Solve this problem by preparing liver extracts and incorporating them into the medium used in the Ames test, meaning chemicals that are pre-mutagens get converted into mutagens

Different mutagens cause different kinds of mutations

The Ames' team generated strains of Salmonella typhimurium with different kinds of mutations in the His gene. These need different kinds of mutations (induced by different kinds of mutagens) to cause reversion to wild-type

WGD in S. cervisiae and K. waltii

The Kellis paper provides direct evidence of WGD in the lineage leading to the yeast Saccharomyces cerevisiae, by sequencing and analyzing a related species Kluyveromyces waltii Kluyveromyces waltii has not undergone the WGD, the divergence of the two species precedes the duplication event. They state that S. cerevisiae lineage (16 chromosomes, haploid) arose from the complete duplication of the genome of ancestral yeast with eight chromosomes Subsequently the duplicated genome returned to a functionally normal ploidy by massive loss of nearly 90% of duplicated genes due to many small deletions (only 10% larger than Kluyveromyces waltii) These losses were balanced and complementary in paired regions, preserving at least one copy of virtually each gene in the ancestral gene set (risky for organism to cut out bits/deleting at random) The WGD occurred after the split leading to Saccharomyces and Kluyveromyces The Candida albicans lineage started using CTG as a serine codon (in most organisms CTG codes for leucine) If the order of genes in the WGD genome and the non-WGD genome are compared they will (over short distances) be conserved. However the WGD genes will occur on two chromosomes The order of the retained duplicates (paralogs) in the WGD genome (genes 3 and 13 in the figure) will be conserved Selection no longer keeps genes functional Over long distances the order of genes may be rearranged by translocations If the retained paralogs on chromosome 4 in S. cerevisiae are compared with the other chromosomes, some paralog blocks occur on different chromosomes. In other words one block of genes on chromosome 4 may have a block of paralogs on chromosome 12, while other blocks of genes on chromosome 4 will have paralogs on different chromosomes. This is experimental data

Nature of mutations

The molecular nature of mutations can be identified by determining the DNA sequences of wild-type and mutant genes. Point mutations are mutations at a single point (often a single base pair) within a genome. The mutations within genes can be substitution mutations or indels (insertion/deletion of base-pairs). Mutations can arise through deletion or large scale rearrangements of chromosomal DNA or insertion of mobile genetic elements

Benzer - results

The outcome showed some pairs of mutants that could form progeny phage (each mutant able to provide function missing from the other). Complementation showed mutations are in different genes. Some pairs didn't give rise to the progeny phage (neither phage could provide the missing function, showed no complementation meaning mutations are in the same gene). The rII mutants had two classes (a & b), all rIIa mutants could complement all rIIb mutants, and vice versa (rIIa could not complement rIIa, rIIb could not complement other rIIb)

Salvador Luria and Max Delbruck - fluctuation test

They found in the individual cultures, some had no T1 resistant colonies, some had low numbers, and some had "jackpot" colonies (large number of T1 resistant colonies). This showed fluctuation in the numbers, agreeing with random mutation theory. They did bulk cultures, making sure the individual culture results was not due to sampling error. Each portion number had similar numbers of T1 resistant colonies

Wollman-Jacob - experiment

They got 6 markers. Mixed their strains, and sampled at specific time intervals by disrupting mating pairs in the blender. Plated samples on media containing Str, killing off HfrH donor, leaving recipient that cannot grow on minimum medium (Thr- Leu-). This selected for the transfer of Thr+ and Leu+, checked each recombinant for other four markers along the way

Robin Holiday Model

This model requires the alignment of DNA sequences that correspond to the chromosomes, followed by breakage of DNA strands (sugar-phosphate bond breakage of one strand per chromosome), strand exchange then rejoining of the DNA. The Holliday junction is the junction where two different chromosomes join. The cross-over branch point can move, giving a segment of hybrid (heteroduplex) DNA where each strand is derived from a different parent molecule, in a step called branch migration. Heteroduplex is a double stranded DNA molecule where each strand is derived from different parent molecule. Can be resolved by a vertical cut (along the V line) causing heteroduplexes and recombinants, or a horizontal cut (along the H line) causes heteroduplexes formed in the middle without recombinants in outside alleles. These cuts occur at a 50/50 rate, as the cell does not distinguish between how it cuts. The resolution of the structures leaves two DNA molecules that represent new combinations of genes, in new combinations of alleles (recombinants) and heteroduplexes. Holliday model is circumstantial, but subsequent information generally supports this model and its basic concepts

George Beadles and Edward Tatum - conclusion

This underpins the one gene, one enzyme hypothesis, with the significance that biochemical reactions in living cell has a series of discrete step by step reactions, each reaction specifically catalyzed by a single enzyme, with each enzyme specified by a single gene Limitations were they couldn't connect the genes to the enzymes

Recombination class frequencies - example

Time of entry showed gene a+ is last gene to enter, have a lot of recombinants for gene c+, and fewer for gene b+, and least for gene a+. Select for a+ as last marker, this means we've selected one cross over on the origin distal side of a+ (furthest away). Haven't selected for b+ or c+ (second cross over can be anywhere along this distance), if bacteria has a+ then merozyote has transiently received b+ and c+ (order of transfer). Some bacteria will only have a+, some will only have b+ and c+, it depends on how close b+ and c+ are to a+ and to each other. All a+ recipient cells will have received b+ and c+ DNA in the cell and have a chance of incorporating b+ and c+ into their DNA (whether they do incorporate b+ and c+ into DNA depends on distance between each of the genes) Theoretically there are 4 recombination classes: ones with a+ b+ c+, ones with a+ b+, ones with just a+ and ones with a+ c+ Recombinant class with a+ b+ c+ has second cross over is between c+ and origin (O), gaining all genes between them. Recombinant class with a+ b+ c- has second cross over between b+ and c+. Recombinant class with just a+ has cross over between a+ and b+. Recombinant class with a+ c+ has second cross over between a+ and b+ with third cross over between b+ and c+ and fourth cross over between c+ and origin (even cross overs), this is the rarest class (can use this to work out gene order, middle gene excised)

Non-composite transposon transposition

Tn3 (non-composite) has a replicative transposition copy and paste system. Transposase makes two ssDNA cuts in donor (one either side of transposon) and a staggered cut in target. The cuts on donor and target sequence are ligased, resulting in a fused replicons (forming cointegrate). DNA synthesis creates dsDNA, resulting in two copies of transposon. The resolution of cointegrate requires the resolvase to catalyse a recombination like event, acting on a res site within resolvase region. The results is transposon pasted with a small direct repeat of target DNA either side of transposon

Transposon

Transposons able to move from one site in genome to another catalyzed by transposases (protein enzymes), independent of host recombination system (jumping genes). Transposases are the most abundant and most ubiquitous genes in nature (reflects success). E. coli devoid of transposons for protein production, transposons cause instability. They cause large fraction of spontaneous mutations and chromosome rearrangements (deletions, inversions, replicon fusions). They enable the rapid acquisitions of multiple antibiotic resistances and facilitate flow of such genes between species. Transposons are powerful tools for molecular genetic studies, helpful selection tool (e.g. Tn5 for kanamycin resistance)

Transposons can

Transposons can generation mutations like antibiotic resistance, with each mutant likely to only have single insertions. Transposons can also tag genes for subsequent isolation, inserted into gene disrupts its function (stable knockout mutation). Also responsible for genomic instability due to multiple identical copies that can recombine Mutation caused by transposon allows isolation of transposon and the surrounding DNA, often using PCR techniques (using primers). This allows identification of mutated gene

Transposon discovery

Transposons first discovered in maize by Barbara McClintock (1050s) and discovered in bacteria in 1968. Transposons are ubiquitous, making up ~50% of human genome (retrotransposons), and up to 70-90% of maize genome

Studying genetics in microorganisms

Useful to study genetics of microorganisms as microbes allow genetic screens that are not possible with higher eukaryotes. Mechanisms and nature of mutations are effectively the same in microorganisms as in higher eukaryotes

Testing for mutagens - microorganisms

Using microorganisms to screen for mutagens. Genes in microorganisms work the same way as eukaryotes, mutagens in eukaryotes should also cause mutations in microorganisms. Able to screen for very large numbers of organisms at once, sensitive screens available to detect occurrence of mutation (detection even possible at a low rate). This means the pathway resulting mutation in microorganisms are well understood. There are also no ethical issues

Repressor in presence and absence of lactose

When no lactose is present, no point producing enzymes to break down lactose. Transcription from DNA to RNA, translation to peptide and folded to form repressor protein. Repressor binds to operator site resulting in RNA polymerase unable to transcribe the operon In presence of lactose, lactose binds to allosteric site on lacI protein, causes conformational change, lacI protein no longer able to bind protein effectively. Unable to bind operators, allowing RNA polymerase to bind and transcribe past operator site through lacZ, lacY and lacA genes Permease molecules are stable, enables lactose to enter cell while system is repressed

Genome duplication

Whole genome duplication has occurred quite often in the eukaryotes Comparative genomes enables us to detect such duplications, also enables us to study the long term evolutionary consequences of such a sudden genomic change Genomic duplication has been proposed as an advantageous path to evolutionary innovation, because duplicated genes can supply genetic raw material for the emergence of new functions through the forces of random mutation and natural selection. Duplicated genes are useful resource Such duplication can involve individual genes, genomic segments or whole genomes. Whole-genome duplication (WGD) is a particularly intriguing but poorly understood situation. In principle, coordinate duplication of an entire genome may allow for large-scale adaptation to new environments. However, polyploidy comes at the cost of major genomic instability, which persists until the genome returns to functionally normal ploidy through mutation, gene loss and genomic rearrangements. Eukaryotes divide through meiosis, problem is two homologous pair up

Whole genome shotgun sequencing

Whole genome shotgun sequencing, cutting many genome copies into random fragments. Sequence each fragment and overlap sequence reads to develop a contig (contiguous sequence). Overlap contigs to give complete sequence Whole genome shotgun sequencing sequences from ends of a large number of random clones, assembles sequences on a computer, filling in gaps with targeted sequencing later. The output is a random collection of short sequences, some of them overlapping, the overlapping reads are assembled into contigs. The contigs are joined together. Faster and cheaper than ordered approach. Automation using sanger sequencing made this approach excellent

Will Hayes discovery

Will hayes discovered the ability to form recombinants was dependent on factors in cell that could be transferred from a recombinant able cell into a recipient cell Conjugation in E. coli K12 depends on plasmid F factor. The F factor is a conjugate of a plasmid that can transfer from F+ cell (donor male, has F factor) to F- cell (recipient female, lack F factor) at high frequency by rolling circle mechanism. F- becomes F+ while F+ remains F+. F+ does not mate with F+ due to surface exclusion, preventing the pili attaching to the recipient surface. F+ can mate with multiple F- cells at one time This shows in Lederberg's and Tatum's experiment, there were F+ and F- bacteria used

Lysogen - drives CI expression

Within a λlysogen, OR1 and OR2 are usually bound to drive more CI expression (auto activating). Good in cell division, keeping concentration of CI high in daughter cells. OR3 is bound when CI levels are too high in the cell, stopping transcription in both directions briefly, allowing cell to dilute CI levels CI binding is affected by intrinsic affinity and co-operativity. Bound CI at OR1 increases the affinity for OR2 (almost immediate binding, aided by protein-protein interactions). Binding at Or3 is weak and has no co-operativity (only binds when concentrations are high) In lysogenic cells, CI bound to OR1 and Or2 to inhibit Cro and maintain expression of CI (transcribe CI). This is a very stable state that is inherited by daughter cells after cell division (epigenetic inheritance, not a change in DNA sequence that is inherited, daughter cells inheriting proteins that maintain a regulatory network)

Wollman-Jacob

Wollman-Jacob (1957) experiment (interrupted mating) established the foundation for the use of conjugation for genetic mapping Will hayes isolated HfrH (Hfr strain) and found it didn't donate F plasmid (fertility) but it donated Thr and Leu at high frequencies compared to other markers Will hayes sent Wollman and Jacobs his strains, they started off with HfrH strain (prototrophic), and mated it with F- strain (autotrophic, Thr- Leu-). HfrH resistant to Azi (poison) and Ton (bacteriophage T1) but sensitive to Str (streptomycin - drug). HfrH strain assumed to be wild type (Gal+ Lac+). F- strain sensitive to Azi and Ton, unable to utilize galactose and lactose (Gal- Lac-) but resistant to Str

cDNA and cDNA library

cDNA is DNA copy synthesized from mRNA using viral enzyme (reverse transcriptase (RT)) cDNA library is a collection of all expressed RNAs (expression library), all expressed sequences use cosmids and plasmids. It generates ssDNA that can be replicated into dsDNA, resulting in DNA sequences lacking introns so genes can be translated into proteins in bacteria. cDNA are smaller fragments, without the requirements of restriction enzymes

Turbit plaques

λ forms turbid (cloudy) plaques in bacteria lawn, due to lysogens being immune to further infections (CI repressor protects lysogen from further λ infection/giving immunity). Sometimes clear plaques are observed, mutants named CI, CII and CIII (clear mutants), dedicated to go down lytic pathway