BIO 472 Final Exam

Bacterial transformation

-F. Griffith demonstrated transformation -uptake of naked DNA by a competent cell followed by incorporation of the DNA into the recipient cell's genome enters as ds DNA and leaves ss DNA

tra genes (type IV SS) (aka pilus)

-LPS outer membrane of gram - -tip -TraA -extension and retraction proteins that hydrolyze ATP to facilitate

SOS response

-RecA protein initiates recombination repair -RecA protein also acts as a protease, destroying LexA repressor protein and thereby increasing production of excision repair enzymes -DNA polymerase IV and V synthesize unrepaired DNA ( some of the genes expressed will be DNA pol IV or V) PIC: left: Normal DNA, with LexA repressor sitting on the SOS box in the promoter of DNA repair enzymes. SOS genes will not express their inducer proteins, so SOS switch is off right: Damage DNA recognized. RecA acts as a protease to destroy LexA. SOS genes will be expressed to repair the damaged DNA. SOS switched on

examples of physical mutagens

-UV light: promotes pyrimidine dimer formation (kink leads to mutation) -X rays: Causes base deletions, single-strand nicks, cross-linking, and chromosomal breaks

simple transposition

-also called cut-and-paste transposition -transposase catalyzed excision -cleavage of new target site and ligation into site Mechanism: 1. TE- a few hundred to several thousand base pairs in length 2. transposase recognizes the inverted repeats and cleaves at both ends of the transposable element, releasing it from its original site 3. transposase carries the TE to a new site and cleaves the target DNA at staggered sites 4. The TE is inserted into the target site 5. DNA gap is filled

generalized transduction

-any part of bacterial genome can be transferred -occurs during lytic cycle of virulent phage -during viral assembly, fragments of host DNA mistakenly packaged into phage head --generalized transducing particle

CRIPSR

-bacterial immune response-protospacer- hunk of DNA cut by cas proteins. integrated into CRISPR locus in front of a repeat that is in front of the leader sequence. then will have a repeat to the right and now you have the repeat-spacer-repeat-spacer pattern-when new phages infect, you get CRISPR locus transcription.-mature CRISPR RNA will association with RNA guide proteins and nucleases. when they detect incoming DNA they can form hybrids. hybrids mark incoming DNA for destruction

sepcialized transduction

-carried out only by temperate phages that have established lysogeny -only specific portion of bacterial genome is transferred -occurs when prophage is incorrectly excised

mobilizing plasmids

-conjugative plasmids (SP) too large (30Kb) to be cloning vectors; contain conjugative genes -conjugative plasmids can move a mobilizable plasmid into another cell -smaller mobilizable plasmids can be good cloning vectors

Excision Repair

-corrects damage that causes distortions in the double helix -remove the damaged portion of the DNA strand and use the intact complementary strand as a template to synthesize new DNA process: 1. starts with UvrAB complex that tracks along the DNA in search of damaged DNA 2. After damage is detected, UvrA is released and UvrC binds 3. UvrC makes cuts on both sides of the thymine dimer 4. UvrD is a helicase that removes the damaged region. UvrB and UvrC are also released 5. DNA polymerase fills in the gap and DNA ligase joins the fragments

nonreciprocal homologous recombination

-incorporation of single strand DNA into chromosome, forming a stretch of heteroduplex DNA -proposed to occur during bacterial transformation Mechanism: 1. association of homologous segments 2. strand separation and pairing 3. endonuclease nicks at the arrow on donor strand 4. endonuclease nicks host strand 5. gaps in strand filled and ligated

Horizontal gene transfer (HGT) in bacteria and archaea

HGT differs from vertical gene transfer -transfer of genes from one independent, mature organism to another --stable recombinant has characteristics of donor and recipient important in evolution of many species -expansion of ecological niche, increased virulence

replica plating

WT exposed to mutagen plate replica plater to collect colonies and stamp onto surface of plate with complete medium (everyone grows) other is one missing lysine (looking for lysine oxotrophs) look for where colony exits on complete medium than on other plate. The lysine plate doesnt have the same colony so its partner isnt present and that mutant is the lysine oxotroph that we can grow and work with

induced mutations

caused by agents that directly damage DNA base analogs: (used in antiviral medicines) -structurally similar to normal bases -mistakes occur when they are incorporated into growing polynucleotide chain DNA modifying agents: -alter a base causing it to mispair intercalating agents: -distort DNA to induce single nucleotide pair insertions and deletions

other types of mutations

conditional mutations: -expressed only under certain environmental conditions auxotrophic mutant: -unable to make an essential macromolecule such as amino acid or nucleotide --has conditional phenotype --WT strain from which it arose is called a prototroph

F-Factor integration

•F factors contain the info for formation of sex pilus -attach F+ cell to F- cell for DNA transfer during bacterial conjugation •F Factors have insertion sequences (IS) -assist in plasmid integration PIC: 1. (round ) F factor plasmid that has been transferred into a recipient and the F factor has insertion sequences that allow it to integrate so it can exist as an episome. 2. integration event 3. result can resolve and jump out

indels

insertions or deletions on the left going down, slippage leads to an insertion (daughter strand gets an extra T) in the right column, slippage leads to a deletion (loss of 2 A's leads to daughter strand lossing two T's)

spontaneous mutations

may result from errors in DNA replication (ir sometimes polymerase messes up) -transition (alteration of similar bases ie a purine for purine or pyrimidine for pyrimidine) and transversion (purine base replaced by pyrimidine) mutations -due to insertion or deletion of nucleotides may also result from the action of mobile genetic elements such as transposons

Deinococcus radiodurans

microbe that can handle high gamma radiation in this experiment, DNA fragments with time post-irradiation -lane one has the control that was not exposed to any radiation (genome cut with RE) -lane 2 expose to radiation, and take that a run cut DNA, leads to smear beacuase DNA is scrambled -lane 3 exposed then waited 2 hours, still scrambled -22 hours after exposure, cells not dead even though their DNA is scrambled, they have slow repair mechanisms to fix the scrambled DNA

creating genetic variability

mutations are subject to selective pressure -each mutant form that survives becomes an allele, an alternate form of a gene recombination is the process in which one or more nucleic acids are rearranged or combines to produce a new nucleotide sequence (recombinants)

mutation detection and selection

observation of changes in phenotype replica plating technique -used to detect auxotrophic mutants use of environmental condition in which only desired mutant will grow -e.g. selection for reverants from auxotrophy to prototrophy

Direct repair

photoreactivation -used to directly repair thymine dimers -thymines separate by photochemical reaction catalyzed by photolyase process: 1. pyrimidine dimer in UV-exposed DNA 2. complex of DNA with photoreactivating enzyme 3. absorption of light (> than 300 nm) 4. Release of enzyme to restore native DNA

SOS repair in E.coli

pol III with Beta-clamp moves down the DNA until it bumps into a thymine dimer. The pol will stop and activate the SOS response. Result is up regulation of error correcting pol V and IV. Pol III and the B-clamp will be removed and either IV or V will repair the lesion. after pol III and the clamp can reattach. pol IV (Din B) pol V (UmuD'2C)

DNA repair

proofreading -correction of errors in base pairing made during replication -errors corrected by DNA polymerase other DNA repair mechanisms also exist

conjugation

some plasmids can move around (mobilizable) but not on their own. Require a conjugative plasmid to be present as well mobilizable type plasmids have origin of transfer (oriT) where they get cut an unwound to initiate the transfer. Typically have a relaxase protein that keeps the end of the DNA from twisting up on itself. A type 4 coupling protein (T4CP) which generates the energy necessarily for the DNA transfer. only the conjugative type plasmids have a type IV secretion system (T4SS) ie pillius responsible for cell-cell exchange conjugative plasmid can move itself or the mobilizable plasmid

Mutation

stable, heritable changes in sequence of bases in DNA -point mutations are the most common --from alteration of single pairs of nucleotide --from addition or deletion of nucleotide pairs -larger mutations are less common --insertions, deletions, inversions, duplications, and translocations of nucleotide sequences mutations can be spontaneous or induced

recombination at the molecular level

three types: -homologous recombination -site specific recombination -transposition

generalized transduction can maifest in may different ways

bacteriophage packaged with shigella toxin that injects it into a commensal E coli that then transduces it into a shiga toxin pathogenic e coli (origin of EHEC)

three main mechanism of horizontal gene transfer

transformation: transfer of free DNA from environment to recipient conjugation: donor cell with plasmid that is able to transfer to recipient transduction: virus acquires genetic info, packages it into phage head, and infects new host with DNA into bacterium

example of a suppressor mutation

two strains: Wildtype is ES114 (V. fischeri), mutant is C10 C10 is defective in cell wall amydase means that this organism must produce amydase in order to divide during cell division C10 isnt motile (can see in photos) the top middle photo is the mutant with the gene added back, but the gene isnt turned on, so the microbe isnt moving in the top left image, gene is added back and turned on by the addition of the chemical IPTG is you let the small spot of mutant strain sit long enough, you see the small clouds form as in the bottom photo -the hypothesis was that those were motile suppressor mutants (ie the clouds spreading out are microbes that have acquired the ability to swim not necessarilty through an alteration or fixing of the gene, but rather something else). -probably an extragenetic suppressor

WGS to ID suppressor Gene Mutation

used whole genome sequencing to id suppressor gene found a point mutation in the OG purple bar at arround 443 there is a T, but in all motile mutants there is an A in not the amydase gene

Effects of mutations

wildtype: -most prevalent form of a gene forward mutation: -wildtype exhibits a mutant form reversion mutation: -("true" vs "suppressor") -mutant phenotype exhibits wildtype phenotype --supressor mutation(can be intrageneic or extragenetic): occurs when the second mutation is at a different site than the original mutation

biofilm example

wt (biofilm stained with crystal violet C10 : deficient in amydase gene, leads to more biofilm (repressor of biofilm because when you take it away, biofilm production is high) 1477: another mutant. lose biofilm (activator of biofilm, take it away means no bioflim)

types of point mutations

-none is WT -silent mutation (base sub, but still encodes same aa) -missense (a different base is laid down, leads to a new aa there) -nonsense mutation (change results in formation of premature stop codon leads to truncated aa seq) -frame shift (insertion or deletion throws frame off, everything down stream is different aa) reverse mutations -forward mutation is change in DNA that encodes new aa. True reversion where it goes back to the OG sequence (think that these are rare) -forward mutation leads to new aa then reversion of that mutation in which ser is replaced -base sub but end up with nonpolar aa exchanged for polar aa leads to pseudo wt (similar chemistry still has most of their functions) frameshift: forward mutation has mutation that changes frame leads to new aa. Then supressor mutation undoes to restore reading frame (intragenetic supressor, changes that happen in the same gene to restore WT ). Similar to WT extragentic suppressor mutations: organism sustains mutation (loses motility), some other mutation somewhere else in the genome restores that ability

Role of R-M systems in recombination

-restriction modification systems-restriction endonucleases cut the DNA/RNA. depending on if the DNA has certain chi sequences, RecB,C,D will act as a nuclease or helicase.-methyl transferases methylate at certain sites to prevent from cutting the DNA.-in theory, phage DNA wouldnt have a chi sequence so it will get chopped up (endonuclease activity)

transposons

-segments of DNA that move about the genome in a process called transposition --can be integrated into different sites in the chromosome -are sometimes called "jumping genes" -the simplest transposable elements are insertion sequences -transposable elements which contain genes other than those used for transposition are called composite transposons

Transduction

-the transfer of bacterial genes by viruses -viruses (bacteriophages) can carry out the lytic cycle in which the host cell is destroyed or the viral DNA can integrate into the host genome, becoming a latent prophage

homologous recombination

-usually involves a reciprocal exchange between pair of DNA molecules with the same nucleotide sequence -RecA proteins carry out PIC: 1. two double helices next to each other 2. double stranded break 3. strand degradation via RecBCD occurs at DSB site to yeild single stranded ends 4. RecA promotes strand invasion and D-loop formation 5. gap repair synthesis fills in the vacant region 6. branch migration and resolution can produce recombinant or non-recombinant chromosomes

transposable elements

DR- direct repeat IR- inverted repeat insertion sequence transposon: insertion sequence + antibiotic resistance replicative transposon: jumps in and back out

bacterial plasmids

•Small, autonomously replicating DNA molecules that can exist independently or, as episomes, integrate reversibly into the host chromosome •Conjugative plasmids such as the F plasmid can transfer copies of themselves to other bacteria during conjugation •F factors contain the info for formation of sex pilus -attach F+ cell to F- cell for DNA transfer during bacterial conjugation •F Factors have insertion sequences (IS) -assist in plasmid integration

DNA uptake in bacterial transformation

•protein system allows DNA to move across gram positive negative cell walls -PilQ aids in movement across outer membrane -Pilin complex (PilE) moves DNA across periplasm and peptidoglycan -ComE is DNA binding protein -N is nuclease that degrades one strand -ComA forms transmembrane channel -comGB and ComGA are extension and retraction proteins