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what are the three main types of genetic exchange systems in bacterial cells?
conjugation-plasmid-mediated transfer of DNA elements (plasmid or chromosome) from one cell to another transformation-cells take-up free DNA directly from the environment transduction-phage mediated transfer of genes from one clel to antoehr. can move any gene
how are plasmids classified in terms of their ability to transfer genetic information?
conjugative-plasmids that carries genes for effective contact between cells. encoded by the tra gene mobiliizable-can prepare dna for transfer, but lacks genes required for conjugation. requires help self-transmissible-plasmids that are both conjugative and mobilizable, ex) F plasmid non-transferable ex) pUC
What is teh outcome of an E. coli F+ x F- cross?
what role does RepE play in F plasmid replication?
replication initiator and autogenous repressor
what are the functions of the relaxase?
1. cuts at oriT and covalently attaches to the 5' end 2. carries DNA into recipient 3. catalyses the reversal of the transesterification reaction closing the ssDNA
what are the two groups of tra genes in F plasmid and what functions do they encode?
mpf (mating pair formation) dtr (DNA transfer and replication)
how is the expression of F plasmid's tra genes controlled?
traJ acts as a trascription activator at PtraY which controls the transcription of the tra operon -traJ mRNA translation is repressed by the interaction with an antisense RNA, finP(fertility inhibition). requires FinO protein
how can a conjugative plasmid assist in the transfer of a mobilizable plasmid? what interactions/recognition between the two systems are most important in this process?
it can provide Mpf functions. recognition by the coupling protein is key in the activation and mobilization. relaxase proteins are specific for their target sequence
what are the two main mechanisms for the integration of F plasmid into the chromosome? which of these processes is most affected by a RecA mutation and why?
homologous recombination using insertion sequence (IS) elements and transposon (gd) mediated insertion. homologous recomb is dependent on RecA b/c it enhances pairing of homologous DNA
what is the phenotype of an Hfr strain with respects to conjugation and mobilization functions? what are the genotype and phenotype, again with respect to conjugative and mobilization functions, of the recipient in an Hfr x F- cross?
tra+ F-tra+
what are the steps in the chromosome mobilization by F plasmid? which genes are transferred first? what functions are needed in the recipient for there to be a change in genotype of the recipient to occur?
F plasmid encodes tra functions, a nick at oriT initiates transfer,rolling circle replication starts w/ transfer of remaining F plasmid followed by transfer of chromsome, replication of 2 strand, Rec A mediated homologous recombination
what are the two mechanisms commonly used to mapping gene order by conjugation? how do these differ in the way or time mating pairs are sampled? why is it necessary to select against the donor and how is this done?
1. Mapping by interrupted mating at a series of time intervals -remove an aliquot of mating cells -agitate to separate any mating pairs -plate on select media -screen for unselected markers 2. mapping by gradient of transfer. the mating is incubated long enough to allow transfer of the entire chromosome -plate on selective media. the furhter the genetic marker is from the origin of transfer, the less likely it will be transferred to the recipient -screen for the unselected markers Donor is selected against because we only want recipients to grow. grow in minimal media + antibitioc resistance the recipient naturally has
how fo F' plasmids arise? what genes are carried on an F'? what effects does the formation of an F' have on the genotype/phenotype of the host strain? what is the phenotype of the recipient cell in an F' x F- cross with respect to F plasmid functions?
occurs when like IS elements align from the F plasmid and chromosome, and circular DNA is excised. contains whichever genes were between the IS elements. host would retain phenotype. recipient would be F'
what approaches have been used to select for the formation of an F'?
1. select for early transfer of distant markers in a mating experiment 2. mate into a recA- strain
how can merodiploid strains be used to determine if two mutations are in the same gene?
an allele test a cis-trans test if you want to determine if the mutant gene encodes a soluble factor
how does the recipient cell signal the pAD1 plasmid-containing donor cell?
recipient cell excretes pheromone-like peptides
what functions are expressing in the donor cell prior to stimualtion by the recipient?
receptors are expressed but tra genes are repressed until phermone stimulation
how does the pheromone cause a change in the gene expression in the donor and what changes occur that facilitate plasmid transfer?
pheromones stimulate expression of tra functions in donor cell. peptide induced signal binds to TraA, TraA repressor is then released form DNA, There is expression of TraE and tra operon so conjugative proteins are made
what changes occur in teh transconjugate that prevent further response to the specific pheromones associated with this plasmid?
the transconjugate expresses iAD1 that blocks TraC receptor preventing further activation by the cAD1 peptide
what are the steps in teh transfer of the conjugative transposon Tn916? what functions are encoded by the Tn916? how is Tn916 maintained in the transconjugate-is it a plasmid or is it chromosomal?
excision, circularize, transfer, integration. it encodes tra and has an oriT function maintained in the chromosome
What is the process of transformation? how does it differ from conjugation?
uptake of free DNA from the environment into a cell. conjugation is the transfer of DNA between cells. Naturally transformable cells can uptake DNA without special treatment Cells must be competent, either naturally or induced by chemical treatment
what might be the physiological functions of transformation systems?
genetic diversity, repair, or food
In the case of B. subtilus, what is the role of the ComEC channel?
to transport DNA into the cell
what are the general features of the B. subtilus DNA uptake system? how is it related to other transport systems?
similar to type II/IV secretion system. com EA - DNA binds and brings it in comG form psuedopilus Nuc A at bottom of Com G makes it ss Com EC brings DNA in comFA ATPase ComK transcription regulator that controls com E comF comG ComA regulates ComK ABC transporter complex- ComEA, ComEC,ComFA
what are the "direct" and "indirect" regulatory mechanisms that lead to competence in B. subtilus? which is a two component system and what is the initial signal molecule?
Direct- ComX activates ComP phosphorylation comP phosphorylates ComA and COmA makes ComK Indirect-PhrC makes CSF CSF activates phosphorylation of ComA 2 component regulatory system is ComP-ComA initial signal molecule is ComX
What is the key difference in the composition of the DNA uptake systems of b. subtilus and Neisseria gonorrhoea? which rquires specific DNA sequences?
G- Neisseria has a hydrophobic outer membrane that DNA must first passs through it only recognizes a specific sequence. B. subtilus is nonspecific
What form of DNA enters these cells? what is meant by the statement that an eclipse phase occurs during transformation of Streptococcus pneumoniae?
ssDNA for both eclipse phase is when ssDNA can't initiate recombination after entry, or before recombination
What signals the development of competence in Haemophilus influenza? what structures form and how does DNA enter into the cells?
competence is regulated internally. transcription controlled by Sxy and CRP transformasomes uptake dsDNA. DNA enters cell, one strand degraded and recombination occurs
What is transduction and how does it differ from conjugation and transformation?
transduction is hte transfer of small DNA fragment from one bacterial cell to anotehr by way of a phage particle generalized-genes for any region (phage P1, P22) specialized-only genes near attachment site of the lysogenic phage (lambda phage)
what features of the P1 life cycle allow this phage to function as a generalized transducing phage?
a measured amount of its DNA is packaged and packaging is not sequence specific
what makes a good transducing phage?
-the phage must not completely degrade the host DNA -packing mechanism must not be so specific that it can't package host DNA -typical packaging mechanisms include head-full packaging and lenient pac site specificity
what is a lysogen and how does this differ from a lytic cell cycle?
a lysogen has phage DNA integrated into the chromosome a lytic cell cycle replicates phage particles and lyses the cell
how does the stability of the CII protein influence the decision of lamba phage to undergo a lytic or lysogenic infection?
High Cii conc. activates P1, so Int is made and lamba DNA integrates. it also activates Pre, and CI is made. C1 outcompetes Or & Ol, represses Pl & Pr and positively autoregulates at Prm. if CII is degraded, little C1 is made, Cro binds at Or & Ol, blocking binding of Ci. N allows O & P replication gene transcription, 2nd auto-terminator Q, allows late-gene transcription so lambda phage particles are made
what mechanisms does lambda phage use to integrate into the host genome? what phage lytic or lysogenic infection?
site specific recombination between attP and attB catalyzed by integrase
what stimulates the excision of lambda from the genome and what host sequences are most likely to be packaged by the phage? how does this relate to the ability of lambda to transduce host genes?
DNA damage -- ssDNA -- RecA-ssDNA binds to CI and this induces autocleavage by CI. CI can no longer bind to OL Or as a repressor. Int & Xis are produced and cleave the attB/attP site to excise lambda.
what role for [|]361 prophage play in the dissemination of toxin genes?
[|]361 encodes shiga toxins. StxA encodes a glycosylase that inactivates eukaryal 28S rRNA StxB is the helper protein that aids in the entry of the toxin into endothelial cells
what is transposition and how is this mechanism of gene transfer/movement different form those requiring RecA
transposable elements are mobile genetic elements that can direct their insertion into DNA using transposase
what are the two main types of transposition mechanisms and how does the number of transposon elements differ after transposition by these mechanisms?
replicate-# of transposable elements increases non-replicative-# of transposable elements remains the same
what are the three classes of transposable elements and how are they related in terms of organization and gene content?
IS - only has transposase Composite-central region, 2 IS elements, 1 w/ a functional transposase noncomposite-central region, transposase and resolvase, IR's that can't transpose
among the different transposons classes, what parts of the element can move?
IS- all can move, composite- all can move or just IS element, Noncomposite- resistance genes can move
what three activities are carried out by the Tn5 transposase? why is this transposition reaction referred to as conservatie or cut and paste? how are the direct repeats at the ends of the element generated during transposition?
TnpA of Tn5 - makes ds breaks at each end of transposon, makes staggered ds breaks at target site, and ligates 3' end of transposon to 5' end of target DNA Considered cut n paste b/c entire transposon excised and the inserted in another place direct repeats generated by the staggered cuts made in the target dna
how do deletions and rearrangements occur during conservative transposition?
transposase cuts at the inside of the IS sequence and there are staggered breaks in target DNA transposase can now join 2 different ways it can creat the loss of a region or it can create an inversion
what three activities are carried out by the Tn3 transposase and how do they differ from those carried out by the Tn5 enzyme?
TnpA of Tn3- makes SS breaks at each end of transposon, makes staggered ds breaks at target site, and ligates 3' end of transposon to 5' end of target DNA
what role does the resolvase, TnpR, play in the transposition of Tn3?
TnpR resolvase - resolves the cointegrate by site specific recombination between the res sites
how is the level of transposition of Tn3 controlled?
Level of transposition of Tn3 is controlled by the level of resolvase. resolvase blocks transcription from TnpA promoter
what sequences are recognized by transposases?
inverted repeats
how is the level of transposition of Tn10 controlled? what roles do antisense RNA interactions and DNA methylation play?
Level of transcription of Tn10 controlled by methylation. Fully methylated no trnsposition b/c RNAP can't bind . Hemimethylated is best for RNAP to bind and there is no Pout yet therefore most active when it first enters the cell Antisense RNA of Pin is Pout and when high amount of Pout it binds to Pin and prevents translation
what are examples of integrase enzymes and how does the process of site specific recombination differ from that requiring RecA?
integrase enzymes- lamda Int and related enzymes of conjugative elements like Tn916 Differs from recombination requiring RecA b/c it requires specific recombinase enzymes
what are the structural and sequence features of an integron? what role do integrons play in the dissemination of antibiotic resistance genes and how might they shape the overall gene content of a genome?
a gene (Intl) encoding an integrase belonging to the tyrosine recombinase family, a primary recombination site , an outward oriented primer a superintegron can hold many cassettes
what are examples of resolvase and invertases? what are the arrangements of the target sequences that are recognized by these enzymes?
TnpR of Tn3, Cre, and XerC,D invertases- phase variation in salmonella
how does invertase lead to a shift in flagellin production by Salmonella sp.?
when HIN invertase is used in salmonella the promoter is turned around causing the flagellin proteins to not be made
what are the two recombinase families (and why are they named after amino acids)?
Tyrosine and Serine- named after amino acid that plays a role in catalysis
what are the basic features of homologous recombination? what role does RecA play in the process of single strand invasion?
- pairing between identical sequences in the crossover region -complementary base pairing between ds DNA molecules -cutting n rejoining by recombination enzymes -Holliday junction
what type of lesion is recognized by the RecFOR (QJ) system and hwo does this system prepare DNA for recombination?
RecFOR recognizes gaps from excision repaira DNA lesion -aids in the binding of RecA abd displacment of SSB
what are Holiday junctions and how are they formed. what roles do the RuvABC proteins play in resolving these structures?
Holliday junctions are double stranded crossover between 2 duplex DNA -RuvABC proteins resolve Holliday Junctions Ruv A n B bind to the junction and Ruv C binds n cuts 2 of the strands then ligase is used
what are common sources of dna damage?
common sources of DNA damage- UV, alkylation, oxidation
what are the two mechanisms of incision in the excision repair process?
Base excision repair and Nucleotide excision repair
what lesion is recognized by the very short patch repair system? what is the basis for the specificity of this system?
VSP recognizes T-G lesion the basis of specificity was dam methylation sites
how do MutY and MutM participate in teh repair of 8-oxoguanine?
MutY glycosylase/ endonuclease removes misincorporated adenine. MutM glycosylase/ endonuclease removes 8-oxoguanine base
how do the functions of AlkA and TagA differ from those of Ada and AlkB in the repair of alkylated DNA? How does modification of Ada effect regulation of the alkylation repair genes?
AlkA- removes 3-mA, 3-mG, 7-mG TagA removes 3-mA AlkA n TagA are n-glyosylases Ada- remove alkyl groups from 06-G and 04-T AlkB and Ada- methyl transferases, repairs DNA w/o strand Cleaveage, inactivated by methylation modification of Ada- converts it into transcription activator for alkylation repair
what roles do the UvrABCD gene products play in DNA repair? what lesion do they recognize and why are DNAP I and ligase activities needed in conjunction with the UvrABCD enzymes?
UvrBCD- help w/ nucleotide excision repair, recognizes distortion in the helix They recognize a thymine dimer lesion DNAPI fills gap n ligase closes
how does photolyase repair cyclobutane dimers? what is an essential co-reactant in this reaction?
Photolyase repairs cyclobutane dimers by complexing the enzyme with the dimer, light is added the dimer is cleaved and the enzyme is released coreactant is light
how does the methyl dependent mismatch repair system give preference to the parental strand during repair? what DNAP is usually involved in the repair synthesis?
during methyl dependent mismatch repair - MutH cuts unmethylated strand of DNA and DNAPIII does repair
how do the homologous repair systems allow leading strand synthesis to continue past a thymine dimer?
Homologous repair allows the leading strand synthesis to continue past a thymine dimer by: allowing a RecA filament on the thymine dimer to invade the sister strand resolution of the junction by RuvABC and then replication continues
what are the key structural features of DNA?
double helix, antiparallel strands, hydrogen bonding deoxyribonucleotides (base-sugar-phosphate) joined by phosphodiester bonds
how does RNA differ from DNA in its chemical composition and structure?
U's instead of T's, higher order structure. 2'-OH
what features of RNA allow, or factilitate, some RNA molecules to act as enzymes?
single stranded, can form intramolecular base pairs
what are examples of non watson-crick pairing in DNA
G-U, G-T
do non watson-crick pairing interactions occur in RNA?
yes. they are important in the function of many RNAs. I-U/A/C
what is meant by the statement that replication of the E. coli chromosome is bidirectional and semi-conservative?
bidirectional-two replication forks moving in opposite direction semi-conservative - each daughter strand contains one parent strand and one newly synthesized strand
what is the polarity, or in which direction does DNA polymerizaiton occur?
5' to 3'. or N to C
what are the general requirements for DNA synthesis?
ori, DNA poly., DNA, dNTPs, ter
what reactions are common to the biosynthesis of all precursors of DNA synthesis and which precursors require specific enzymes?
ribonucleotide reductase - removes 2'-OH to make deoxyribonucleotide diphosphates kinase - adds phosphate to make NTP phosphatase - dUTP to dUMP thymidylate synthetase with THF (methyl donor) - dUMP > dTMP kinase - dTMP > dTTP
what reactions are common to DNAPI and DNAPIII?
5' to 3' synthesis and 3' to 5' exonuclease activity
what reactions are unique to DNAPI?
5' to 3' exonuclease activity
what events occur at the replication fork that allow the discontinuous synthesis of DNA on the lagging strand?
primase makes a short RNA primer on the lagging strand. DNAPIII synthesizes short fragments DNAPI removes primers w/ 5' to 3' exonuclease DNA ligase synthesizes phosphodiester backbone
what is "trombone" model of replication tell us about the coordination of leading and lagging starnd synthesis?
DNAPIII forms a complex so leading and lagging strand synthesis run simultaneously. leading is faster
how does DNAPIII detect and remove an incorrectly inserted nucleotide?
DNAQ stimulates 3' exonuclease activity when a mismatch is formed. the mismatch is removed, 5' to 3' synthesis fixes error
how does the DNA repair machinery give preference to the parental DNA during repair?
only the parent strand is methylated after DNA synthesis until Dam methylase adds methyl group. the repair system recognizes the methylated strand as the template
how are interstrand cross-links repaired? what roles do UvrABC and RecA play in the process?
Interstrand crosslinks are repaired by: Excision and recombination UvrABC cuts a side out- recombination repair w/ RecA occurs then nucleotide excision repair cleaves the link from the 2nd strand
how does the UmuD'2C complex function in the repair of thymine dimers? why is this system the "last choice" for repair?
UmuD'2C complex replicates over the damaged DNA in translesion synthesis. Last choice b/c makes a lot of mutations it is error prone
what is the SOS response?
The SOS response- Induction of transcription of the SOS genes in response to DNA damage - physiological responses that aid in the cell's survival
how is the expression of SOS associated genes regulated? what roles do RecA and LexA play in the control mechanism?
expression of SOS genes is regulated by LexA repressor. LexA dimer binds to SOS box and the RecA-SSDNa complex induces LexA to cleave itself
what are the three time intervals in the E. coli cell cycle?
I - time from when the last round of chromosome replicatin initiated until the next round begins C - time it takes to replicate the entire chromosome D - time from when a round of replication is complted until cell division occurs
which intervals vary w/ growth conditions and how does this affect the copy number of some genes?
I can vary, fires off oriC at different rates
what are the functions of oriC and ter?
oriC - DnaA binding sites. dam methylase target sites. DnaB helicase loading site ter - slow replication fork. bind the Tus protein (termination utilization substance) that stops replication
what are the sequence features of E. coli oriC and what are the events that lead to the initiation of replication?
GATC methylation sites. 13 mer AT rich sites. DnaA-ATP bind to DnaA boxes sites. A-T rich regions open up and DnaC/DnaB bind, DnaC releases
what physiological signals or regulatory signals control activation of oriC?
DnaA concentraation, ATP/ADP ratio, DnaA completition for binding with SeqA
how are chromosome dimers and inter-locking circles resolved following replication?
XerCD cleaves at dif -interacts with FtsK
what protein coordinates replication with cell division?
what proteins specify chromosome organization in bacterial nucleoids?
small nucleoid-associated (histone-like) proteins structural meaintenance of chromosomes (SMC) complexes
what are the two classes of topoisomerases in bacterial cells and what role(s) do they play in controlling DNA structure?
-topoisomerase I/III. cut 1 strand, fix and ligate. top I relaxes supercoil -topoisomerase II/IV (gyrase) breaks both strands, rotate and ligate. top II introduces negative supercoils
what are the targets of the replication inhibitors novobiocin and nalidixic acid?
novobiociin=gyrB subunit of gyrase. blocks ATP hydrolysis by the enzyme -nalidixic acid - gyrA subunit of gyrase. blocks topoisomerase activity
what is hydroxyurea an effective inhibitor of replication in living cells?
inhibits ribonucleotide reductase. can't convert GDP to dGDP
what are the two stages of rolling circle replicatrion for a plasmid DNA?
1. dsDNA replicates to produce a cccDNA and a ss circular DNA 2. the complementary strand of the ssDNA is synthesized to form a second cccDNA
how is the priming reaction different from that which occurs during bidirection replication of the chromosome?
Rep binds, nicks 1 strand, attaches 5'-P. 3'OH is primer for DNAPIII
what are the two stages in lambda replication?
early(bidirectional) and late (rolling circle)
what phage specific proteins are needed and how does lambda "pirate" the host replication machinery?
O acts as DnaA. P acts like DnaC, pirates DnaB to bind to O and begin replication lambda Gam blocks RecBCD from stopping r.c.r. lambda cro represses transcription from Pr

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