Micro Test II – Flashcards
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| Topoisomerases |
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| Type I: reactive, cut one strand, and reattach to reduce torsion Type II: preemptive, cut both strands and twist negatively, to preempt torsion |
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| Prokaryotic DNA Replication Regulation |
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| 1) Methylation of origin (Hi = go) 2) SeqA (association = no go) 3) clamp loader concentration (Hi = go) |
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| helicase loader |
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| recruits helicase and allows it to bind origin |
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| DNA organization types in Bacteria |
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| linear/circular/mix; also plasmids, and/or chromids (sinorhizobia) |
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| Regulon |
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| all the operons/genes affected by a regulatory element |
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| rolling circle model |
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| DNA pol displaces one strand, synthesizing a new one in its place. Displaced strand gets replicated independently |
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| shuttle vector |
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| usually a plasmid, built to propagate in 2+ species |
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| metagenomics |
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| the study of multiple genomes found in a single environment simultaneously, helps study diversity |
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| PCR |
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| requires two polymerases, gene in question, NTPs, and 2 primers; melt (95), anneal (55), synthesize (72), repeat |
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| DNA melting point |
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| depends on GC content: more GC higher melting point |
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| Shot-gun sequencing |
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| Dideoxynucleotides added to truncate at random places, giving you oligo’s that overlap, giving you contigs and possibly the full sequence |
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| Open Reading Frame (ORF) |
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| gene or series of genes (mono/polycistron), which code for protein(s) |
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| promoter |
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| prokaryote: -35 and -10 consensus sequences that recruit sigma factor |
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| sigma factor |
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| RNP which locates/binds promoter and recruits holoenzyme; different sigmas for different kinds of genes |
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| Initiation (translation) |
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| 1) 30S unit binds start codon with help 2) Met-charged tRNA is recruited 3) 50S subunit encompasses tRNA w/ help |
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| Elongation (translation) |
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| 1) new tRNA enters A site, peptidotransferase acitivity moves protein chain to its amino acid (ATP) 2)50S unit is shifted down one codon, tRNAs are shifted down one site (ATP) 3) 30S unit is shifted afterward |
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| Termination (translation) |
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| 1) RF1/2 enters A site, PTase releases protein (ATP) 2) 50S and 30S are separated (ATP) |
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| how antibiotics affect transcription |
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| intercalation into DNA proper (actinomycin D) binding active sites in RNA pol (rifamycin B) |
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| tmRNA |
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| adds tags to proteins of stop-less mRNA; chaperones take tagged proteins to ATP-dependent proteases |
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| TAT |
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| secretory protein complex; secretes entire folded proteins to periplasm |
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| direct secretion |
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| three part secretory protein complex that transports amino acid chains through both cell membranes without it ever coming in contact with the periplasm |
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| proteosomes |
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| hetero (eu), or homoheptameric (pro) rings of protein which degrade tagged proteins. Hetero has an unwinding cap (sometimes) |
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| list the major types of RNA in order of increasing half-life |
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| mRNA, tRNA, then rRNA |
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| how we know codons are 3 bps |
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| alternating bp's gave two different amino acids: ATA and TAT; if 2: it would be the one, over and over: AT and AT |
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| Merodiploid |
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| haploid organism with an extra copy of part of its own genome; occurs through Hfr conjugation |
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| Competance and quorum sensing |
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| CF excreted and, at high enough concentration, reenters, begins pathway for gene transcription of transformation |
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| Pheromones and conjugation |
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| pheromones produced by recipient, enter donor, induce genes which code for enzymes on conjugation pathway |
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| specialized vs. generalized transduction |
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| S: few close genes are transduced (lysogenic virions) G: any set/number of gense can be transduced (lytic virions) |
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| Holliday Structure |
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| Rec D causing the recombination of donor and recipient DNA such that one strand of the donor is paired with the complementary strand of the recipient and vice versa (+) |
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| Cointegrate (noun) |
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| intermediate molecule during generalized recombination; D + R (Holliday) |
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| Cotransduction |
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| two genes are simultaneously added to host genome during transducive recombination: the closer they are on the donor genome, the more likely they are to be cotransduced |
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| Six mutation repair mechanisms |
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| NER, Methylation mismatch, BER, Recombination, Translesion bypass synthesis, Photoreactive Mnemonic: NuMBeR TraP |
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| Griffith |
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| Microbiologist who discovered transformation: live non-virulent strain + dead virulent strain = death |
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| Competence of Bacteria Based on Gram Staining |
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| Gram positive: naturally competent Gram negative: requires membrane compromising mechanisms |
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| Transformation |
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| CFs --> dsDNA --> recombination |
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| Starved F+ E. Coli... |
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| ...will cease maintenance of unnecessary mechanisms (including plasmids) |
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| Sex Pilus |
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| DNA is transferred through hollow tube rather than relaxosome |
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| macrolesions (5) |
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| Inversions, mass insertions/deletions, translocations, and duplications |
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| Positive selection |
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| Poisoning a population to select for mutants resistant to poison (pen + screening) |
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| Indirect selection example |
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| tryptophan auxotrophs, replica plating |
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| Ames test |
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| His - in limited Histidine, healthy colonies are REVERTANTS; mutagen testing: more revertants, more mutagenic; Benzpryene, only mutagenic when metabolized: add rat liver homogeny |
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| Recombination |
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| generalized: considerable homologous DNA, RecA mediated specialized: site specific, recombination enzyme mediated (no RecA) |
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| Transition |
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| substitution of purine for purine, or pyrimidine for pyrimidine |
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| Transversion |
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| substitution of purine for pyrimidine, or pyrimidine for purine |
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| Transcriptional Attenuation (example) |
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| as w/ trp attenuation: high [trp], ribosome stalls to polymerize, allowing 3:4 attenuation loop to form, and release RNA pol; low [trp] 2:3 loop forms allowing time for RNA pol to begin transcription of functional genes |
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| Sigma Factor Regulation |
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| Transcriptional regulation, Anti-/anti-anti-sigma factor activity (sporulation), Methylation, conditional Proteolysis Mnemonic: TAMP(on) |
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| Major pro and con to transcriptional modification |
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| ideal energetically, very slow |
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| operon |
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| regulator--operator--promoter--SG--SG--SG |
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| Synthesize protein from one prokaryotic org's gene in another prokaryotic org (6) |
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| PCR, restrict stock plasmid and gene with same RE, ligate gene into plasmid, detect its presence, denature wall of recipient (if incompetent) and transform plasmid, select for F+ strain |
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| Ways to detect presence of gene in plasmid |
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| mass spec before and after or a good gel; re-restrict: 1 or 2 pieces? |
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| Polyethylene glycol |
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| induces competence in E. coli by denaturing outer wall |
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| Specify the gene for a particular protein |
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| Sequence the amino acids of protein, predict the sequence, probe endonuclease polymorphisms for the sequence, PCR that morphism (use same endonuclease to introduce said morphism into a plasmid, transform) |
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| synthesize a eukaryotic protein in a prokaryote |
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| buy stock mRNA for that protein(no introns), use viral reverse transcriptase to create ds cDNA, PCR, plasmid, transform |
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| Mechanism of Gene Therapy |
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| buy cDNA for gene, transduce; may kill patient |
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| Mechanism of Vaccine Therapy |
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| specify the gene for the primary antigen (a.a. sequence, predict, probe), insert into plasmid for E. coli and replicate (denature, transform, conjugate), isolate protein from E. coli, innoculate |
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| RNA dependent DNA polymerase |
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| used to make cDNA from mRNA in gene studies |
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| Steps of Plasmid Genetic Engineering |
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| 1: Restrict/Digest DNA in question 2: Isolate Gene 3: cleave plasmid 4: transform 5: ligate |
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| Promoters Serve what specific purpose? |
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| To attract RNA Pol, to synthesize RNA strands. Period. |
