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30 Cards in this Set
- Front
- Back
Describe Recombination bya tyrosine recombinase |
(a) the R1 and R3 subunitscleave the DNA, proteinbecomes linked to the cut DNAby a 3´ P-tyrosine bond.
(b) Exchange of the firstpair of strands occurs when thetwo 5´-OH groups at the breaksites each attack the protein-DNAbond on the other DNA molecule. (c, d) The second-strand exchangeoccurs by the same mechanism,using the R2 and R4 subunits.Cleavage of the bottoms strandsresolves the Holliday junction. |
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Bacterial transposons were firstdiscovered as ? What experiment?
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insertional mutations, Used λ phage to pick up hostDNA:either λgal+ or λgal- • gal- = mutant form of gal gene • λgal- density was greater inCsCl centrifugation • Because gal- seq containsmore DNA (~1kb) than gal+• gal- mutants contain extraDNA, i.e. insertion sequence |
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What are the three classes of transposable elements? |
DNA-only transposons/ IS elements, Retroviral-like retrotransposons,
Nonretroviral retrotransposons |
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Describe DNA transposons/ Insertion Sequence (IS) |
transposase gene and inverted terminal repeats and generate direct repeats of flanking DNA at target site, Duplicates target sitesequences of the host DNAupon insertion |
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What GENERATES DIRECT REPEATS IN HOST DNA ? |
Staggered cuts in targetDNA permit ligation oftransposable element intohost DNA single-strandedoverhangs |
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What is a composite transposon? What orientation is the IS element in? how is it created? whats an example? |
flanked by 2 IS elements indirect or inverted orientation- contain IRs, transposase genes plus additional genes,i.e. antibiotic resistance genes
created when any two IS pick up DNA between them, example Tn10 plasmid
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What are the types of transposition? |
1. REPLICATIVE: element is duplicated
2. NONREPLICATIVE: element movesfrom one to another site leavingbreak at donor site 3. CONSERVATIVE: element moveswithout leaving break at donor site |
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Whats an example of Replicative transpositon? Whats the structure of it? |
Tn3, tnpA and tnpR are required fortransposition; res is the site of recombination;bla encodes Ampr (β lactamase); inverted repeats (IR); (can track it with antibiotic resistance) |
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Whats the process of transposition in Tn3? |
Transposon ends and targetDNA are nicked (1) and joined(2) - strand transfer complexII. 3’ ends (b & c) of strandtransfer complex are used asprimers for transposonreplication (3), ligate (with e &h) forming a COINTEGRATE(4)III. Cointegrate resolution isachieved by homologousrecombination between twocopies of the transposon (5)and catalyzed by resolvase (6
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Describe non-replicative transposition |
1. Generation of strand transfercomplex as in replicativetransposition
2. But, NO REPLICATION: newnicks in donor DNA flankingtransposon - 2 plasmids released3. Gaps in recipient DNA filled (inconservative transposons, donorDNA may be repaired) |
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Describe Conservative Transpositon |
concerted movement of all nucleotide bonds and transposable element to donor to recipient |
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Nobel prize winner for transposition in eukaryotes? (corn) |
Barbara McClintock |
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Spotted kernel is the result of what? |
The spotted kernel shows the results ofreversion in some of the kernel’s cells.Wherever the mutation has reverted, therevertant cell and its progeny will be able tomake pigment, giving rise to a dark spot on thekernel |
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What kind of transposon is Ac and Ds? Desribe the type briefly |
Ac is an autonomous transposon (can transpose itself), Ds is nonautonomous(can transpose but only with the help of Ac) |
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What kind of mechanism does Ac/Ds transposon use? |
Transposition of Ac/Ds occurs by a non-replicative mechanism |
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Describe the Ac transposase element |
encodes transposase and is flanked by imperfectinverted terminal repeats |
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How are acentric and dicentric chromosomes made? what are they? |
acentric fragment is a segment of a chromosome that lacks a centromere, formed at Ds break sites, dicentric is when they have two centromeres, this is the fusion of two chromosome fragments
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How does P-M HYBRID DYSGENESIS in drosophilia occur? |
M in females, P in males, P-M hybrid dysgenesis iscaused by P-elementtransposition and insertioninto genes |
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What color eyes are Drosophilia wildtype vs with P element? |
White eyes if they have P element, Red eyes are wild type |
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Why do P-elements transpose andcause dysgenesis only in hybrids? specifically describe P females vs M females
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P elements are onlyactive in germline, P Females have a repressor that will repress the P element, M females do not, thus they only transpose in M females, P mothers deposit the 66kDa repressor protein intoeggs, which preventstransposition in her descents |
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Describe P element, and their exons? |
P elementshave inverted repeats,generate target siteduplications, and encodetransposase, P element contains 4 exons(ORF0, 1, 2, and 3)• First 3 exons are splicedtogether in somaticexpression – makes a 66kDrepressor • All 4 exons spliced in germline expression – makes87kD transposase |
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What was the experiment for reverse transcriptase? |
they incubated retrovirus particles with 4 DNTPS, thenacid-precipitated the product (DNA) andmeasured radioactivity by liquidscintillation counting, RNA template -> DNA product,RNase-sensitive, RT enzyme is inside theviral particle |
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what're the two groups of retrotransposons? |
– LTR-Retrotransposons: replicate like retroviruses (RNA intermediate) butdo not pass from cell to cell in viral particles – Non-LTRs retrotransposons: replicate by nicking thetarget DNA and reverse transcription |
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Describe LTR-Retrotransposons |
replicated like retroviruses through an RNA intermediate, have gag-like genes that codes for the nucleo-protein coat and pol-like gene encoding reverse transcriptase, integrase, RNAse H polyprotein |
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Whats the name of the LTR-retrotransposon in fruitflies and yeast? |
copia in fruit flies, Ty (transposon yeast) in yeast |
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Describe Ty1 element and its mechanism of transposition |
Ty element has long flanking direct repeats that flank gag-like and pol-like genes
Ty element is first transcribedwith its experimentally suppliedintron, spliced, the spliced RNAis reverse transcribed intodsDNA, and then inserted backinto the yeast genome at a newlocation. |
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What are some env encoding retrotransposons in yeast/fruitflies |
Yeast Ty3 & Drosophilagypsy retroposons DOencode env-like proteins |
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Describe Non-LTR (long terminal repeat) RetroTransposons, (whats the most abundant, what're they missing, ORFS) ? |
Most abundant are the Long interspersed elements (LINEs), But most missing their 5’-ends and have mutations that preventtransposition, Two OpenReadingFrames: • ORF1 encodes an RNA-binding protein p40 • ORF2 encodes a protein with two activities: an endonuclease (EN)and a reverse transcriptase (RT). |
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What is L1 transposition (Line1) or Target-primed retrotransposition mechanism? |
Transposon is transcribed and processed(mainly from a neighbouring promoter) Translated in the cytoplasm producing ORF1product (p40) and ORF2 product(endonuclease and RT activities)– these proteins associate with the mRNAto form a RNP (ribonucleoprotein)c) RNP reenters the nucleus; endonucleasenicks the target DNA; RT uses the 3’-end toprime synthesis of the reverse transcriptd) Second strand of L1 is made, second strandof target is cleaved, L1 is ligated into thetarget DNA |
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what are SINES? |
SINES (short interspersed elements) • Nonautonomousretrotransposons • 100-300bplong • Do not encodeproteins • No introns • Noterminalrepeats • Types:pseudogenes–DNAsequencesthatresemblegenes but have no function for a variety of reasons – probably arose by reverse transposition throughan RNA intermediate – Some have inactive or missing splicing signalswhile others inactive promoters • Usually a combination of these problems and others,prevents their expression |