Mbb 331 Lec 11

Front 1

Describe Recombination bya tyrosine recombinase

Back 1

(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.

Front 2

Bacterial transposons were firstdiscovered as ? What experiment?

Back 2

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

Front 3

What are the three classes of transposable elements?

Back 3

DNA-only transposons/ IS elements, Retroviral-like retrotransposons,

Nonretroviral retrotransposons

Front 4

Describe DNA transposons/ Insertion Sequence (IS)

Back 4

transposase gene and inverted terminal repeats and generate direct repeats of flanking DNA at target site, Duplicates target sitesequences of the host DNAupon insertion

Front 5

What GENERATES DIRECT REPEATS IN HOST DNA ?

Back 5

Staggered cuts in targetDNA permit ligation oftransposable element intohost DNA single-strandedoverhangs

Front 6

What is a composite transposon? What orientation is the IS element in? how is it created? whats an example?

Back 6

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

Front 7

What are the types of transposition?

Back 7

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

Front 8

Whats an example of Replicative transpositon? Whats the structure of it?

Back 8

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)

Front 9

Whats the process of transposition in Tn3?

Back 9

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

Front 10

Describe non-replicative transposition

Back 10

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)

Front 11

Describe Conservative Transpositon

Back 11

concerted movement of all nucleotide bonds and transposable element to donor to recipient

Front 12

Nobel prize winner for transposition in eukaryotes? (corn)

Back 12

Barbara McClintock

Front 13

Spotted kernel is the result of what?

Back 13

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

Front 14

What kind of transposon is Ac and Ds? Desribe the type briefly

Back 14

Ac is an autonomous transposon (can transpose itself), Ds is nonautonomous(can transpose but only with the help of Ac)

Front 15

What kind of mechanism does Ac/Ds transposon use?

Back 15

Transposition of Ac/Ds occurs by a non-replicative mechanism

Front 16

Describe the Ac transposase element

Back 16

encodes transposase and is flanked by imperfectinverted terminal repeats

Front 17

How are acentric and dicentric chromosomes made? what are they?

Back 17

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

Front 18

How does P-M HYBRID DYSGENESIS in drosophilia occur?

Back 18

M in females, P in males, P-M hybrid dysgenesis iscaused by P-elementtransposition and insertioninto genes

Front 19

What color eyes are Drosophilia wildtype vs with P element?

Back 19

White eyes if they have P element, Red eyes are wild type

Front 20

Why do P-elements transpose andcause dysgenesis only in hybrids? specifically describe P females vs M females

Back 20

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

Front 21

Describe P element, and their exons?

Back 21

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

Front 22

What was the experiment for reverse transcriptase?

Back 22

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

Front 23

what're the two groups of retrotransposons?

Back 23

– 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

Front 24

Describe LTR-Retrotransposons

Back 24

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

Front 25

Whats the name of the LTR-retrotransposon in fruitflies and yeast?

Back 25

copia in fruit flies, Ty (transposon yeast) in yeast

Front 26

Describe Ty1 element and its mechanism of transposition

Back 26

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.

Front 27

What are some env encoding retrotransposons in yeast/fruitflies

Back 27

Yeast Ty3 & Drosophilagypsy retroposons DOencode env-like proteins

Front 28

Describe Non-LTR (long terminal repeat) RetroTransposons, (whats the most abundant, what're they missing, ORFS) ?

Back 28

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).

Front 29

What is L1 transposition (Line1) or Target-primed retrotransposition mechanism?

Back 29

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

Front 30

what are SINES?

Back 30

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