Transposons and insertion sequences

Question 1

Polar mutations

Answer 1

A polar mutation affects expression of downstream genes or operons. It can also affect the expression of the gene in which it occurs, if it occurs in a transcribed region. These mutations tend to occur early within the sequence of genes and can be nonsense, frameshift or insertion mutations. Polar mutations are found only in organisms containing polycistronic mRNA.

Question 2

Transposition

Answer 2

the process by which transposable elements (interspersed repeats) are copied and move to another site in the genome

Question 3

Insertion Sequences Bacterial Mobile elements

Answer 3

Inverted repeat of ~50bp on each end (5’-3’ sequence repeated at both ends). Between these repeats is a region encoding transposase. Either side of the IS element are direct repeat sequences that are characteristic of each IS

Question 4

Frequency of transposition in E. coli

Answer 4

10^5-10-^7 per generation. higher rates may interfere with genes and kill individual. Less than 10 IS sequences in E. coli

Question 5

Transposase

Answer 5

coded for by IS element. Cuts the target DNA with sticky ends and the donor DNA with blunt ends

Question 6

Composite transposons

Answer 6

IS elements flank a marker gene. E.g. two Tn9 for chloramphenicol resistance or Tn10 and Tn5 for other markers

Question 7

Contol of transposase

Answer 7

Over production of RNA prevents in one direction prevents the RNA forming transposase in the other direction. The RNAs bind and so cannot form protein. Methylation also plays a part (out tn10 and in tn10). Tn5 Repressor of 58kDa In-frame with transposase.

Question 8

Tn10 and Tn5

Answer 8

replication of transposon non-replicative. destruction of donor site instead of repair would be lethal

Question 9

Transposition intermediate

Answer 9

the form the element takes before it has bound to any target sites and after it has been cleaved from donor DNA

Question 10

Tn3 family

Answer 10

family that replicate via replicative transposition. Tn3, Tn1 encodes ampicilin resistance, Tn501. Encodes resistance to mercuric ions. Contain genes for tnpA and tnpR

Question 11

Replicative transposon

Answer 11

the transposon is both replicated and inserted into a new site

Question 12

Cointegrate

Answer 12

Two dna loops. Donor and reicipient DNA fuse. Single strands filled in by DNA replication. Cointegrate is figure of 8 at this point. Breaks apart after recombination between transposons

Question 13

Ty elements

Answer 13

resemble retroviruses so are known as retroposons. In yeast there are 33 ty1 elements and 13 ty2 elements

Question 14

Ty element structure

Answer 14

It is a LTR retrotransposon. Target direct repeats flank LTR (250-600bp) and these LTRs flanka protein coding region. This protein coding region. Coding regions TyA and TyB have properties of a DNA binding
protein. c.f. gag of retroviruses and properties similar to the reverse
transcriptase, protease and integrase
functions of retroviruses c.f. pol respectively

Question 15

Ty element mechanism

Answer 15

Transcription –> reverse transcription (to form DNA) –> integration

Question 16

Integration of viral DNA

Answer 16

viral dna cut by integrase, attacks target dna, gap filling by dna repair,

Question 17

Transposons in Drosophilia

Answer 17

Same mechanism as in yeast. Retrotransposons (e.g. copia). Also similar mechanism to bacteria (p-element). This causes hybrid dysgenesis

Question 18

P-element

Answer 18

alternate splicing can lead to somatic mRNA which is a repressor or germline mRNA which codes for transposase. Therefore the transposition only occurs in germ-line cells because the splicing event only happens here. Hybrid dysgenesis refers to the high rate of mutation in germ line cells of Drosophila strains resulting from a cross of males with autonomous P elements (P Strain/P cytotype) and females that lack P elements. The eggs of wild-type mothers repress the transposase splicing

Question 19

Human transposons

Answer 19

retrotransposons that lack LTR. LINEs 20% of human genome, L1 common family, more than 5kb in length. SINEs, best known alu family, require LINE for transposition, about 300bp in length, 300,000 to 500,000 alu in human genome

Question 20

Mechanism of LINE integration

Answer 20

RNA synthesis, ORF1 and ORF2 are translated from the RNA. In complex with ORF 1 and ORF 2 (binds to poly A tail) it is transported back into the nucleus where it is reverse transcribed into LINE DNA by ORF2. TTT, AAA used as primer (d Insertion is completed by cellular enzymes and it is inserted into new location.

Question 21

Plant transposons

Answer 21

Several families of transposons in plants. Best studied are found in Maize. All families are unrelated, but closely resemble bacterial transposons. They have IR ends and create DR insertion sites. Each family consists of a complete fully functional transposon, or autonomous element (eg. Ac) and a non-functional nonautonomous element (eg.
Ds).

Question 22

Maize Kernal

Answer 22

Ac activates Ds which transposes into C (gene for colour). It can then activate transposition of Ds out of C. A reversion of Cmutant to C during development results in spotted kernel

Question 23

Ac replication

Answer 23

if Ac transposes to an unreplicated donor site during replication there will be increase in number of transposons