Transposable genetic element

Change in genetic makeup

Introduction

•Segments of the genome that are capable of moving around to different location.

•Transposable elements usually are flanked (Be situated on each side of or on one side of something) by repeated sequences .

•Carry transposase genes that confer the transposition ability .

•Variety of names controlling elements , jumping genes , mobile genes , mobile genetic elements , transposons.

•Found in phages ,viruses , prokaryotes ,eukaryotes and Insects.

Types of transposable elements

In prokaryotes

• Insertion sequence (IS) elements

• Transposons (Tn)

• Bacteriophage Mu

Insertion Sequence• segments of bacterial DNA that can move from one position

to a different position• Range in size from 768 bp to 5 kb• Disrupt coding sequences or regulatory regions.

• Alter expression of nearby genes.

• Cause deletions and inversions in adjacent DNA.

• Result in crossing-over.

Physical demonstration of DNA insertion

• IS (insertion sequences) were first discovered in the gal operon of E. coli

•Mutation by insertion is demonstrated with phage lambda particle carrying the bacterial gene for galactose utilization (gal+) or the mutant gene gal-

The increased density of gal- particle was caused by the insertion of DNA

Direct visualization

•Wild type lambda d gal•Mutated lambda d gal •Electron microscopy heteroduplex,single

stranded loop,no complementry sequence to wild type

•800 nucleotide•4 single stranded tail

Physical demons. Contd.:

Direct visualization of inserted DNA

The single stranded loop is caused by the presence of an insertion sequence in dgalm

Electron micrograph of a dgal+/ dgalm DNA heteroduplex

Each heteroduplex shows a single stranded buckle, or loop

Insertion sequence

Transposition of insertion sequence (IS) elements:

1. Original copy remains in place; new copy inserts randomly.

2. IS element uses host replication enzymes for replication.

3. Transposition requires transposase, coded by the IS element.

4. Transposition initiates when transposase recognizes ITRs(inverted terminal repeats)

5. Site of integration = target site.

6. Staggered cuts are made in DNA at target site, IS element inserts, DNA polymerase and ligase fill the gaps.

7. Small direct repeats (~5 bp) flanking the target site are created.

Integration of IS element in chromosomal DNA

Transposons

• Structure formed when denatured drug resistance plasmid DNA is re annealed(Reannealing is the process by which two single strands of DNA combine to form double-stranded DNA)

• Double stranded IR region separates a large circular loop from the Small “lollipop” loop

•Gene for drug resistance by plasmid between IR of lollypop called Transposons

Transposons

Movement of Transposons

a)Transposons before denaturation ds

Insertion of Transposons in Plasmid

Transposable elements in the evolution of drug resistance

Phage mu• a normal-appearing phage• 36,000 nucleotide long• can insert itself anywhere in a bacterial or plasmid genome in either orientation; mutation in the genome like IS • each mature phage particle has on each end a piece of

flanking DNA• from its previous host no insert into genome in next

generation• contain its own IR sequences; but not in the chromosome

ends

•genetic snap fastener: phage mu can mediate the insertion of phage or drug resistant gene into a bacterial chromosome using 2 mu genome

Bacterial genes into a plasmid by phage mu

Deletion & insertion of adjacent bacterial segments by phage mu

Mechanisms for transposition

• Replicative transposition: The element moves a copy of itself to a new site via a DNA intermediate

• Conservative transposition: (Non replicative) The element itself moves from the donor site into the target site

Replicative

•A new copy of the transposable element is generated in the transposition event

•The Transposition of Tn3 takes place through a cointegrate intermediate

Co integrate intermediate transposition

Replicative transposition

Conservative Transposition

•Some transposons excise from the chromosome and integrate Into the target DNA

- Generation of heteroduplex and • homoduplex Tn10 elements

Conservative Transposition

Consequences of Conservative & Replicative Transposition

Contd.:

Transposable Elements in Eukaryotes

•Yeast•Drosophila•Retroviruses•Maize

Transposable elements in yeast

•Ty elements•Ty1 sequence •35 copies in yeast genome•38 basepair long terminal sequence -- delta sequence

-100 copies•Delta sequence are present in direct –repeat orintation•Ty element are transposases through an RNA

intermediate

Ty element

The mechanism for transposition by RNA intermediate

- The addition of galactose greatly increases• the frequency of transposition of the altered Ty

element

- The transpose Ty DNA contains no intron- Transposition occurred through RNA intermediate

Contd.:

Retrotransposition

Retroviruses•SSRNA animal viruses•Reverse transcriptase•E.g: mouse mammary tumor virus (MMTV) , Rous

Sarcoma Virus (RSV) –cancer • Integrated into host chromosome as DSDNA called

provirus •Like mu phage considered as transposable element ,

can infect , transposes.• Integration results in duplication of short target

sequence in host chromosome

Retroviruses

Contd.:

Transposable elements in Drosophila

•10% of the drosophila chromosome consists of family of dispersed ,repetitive sequences that moves as discrete elements called transposable elements.

•Copia like element•FB element•P element

Copia like element

•7 families•5-8.5 kb•Appear at 10 -100 positions at drosophila

chromosome•Long direct •Upon insertion causes duplication of

number of base pairs in drosophila DNA

FB element

•FB --- fold back•Few hundreds to few thousands base pairs•Long inverted terminal repeats •Some time entire elements consists of inverted

repeats ,central sequence separate the inverted repeats•Mutation due to interruption by FB element in gene

coding sequence or near a control region •Excise them from the genome and promote

chromosomal rearrangements at high frequencies

Transposable elements in drosophila

P-elements•Hybrid dysgenesis •Drosophila Melanogaster•Lab- M Cytotype female•Natural P- Cytotype male•Sterility, high mutation rate ,high

frequency of chromosomal aberation and non disjunction

•Result –dysgenic or biologically defected•Vice versa no mutation•Large no of mutations are unstable---wild

type

Contd.:

•Eye –color locus• Insertion of genetic element in to the middle of the

white gene•Called P-element•30-50 copies per genome in P-strain•0.5 -2.9 kb •31 bp perfect inverted repeats at their ends

P-Element

Using P elements to insert genes- P elements mobilize only in germ-line cells

Controlling element of Maize

• 1938: Marcus Rhoades reported an odd finding in phenotypic ratios of corn...

• Self pollination of a true-breeding pigmented corn kernel yielded 12 : 3 : 1 ratio of pigmented : dotted : un pigmented kernels

Contd.:

• He first hypothesized that two events had occurred at unlinked loci: ▫1. Pigment gene A1 had mutated to colorless mutant a1

▫2. At another locus, a dominant allele for dotting (Dt) had appeared

• What was causing the dotted phenotype?• - Reverse mutation of a A some are completely pigmented type- a : unstable mutant allele (high reversion rate)- the allelic instability is dependent on the unlinked Dt gene

Contd.:

Contd:

McClintock’s experiments: the Ds element

• 1950's: McClintock reported the presence of a genetic factor Ds (for "Dissociation")

• whose presence caused a high degree of chromosome breakage wherever it appeared

• The action of Ds is another type of instability• The instability of Ds turned out to depend on the

presence of an unlinked gene Ac (for "Activator") • Ac & Ds locus was constantly changing position

McClintock’s experiments: the Ds element

Contd:

Kernel color in corn and transposon effects

The transposition of Ds into the C gene in corn

General characteristics of controlling elements