Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 17 RECOMBINATION AND TRANSPOSITION AT THE MOLECULAR LEVEL

Transposition integration of small DNA segments into chromosomes Can occur at many locations within genome transposable elements (TEs) “jumping genes” DNA segments that move 1 st identified by Barbara McClintock in corn Transposable Elements And Transposition

Babara McClintock identified many unusual features of corn chromosomes She noticed that in one strain of corn, chromosome 9 tended to break at a high rate at the same site termed this a mutable site or mutable locus This observation initiated a six-year study that culminated in 1951 with the following proposal Mutable sites are actually locations where transposable elements have been inserted into the chromosomes She received the Nobel Prize in 1983 for this work McClintock Discovers Moving Loci in Corn

3 general types of transposition Simple transposition Replicative transposition Retrotransposition Transposition Pathways

Figure Known as Insertion Sequences - IS bacterial Tn10 eukaryotic Ac/Ds Simple Transposition

Involves replication of the TE and insertion of the copy into another chromosomal location Only found in bacteria Figure Replicative Transposition

Very common but only occurs in eukaryotes These types of elements are termed retroelements or retrotransposons Similar organization to retroviruses Figure Retrotranposons & Retrotransposition

Simple & Replicative Transposons Both contain a gene encoding a transposase enzyme Transposase function recognizes direct and indirect repeats cuts DNA for both excision and insertion

Figure Direct repeats – DNA sequences that are identical and run in the same direction (5’  3’) Inverted repeats - DNA sequences that are identical (or very similar) but run in opposite directions 5’ CTGACTCTT 3’ 3’ GACTGAGAA 5’ 5’ AAGAGTCAG 3’ 3’ TTCTCAGTC 5’ and Regulatory Sequences of Transposable Elements 5’ ATGACTGAC 3’ 3’ TACTGACTG 5’ 5’ ATGACTGAC 3’ 3’ TACTGACTG 5’ and transposon

Figure Contain additional genes that are not necessary for transposition per se Only the two inverted repeats at the ends of the transposon are involved in the transposition event Only these are adjacent to direct repeats Composite Transposons

Organization is similar to insertion sequences Resolvase gene is found between the inverted repeats Both enzymes are needed to catalyze the transposition of these types of elements Figure Elements of Replicative Transposons

Evolutionarily related to known retroviruses Retroviruses - RNA viruses that make a DNA copy that integrates into the host’s genome LTR – long terminal repeat act as promoters to transcribe viral genes – in this case RT and Int genes RT – reverse transcriptase uses RNA as a template to synthesize a cDNA (complementary DNA) Int – integrase recognizes DR sequences, cuts host DNA and insert retroelement sequences There is no excision of retroelements or retroviruses! There are ~100,000 copies of the L1 retroelement in humans Virtually all have lost function of RT and/or Int genes Retrotransposons – Retroviral-Like Elements

Figure Share little sequence similarity with retroviruses derived from normal eukaryotic genes some have RT or RT-like gene, many such genes are not functional Alu family of non-viral retroelements derived from a single ancestral gene known as the 7SL RNA gene has been copied by retroposition to > 500,000 copies ~ 6% of the human genome An example of a SINE – short interspersed element Non-viral Retroelements

Describing Function of Transposable Elements Autonomous contain all the information necessary for transposition to occur functional transposase, RT, Int etc… DNA elements – DRs, IRs, LTR, etc… Nonautonomous lack a gene or sequence element necessary for transposition If element is missing – transposon will not transpose if Transposase is mutated, element can still transpose if enzyme from another transposon “helps” it McClintock’s Ds element is nonautonomous lacks transposase gene McClintock’s Ac locus (Activator) is autonomous has functional transposase enzyme If Ds and Ac are both present in genome, transposition of Ds can occur

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The enzyme transposase catalyzes the removal of a TE and the its reinsertion at another location Transposase recognize the inverted repeats at the ends of a TE and bring them closer together The remainder of the general scheme for simple transposition is shown in Figure Transposase Catalyzes Excision & Insertion 17-65

Figure Transposase Catalyzes Excision & Insertion

Figure They are in the same direction and are repeated at both ends of the element

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Over the past few decades, researchers have found that transposable elements occur in the genomes of all species Transposable Elements Influence Mutation & Evolution 17-74

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In some cases, repetitive sequences in eukaryotic genomes are due to the proliferation of TEs In mammals, for example LINEs Long interspersed elements Usually 1,000 to 5,000 bp long Found in 20,000 to 100,000 copies per genome SINEs Short interspersed elements Less than 500 bp in length Example: Alu sequence Present in 500,000 to 1,000,000 copies in the human genome 17-76

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The biological significance of transposons in evolution remains a matter of debate There are two schools of thought 1. TEs exist because they simply can! In other words they are like parasites They can proliferate within the host as long as they do not harm the host to the extent that they significantly disrupt survival This has been termed the selfish DNA theory 2. TEs exist because they offer some advantage Bacterial TEs carry antibiotic-resistance genes TEs may cause greater genetic variability through recombination TEs may cause the insertion of exons into the coding sequences of structural genes This phenomenon, called exon shuffling, may lead to the evolution of genes with more diverse functions 17-77

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transposable elements can rapidly enter the genome of an organism and proliferate quickly Drosophila melanogaster A TE known as the P element was introduced into the species in the 1950s Remarkably, in the last 50 years, the P element has expanded throughout D. melanogaster populations worldwide The only strains without the P element are lab stocks collected prior to 1950 Transposable elements have a variety of effects on chromosome structure and gene expression 17-78

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 17-79

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The features of transposons have made them an important experimental tool in molecular biology 1. The introduction of transposons into a cell is a convenient way to abolish the expression of a gene 2. It can be used to clone a particular gene in an approach known as transposon tagging Transposons Have Become Important Tools in Biology 17-81

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display An early example of transposon tagging involved an X-linked gene in Drosophila that affects eye color Wild-type = red ; Mutant = white In 1981, Paul Bingham, Robert Levis and Gerald Rubin use transposon tagging to clone this gene They started with a wild-type population of Drosophila that carried a transposon called copia From this red-eyed strain, a white-eyed strain was obtained The copia element transposed into the X-linked eye color gene, thereby inactivating it 17-82

17-83 Figure 17.17

17-84 Figure 17.17