Transposable Genetic Elements

1. Transposable Genetic Elements

2. 미국 코네티컷주(州) 하트포트 출생 • 코넬대학교 가정학과에 들어갔으나 식물배양에 관심이 쏠려 곧 식물학과 • 로 옮겨 유전학에 몰두 • 25세에 식물학박사 학위를 얻고, 유전자와 염색체의 관계를 발견 • 41세 때 콜드 스프링 하버연구소의 연구원이 되어 40여년간 연구 • 43세 때 사상 세 번째로 국립과학원 여성회원에 선출되었고, 이듬해에 여 • 성으로는 사상 최초로 미국 유전학회 회장역임 • 하버드대학교 ·록펠러대학교 ·뉴욕대학교 등에서 잇달아 박사 학위수여 • 1967년 미국과학원이 수여하는 킴버유전학상을, 1981년 엘버트 라스카 • 의학연구상을 각각 수상 • 40대에 옥수수의 유동유전인자를 발견하였으나, 관심을 끌지 못했는데, • 1970년대에 들어서 이 인자가 가진 생물학적 ·의학적 중요성이 인정됨 • 1983년 여성으로는 세 번째, 여성 단독으로는 첫번째로 노벨생리 • 의학상을 수상하였다 Babara McClintock

3. Transposable Genetic Elements Contents • Controlling Elements in Maize • Bacterial Insertion Sequences • Prokaryotic Transposons • Mechanism of Transposition in Prokaryotes • Review of Transposable Elements in Prokaryotes • Molecular Nature of Transposable Elements in Eukaryotes • Review of transposable Elements in Eukaryotes

4. Transposable Elements o Segments of the genome that are capable of moving around to different locations - Transposable elements usually are flanked by repeated sequences - often carry transposase genes that confer the transposition ability Two basic types of transposition oConservative transposition – An element leaves one locations and inserts in another Does not change overall copy number oReplicative transposition – A copy of a transposable element is made and this inserts in a new place This is the most common form of transposition

5. Controlling elements in maize • McClintock’s experiments: the Ds element • The wx (waxy) locus • General characteristics of controlling elements

6. Controlling elements in 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 : unpigmented kernels [dominant epistasis] • 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 • Pollen from a/a;Dt/- plants X a/a tester •  some are completely pigmented type • a : unstable mutant allele (high reversion rate) • the allelic instability is dependent on the unlinked • Dt gene

7. 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

8. The transposition of Ds into the C gene in corn • CDs/CDs;Ac/Ac+ x cDs+/cDs+;Ac+/Ac+ • C means pigment expressed; • Ds+ and Ac+ indicate the lack of the element • Cu indicates the insertion of Ds into the C locus, which converts • it to UNSTABLE colorless! • aDt system  CuAc system • ; some specificity prevents this cross-activation of • mutational instability (aAc or CuDt)

9. General characteristics of controlling elements Target gene / receptor element / regulator gene controlling elements Nonautonomous unstable allele ; revert only in the presence of the regulator Autonomous unstable allele - Ds is an incomplete version of Ac itself Summary of the main effects of controlling elements in corn

10. Mosaicism through transposon mutagenesis snapdragon corn

11. Bacterial insertion sequence • segments of bacterial DNA that can move from one • position to a different position 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 lamda particle carrying the bacterial gene for galactose utilization (gal+) or the mutant gene gal- - The increased density of gal- particles was caused by the insertion of DNA

12. Bacterial insertion sequence 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

13. Bacterial insertion sequence Identification of discrete IS elements • * Are the segments of DNA that insert • into genes merely random DNA fragments • or are they distinct genetic entities? • Hybridization experiments • Cross-hybridization • Distinct elements • * The genome of the standard WT E.coli • is rich in IS elements • - these elements are mobile Insertion sequences are also commonly observed in the F factor

14. Bacterial insertion sequence Orientation of IS element Heteroduplex DNA structure The hybrid can be explained by assuming that the IS1 sequence is inserted in opposite directions in the two mutants

15. Prokaryotic transposons • Physical structure of transposons • Movement of transposons • Phage mu R factors Mode of action of plasmids

16. Prokaryotic transposons Physical structure of transposons • Peculiar structure formed when denatured plasmid DNA is reannealed • Double stranded IR region separates a large circular loop from the • Small “lollipop” loop

17. Prokaryotic transposons Physical structure of transposons • The IR sequences are a pair of IS elements in many cases • The IR sequences together with their contained genes •  transposon (Tn)

18. Prokaryotic transposons Physical structure of transposons The insertion of a Tn into a plasmid RTF: resistance-transfer functions

19. Prokaryotic transposons Movement of transposons Each transposon can be transferred independently

20. Prokaryotic transposons 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

21. Prokaryotic transposons Phage mu Phage mu can mediate the transposition of a bacterial gene into a plasmid Phage mu can cause deletion or inversion of adjacent bacterial segment

22. Mechanism of transposition in prokaryotes Replicative transposition * A new copy of the transposable element is generated in the transposition event * The structure of transposon 3 * Transposition of Tn3 takes place through a cointegrate intermediate

23. Mechanism of transposition in prokaryotes Conservative transposition • Some transposons excise from the chromosome and integrate • Into the target DNA • Generation of heteroduplex and • homoduplex Tn10 elements

24. Mechanism of transposition in prokaryotes Conservative transposition • Consequences of conservative & • Replicative transposition • Tn10 transpose by excising themselves from the donor DNA & integrating directly into the recipient DNA

25. Mechanism of transposition in prokaryotes Molecular consequences of transposition • Duplication of a short sequence of nucleotides • in the recipient DNA is associated with the insertion of a • Transposable element • The number of base pairs is • a characteristic of each • element

26. Mechanism of transposition in prokaryotes Rearrangements mediated by transposable elements • Transposons generate a high incidence of deletions in their vicinity • When varying lengths of the surrounding DNA are excised • along with the transposon; IMPRECISE EXCISION • When the transposon is excised and the gene that was disrupted by the insertion • are restored; PRECISE EXCISION • (This occurs rarely, compared to imprecise excision)

27. Review of transposable elements in prokaryotes • 1. There are several different types of transposable elements • ; IS, transposons and phage µ • 2. Two copies of a transposable element can act in concert to transpose • the DNA segments in between them • (the transposon containing antibiotic resistance gene) • 3. Most transposable elements have recognizable inverted repeat structures • 4. Transposable elements are found in plasmids, and in bacterial chromosomes • 5. Transposable elements usually generate repeats after insertion • 6. Detailed mechanism of transposition isn't yet known! • (replicative & conservative)

28. Molecular nature of transposable elements in eukaryotes • Transposable elements are even more prevalent in • eukaryotic chromosomes than in bacterial chromosomes • 1. Retroviruses; ss RNA animal virus The life cycle of a retrovirus Transposition by retrovirus

29. Molecular nature of transposable elements in eukaryotes • 2. Retrotransposons • - transposable elements that • utilize reverse transcriptase to • transpose through an RNA • intermediate • - two types • ; viral & nonviral • Viral retrotransposon • ; similar to retrovirus • - Yeast Ty elements • ; 35 copies in yeast genome • generate a repeated sequence of target DNA during transposition Schematic representation of a viral retrotransposon The structure of a yeast transposable element

30. Molecular nature of transposable elements in eukaryotes The mechanism for transposition of retrotransposon • 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

31. Molecular nature of transposable elements in eukaryotes - copia-like elements ; in Drosophila LTR at least, seven families generate a repeated sequence of target DNA during transposition - A comparison of the genes of integrated retrovirus DNA and the yeast Ty elements and Drosophila copia elements

32. Alus SINES LINES LTR Molecular nature of transposable elements in eukaryotes Nonviral retrotransposons: LINES and SINES - Frequent in mammals - Some sequence divergence Repetitive elements found in the human gene (HGO) encoding Homogentisate 1,2-dioxygenase, the enzyme whose deficiency cause alkaptonuria

33. Molecular nature of transposable elements in eukaryotes Function of transposable elements - Mutation of genes by insertion - Regulation of gene expression by insertion - Transposable element-mediated rearrangements ; deletion, duplication, and inversion  new genome, new function during evolution Uses of transposable elements - prokaryotes; conventional antibiotic-resistance marker - eukaryotes; generation of insertion mutations, mapping, gene cloning, transgenic organism

34. Molecular nature of transposable elements in eukaryotes • P elements in Drosophila • - 2.9kb with 31 base inverted repeats • encodes a transposase • - Do not utilize an RNA intermediate during transposition • - Can insert at many different positions • - Transposition is controlled by • repressors encoded by the element • - P elements with internal deletions • can be mobilized and then remain • fixed in the new position • ; transposon tagging

35. Using P elements to insert genes P-element mediated gene transfer in Fly. - P elements mobilize only in germ-line cells ry-/ry- Phenotype observation

36. Review of transposable elements in eukaryotes • Transposable elements exist in all cells • (yeast, drosophila, maize, and mammals) • Some elements can be used as tools for cloning and gene manipulation • (P element: insertion of genes into germ lines of recipient cells) • Short repeated sequences are generated at the site of insertion in the • target site • Some eukaryotic transposable elements use an RNA intermediate during • insertion; this is not seen in prokaryotes