《分子遗传学》Chapter 5 Mobile Genetic Elements.ppt

1、Chapter 5 Mobile Genetic Elements,Mobile genetic elements are segments of DNA able to move from site to site in the genome, or between genomes in the same cell. Found in both prokaryotes and eukaryotes, they are a diverse group differing in structure, mechanism of mobilization, distribution, freedom

2、 of movement and level of autonomy.,Mobile elements can be placed into three broad categories according to their level of independence: Episomes: Be able to exist in two states either integrated into the host genome or as autonomous extrachromosomal replicons - are termed episomes (e.g. bacteriophag

3、e , the F plasmid). Cassettes: Elements whose mobility is controlled by the cell to mediate specific genomic rearrangements are termed cassettes (e.g. yeast mating type cassettes, trypanosome VSG cassettes). Transposable elements: Those elements which cannot replicate outside the host genome, but wh

4、ich control their own mobility within it, are termed transposable elements (e.g. P-elements, retroviruses),Transposable elements fall into two major classes based on their active transposition mechanism. Class I transposable elements (retroelements) utilize an RNA intermediate during the transpositi

5、on process - an integrated element is transcribed, then reverse transcribed to generate a cDNA copy which is integrated at a new site. Class II transposable elements (transposons) transpose directly as DNA, either by excision and integration or by a replicative process whereby one copy of the elemen

6、t is left at the original site and another is integrated at a new site.,5.1 Mechanisms of transposition,Overview of transposition mechanisms. Transposable elements can be divided into two families according to their general mechanism of transposition. Transposons mobilize directly as DNA, whereas re

7、troelements employ an RNA intermediate. Within each family, several specific types of transposition mechanism can be used. (1) Proliferation. Increase the number of copies of the transposable element in the genome; this can be accomplished actively or passively.,(2) Stages of transposition. Transpos

8、ons actively mobilize in three stages: (i) the donor cut stage, an endonucleolytic cleavage reaction catalyzed by transposase which separates the transposable element from the host DNA; (ii) the strand exchange stage, a pair of transesterification reactions also catalyzed by transposase which join t

9、he 3 ends of the transposable element to host DNA at the target site; (iii) the repair stage, DNA synthesis undertaken by the cell which fills any remaining gaps. Retrotransposons precede these three stages by transcription and reverse transcription to generate a cDNA for processing and integration.

10、 Nonviral retroelements integrate into host DNA essentially utilizing only the DNA repair stage of transposition.,Overview of the three major active transposition mechanisms which are utilized by class II, class I.1 and class I.2 elements, respectively.,(3) Target site duplication. For most transpos

11、able elements, the strand exchange stage of transposition occurs at a staggered break in the target DNA, so that the freshly integrated element is initially flanked by single-stranded DNA tails. Repair synthesis over the gaps generates direct repeats of the sequence at the target site, so-called tar

12、get site duplications (TSDs). Most transposable elements generate TSDs of specific length, but the heterogeneous class I.2 retroelements generate variable sized TSDs even within the same genome, probably because they utilize broken DNA ends adventitiously.,Mechanism for the generation of target site

13、 duplications (TSDs), a hallmark of transposition but not of other mechanisms of recombination, which conserve the recombining sequences exactly. Staggered nicks are introduced at the target site and the transposable element joined to the 5 overhangs. Repair synthesis over the resulting gaps generat

14、es direct repeats of the sequence between the nicks. Almost all elements generate TSDs, but those that do not (e.g. IS91) probably introduce a blunt-ended break at the target site,Flanking direct repeats are generated when a transposable element inserts into DNA.,All types of transposition have seve

15、ral features in common: (1) staggered breaks are made in the target DNA; (2) the transposable element is joined to single-stranded ends of the target DNA; and (3) DNA is replicated at the single strand gaps.,(4) Target preference. Transposable elements vary in their preference for target sites, some

16、 integrating at specific sequences, others appearing to integrate at random. (Tn7) Most elements avoid integrating into preexisting elements of the same type, i.e. they display cis immunity. Many elements demonstrate topographical preference. Some eukaryotic transposons move only a short distance (1

17、-10 kbp) from their donor site; this is regional reintegration. Bacterial replicative transposons prefer to move to plasmids rather than from plasmids to the bacterial chromosome.,Conservative transposition (also called nonreplicative or simple transposition, or the cut-and-paste mechanism) involves

18、 the excision of the element from one site and its integration at another. The active transposition mechanism does not increase the copy number of the element, although this may be achieved passively. Replicative transposition (also called nonconservative or complex transposition) involves duplicati

19、on of the element, one copy remaining at the donor site and one copy integrating at the target site. Retroelement transposition is necessarily replicative because the element yielding the transcript which is converted into a cDNA for integration remains in the genome.,Conservative and replicative tr

20、ansposition,Conservative transposition,Replicative transposition,Retrotransposition,Replicative transposition increases the number of copies of the transposable element.,Replicative transposition requires single-strand breaks, replication, and resolution.,A unified model of conservative and replicat

21、ive transposition,Following replicative transposition between, for example, two plasmids, the circles become joined to form a composite structure termed a cointegrate, with copies of the transposable element marking the boundary between each contributing replicon. Transposons which move in this mann

22、er also carry functions which separate the cointegrate into its two constituent replicons, a process termed resolution. Each transposon encodes resolvase, a site-specific recombinase which acts on a short sequence within the transposon, the internal resolution site (res).,Resolution,Passive transpos

23、ition describes any process where the mobilization of a transposable element is not self-controlled. There are two types of passive transposition. (1) In-trans passive transposition occurs by the same mechanism used by active elements and is facilitated by enzymes (e.g. transposase, reverse transcri

24、ptase) supplied in trans by an active element. Only nonautonomous elements move by in-trans passive transposition. (2) Cell-controlled passive transposition is an apparent transposition event caused by cellular DNA replication. Both autonomous and nonautonomous elements move by this process.,Passive

25、 transposition,Increasing copy number during conservative transposition by cell-controlled passive Transposition. Two mechanisms are shown, each divided into pretransposition stage (A), active transposition stage (B) and passive transposition stage (C). Homing introns and inteins use the second mech

26、anism without actively transposing: intron- or intein-encoded endonuclease cleaves homologous, intronless DNA and the sequence is copied to the intronless allele by gene conversion.,All elements must increase their copy number by transposition to avoid extinction. For those elements that transpose c

27、onservatively, amplification must be achieved passively by the cell. Cell controlled passive transposition occurs in two ways: (1) Transposition from replicated to nonreplicated DNA - this method is utilized by, for example, the Ac-Ds elements of maize. (2) Repair of excision breaks by gene conversi

28、on - this method is utilized by Drosophila P-elements, for example, and is also the sole transposition mechanism of homing introns and inteins. Additionally, copy number may be increased by increasing the copy number of the host replicon - this occurs for bacterial transposons which integrate into p

29、lasmids and bacteriophage genomes,Retrotransposition and retroposition,Overview of retrotransposition (left) and retroposition (right) mechanisms. The integrated element is shown as an open box, RNA as a thin line and DNA as a thick line (that generated by repair synthesis as a broken line, correspo

30、nding to position of target site duplications,Retrotransposons transpose through RNA intermediates.,Regulation of transposition,Behaving as selfish DNA, successful transposable elements mobilize often and increase their copy number wherever possible. However, unchecked transposition would destroy th

31、e host genome and any transposable elements contained within it. Most transposable elements therefore regulate their own transposition. Transposition may be regulated in a number of ways, and the Drosophila P-element and bacterial transposon Tn10 are well understood in this respect. 1. Many transpos

32、ons encode a transposase repressor colinear with part of the functional transposase. P-element,2 Tn10 represses its transposition by transcription from a promoter called pOUT having opposite polarity to the transposase promoter pIN. Transcription from pOUT represses transposition in several ways: by

33、 counter transcription against the pIN product, by production of antisense RNA, and by preventing the transposase binding to its recognition site. 3 Both the binding of transposase and transcription from pIN are inhibited by DNA methylation.,5.2 Consequences of transposition,Genetic consequences of

34、transposition 1 Insertion mutation a Insertional mutagenesis can affect single genes or any genes at once (hybrid dysgenesis). The classic effect of transposition is the generation of an unstable mutant allele, a mutant allele that reverts to wild-type with high frequency, but whose reversion rate i

35、s not influenced by mutagens (because the reversion represents an excision event).,b The modulation of gene expression without direct insertional inactivation occurs if the element carries an endogenous gene whose expression is controlled within the element, or if it carries an endogenous regulatory

36、 element which influences the expression of adjacent genes. Such phenomena are often seen in retroviruses and have led to the isolation of many oncogenes. c The element may possess a sequence which creates a hybrid regulatory element upon insertion, e.g. many IS elements carry sequences which resemb

37、le the -35 box motifs of bacterial promoters and may activate repressed operons such as lac, which lack such motifs. d Gene expression may be influenced by modulation of the structural properties of DNA.,2 New genes 3 Chromosome aberration 4 Evolution,Chromosome rearrangements are often generated by

38、 transposition.,Structural consequences of precise transposition,Transposition events mediate a range of structural rearrangements in the flanking host DNA. Such events are simplest when the donor and target sites lie on different molecules, e.g. on separate plasmids (intermolecular transposition).

39、Conservative transposition results in the integration of the element into the target replicon, leaving a gap in the donor replicon (which may be lost if the gap is not repaired).,Replicative transposition generates a cointegrate, a replicon fusion containing two copies of the element, and this may b

40、e resolved by site specific recombination, producing an unchanged donor replicon and a target replicon with a new insertion.,The consequences of intermolecular transposition. D, donor molecule: T, target molecule: C, cointegrate. The cross shows site-specific recombination (resolution).,Structural c

41、onsequences of intramolecular replicative transposition. The donor molecule(D) has four loci a, b, c, d, where c is origin of replication. The middle row shows the alternative Shapiro intermediates which can form when the donor and target sequences lie within the same circular molecule. In one confi

42、guration, DNA synthesis across the intermediate generates a duplicative inversion. In the other, DNA synthesis across the intermediate divides the original replicon in two. The circle lacking the origin of replication, c , is lost. The other circle persists as a deletion mutant,Consequences of aberr

43、ent transposition,One-ended transposition, where only one end of the transposon is integrated at the target site. Partial transposition, when only part of the transposon actually moves, leaving a large footprint at the donor site. Cryptic-site transposition, where flanking host DNA is mobilized alon

44、g with the transposon because it is recognized by the transposase.,Cooperative transposition, Two transposons can also facilitate adventitious cooperative transposition to mobilize a segment of intervening DNA, the efficiency of this process being inversely proportional to the distance between the i

45、ndividual elements. Inverse transposition, small circular replicons can sometime transpose the wrong central region, i.e. the rest of the replicon instead of the central region of the transposon.,Consequences of host-cell activity,The host cell can facilitate passive transposition of integrated elem

46、ents as part of normal DNA metabolism. However, cellular DNA metabolism can also mediate aberrant rearrangements which occur by three major processes: (1) attempts to repair gaps left by excision; (2) recombination within elements; (3) recombination between elements.,Excision of a transposon leaves

47、a double- stranded break at the donor site. The cell often attempts repair by homologous recombination using as the template either a sister chromatid (or a sister genome in bacteria) or, in eukaryotes, a homologous chromosome. Alternatively, the broken ends may be directly end-joined, a process whi

48、ch may be perfect (resulting in a simple target site duplication footprint) or may be preceded by some degradation of free ends, generating a deletion. An alternative response to the appearance of a double-strand break is the formation of P-DNA, inverted repeats of host DNA at the donor site. P-DNA

49、is generated by strand-to-strand sister chromatid ligation at the broken chromosome ends following replication, generating a hairpin which effectively joins the two chromatids together.,Recombination within transposable elements can result in their (passive) excision from the donor site. This occurs

50、 by homologous recombination between inverted terminal repeats or direct repeats, the former often resulting in perfect excision (but leaving a target site duplication footprint), the latter often leaving a larger footprint comprising a single copy of the direct repeat. Similar processes occur which

51、 are independent of host and transposon-encoded recombination systems, and generally leave a footprint of the element involved. In this case, intrastrand hairpin or stem-loop structures may form between inverted terminal repeats: replication across the looped out region results in a deletion in the

52、daughter strand. This is an extreme example of strand slipping as a form of illegitimate recombination.,Finally, transposable elements with moderate-to-high copy numbers represent portable recombinogenic sequences. Recombination between dispersed homologous elements on the same chromosome can cause

53、deletion, inversion or (for linear chromosomes) circularization. Recombination between two elements on different chromosomes can cause terminal deletions, chromosome fusions (cointegration), and translocations.,Transposable elements as research tools,5.3 Transposons,Structure and subclassification o

54、f transposons. The term transposon was coined to describe a bacterial transposable element carrying genes for antibiotic resistance as well as those concerned with transposition functions. More recently, the term has been adopted to encompass all transposable elements which mobilize directly as DNA,

55、 i.e. all class II transposable elements,All transposons have a conserved structure comprising one or more open reading frames (one encoding transposase) flanked by inverted terminal repeats( ITRs). The terminal repeats are necessary for transposition and are the substrates recognized by the transpo

56、sase. However, they are often not sufficient, and further internal sequences are required.,Three major groups of transposons are recognized in bacteria: class I transposons include the IS elements (simple transposons) and composite transposons; class II transposons are the complex transposons; class

57、 III transposons are the transposing bacteriophages related to Mu. A fourth class has been proposed to represent transposons which do not fit into any of the above classes.,All eukaryotic transposons resemble bacterial IS elements in that they encode only the functions required for transposition. Mo

58、st have canonical structure comprising a central region flanked by short ITRS. A special class of transposons termed foldback elements consist mainly of large inverted repeats. Little is known of how the structures mobilize, but they are not thought to utilize an RNA intermediate.,The structure of d

59、ifferent classes of transposons. (A) A bacterial IS element. (B) A compound transposon with IS elements in same orientation. (C) A compound transposon with IS elements in opposite orientations: ISL and ISR are assigned according to the polarity of the genetic map of the internal region. (D) A complex transposon. (E) A typical eukaryotic transposon. (F) A eukaryotic foldback element. tra is a gene for transposase; only one of the tra genes needs to be functional in B and C. ORF is a gene for nonessential function, e.g.