分子生物学Chapter6MobileGeneticElements.doc

Chapter 6 Mobile Genetic Elements IntroductionMuch of the discussion throughout this book deals with the functions of the gene products expressed from proteincoding genes and RNA genes. However, noncoding repetitious DNA, consisting of simple-sequence DNA and moderately repeated DNA, constitutes a significant fraction of the genomic DNA in higher eukaryotes. In this section, we focus on moderately repeated DNA sequences, or mobile DNA elements, which are interspersed throughout the genomes of higher plants and animals. Although mobile DNA elements, ranging from hundreds to a few thousand base pairs in length, originally were discovered in eukaryotes, they also are found in prokaryotes. The process by which these sequences are copied and inserted into a new site in the genome is called transposition. Mobile DNA elements (or simply mobile elements) are essentially molecular parasites, which appear to have no specific function in the biology of their host organisms, but exist only to maintain themselves. For this reason, Francis Crick referred to these sequences as “selfish DNA.”The transposition of mobile DNA elements is believed to have resulted in their slow accumulation in eukaryotic genomes over evolutionary time. These elements also are lost at a very slow rate by deletion of segments of DNA containing them and by accumulation of mutations until they can no longer be recognized to be related to the original mobile DNA element. Since mobile elements are eliminated from eukaryotic genomes so slowly, they have accumulated to the point where they now constitute a significant portion of the genomes of many eukaryotes.As research on mobile elements progressed, they were found to fall into two categories: (1) those that transpose directly as DNA and (2) those that transpose via an RNA intermediate transcribed from the mobile element by an RNA polymerase and then converted back into double-stranded DNA by a reverse transcriptase. Mobile elements that transpose through a DNA intermediate are generally referred to as transposons. (As discussed below, this term has a more specific meaning in reference to bacterial mobile elements.) Mobile elements that transpose to new sites in the genome via an RNA intermediate are called retrotransposons because their movement is analogous to the infectious process of retroviruses. Indeed, retroviruses can be thought of as retrotransposons that evolved genes encoding viral coats, thus allowing them to transpose between cells. Both transposons and retrotransposons can be further classified based on their specific mechanism of transposition, as summarized in Table 9-3. We describe the structure and movement of the various types of mobile elements and then consider their likely role in evolution.Bacterial Genetic Mobile Elements: Insertion Seqs (IS) - Transposons (Tn)Eukaryotic Genetic Mobile Elements: Transposons (Tn) - RetrotransposonsBacterial and eukaryotic Transposons are similar in mechanisms of transpositionRetrotransposons or retroposons transpose via an RNA intermediate, and thus mimic retroviruses in their genomic duplication: need Reverse Transcriptase activity Genetic elements that are Mobile - can self-transport or translocate or transpose from one location to another on the organismal DNA molecules, or . hopping genesOften hop into other genes: source of MutationsGenome evolution: acquire new sequences (plasmids, phage) or rearrange existing onesTransposons can cause deletions, insertions, inversions, rearrangementsLodish, pgs 320-338Most mobile elements in bacteria transpose directly as DNA. In contrast, most mobile elements in eukaryotes are retrotransposons, but some eukaryotic transposons have been identified. Indeed, the original mobile elements discovered by Barbara McClintock are transposons. found in the wild-type1. Bacterial Mobile Elements 1.1 Insertion Sequences The first molecular understanding of mobile elements came from the study of certain E. coli mutations resulting from the spontaneous insertion of a DNA sequence, 1 2 kb long, into the middle of a gene. These inserted stretches of DNA called insertion sequences, or IS elements were first visualized by analyzing hybrids (heteroduplexes) of wild-type and mutant DNAs in the electron microscope. Because the IS element integrated into the mutant strand has no complement in the wild-type strand, it cannot hybridize and forms a visible single-stranded loop extending from the rest of the double-stranded heteroduplex. So far, more than 20 different IS elements have been found in E. coli and other bacteria.IS elements appear to be molecular parasites of bacterial cells. Transposition of an IS element is a very rare event, occurring in only one in 105 107 cells per generation, depending on the IS element. Higher rates of transposition would probably result in too great a mutation rate for the host cell. At a very low rate of transposition, most host cells survive and therefore propagate the parasitic IS element. Even though many transpositions inactivate essential genes, killing the host cell and the IS elements it carries, other host cells survive. Since IS elements transpose into approximately random sites, some transposed sequences enter nonessential regions of the genome (e.g., regions between genes), thereby expanding the number of IS elements in a cell. IS elements also can insert into plasmids or lysogenic viruses, which can be transferred to other cells. When this happens, IS elements can transpose into the chromosomes of virgin cells.The general structure of IS elements is diagrammed in Figure 6.1. An inverted repeat, usually containing 50 base pairs, invariably is present at each end of an insertion sequence. Between the inverted repeats is a protein-coding region, which encodes one or two enzymes required for transposition of an IS element to a new site. In either case, IS-encoded proteins are expressed at a very low rate, accounting for the very low frequency of transposition. An important hallmark of IS elements is the presence of short direct repeats, containing 5 11 base pairs, immediately adjacent to both ends of the inserted element. The length of the direct repeat is characteristic of each type of IS element, but its sequence depends on the target site where a particular copy of the IS element is inserted. When the sequence of a mutated gene containing an IS element is compared to the sequence of the wild-type gene before insertion, only one copy of the short direct- repeat sequence is gene. Duplication of this target-site sequence to create the second direct repeat adjacent to an IS element occurs during the insertion process.Figure 6-1. General structure of bacterial IS elements. The central region, which encodes one or two enzymes required for transposition, is flanked by inverted repeats whose sequence is characteristic of a particular IS element. The 5 and 3 short direct repeats are generated from the target-site DNA during insertion of a mobile element. The length of the direct repeats is constant for a given IS element, but their sequence depends on the site of insertion and is not characteristic of the IS element. Arrows indicate sequence orientation. The regions in this diagram are not to scale; the coding region makes up most of the length of an IS element. The enzyme that catalyzes transposition of an IS element is called a transposase. In the simplest mechanism of transposition, an IS element is excised from one location and inserted at a new position in the bacterial chromosome by a nonreplicative process (Figure 6-2). In this mechanism, transposase molecules bind to the inverted-repeat sequences present at each end of the IS element in the donor DNA and cleave the DNA, precisely excising the element. Transposase molecules also bind to and make staggered cuts in a short sequence in the target DNA, generating single-stranded tails. This remarkable enzyme then ligates the 3 termini of the IS element to the 5 ends of the cut donor DNA. A DNA polymerase encoded by the host cell then extends the 3 ends of the target site, filling in the single-stranded gaps and generating a short repeat of the target-site sequence at either end of the newly inserted IS element. This is the origin of the short direct repeats that flank IS elements. Some IS elements transpose by a more complicated replicative mechanism; in this case, a copy of the original IS element is generated in the target DNA and the original copy is retained in the donor DNA.Figure 6-2. Model for nonreplicative transposition of bacterial insertion sequences. Step 1: A transposase, which is encoded by the IS element (IS10 in this example), cleaves both strands of the donor DNA between the terminal direct repeats (light blue) and the inverted repeats, excising the IS10 element. At a largely random target site, the transposase makes staggered cuts in the target DNA. In the case of IS10, the two cuts are 9 bp apart. Step 2: Ligation of the 3 ends of the excised IS element to the staggered sites in the target DNA also is catalyzed by transposase. Step 3: The 9-bp gaps of single-stranded DNA left in the resulting intermediate is filled in by cellular DNA polymerase; finally cellular DNA ligase forms the 3 5 phosphodiester bonds between the 3 ends of the extended target DNA strands and the 5 ends of the IS10 strands. This process results in duplication of the target-site sequence on each side of the inserted IS element. Note that the length of the target site and IS10 are not to scale. See H. W. Benjamin and N. Kleckner, 1989, Cell 59:373 and, 1992, Proc. Natl. Acad. Sci. USA 89:4648.1.2. Bacterial Transposons In addition to IS elements, bacteria contain composite mobile genetic elements that are larger than IS elements and contain one or more protein-coding genes in addition to those required for transposition. Referred to as bacterial transposons, these elements are composed of an antibiotic-resistance gene flanked by two copies of the same type of IS element (Figure 6-3). Insertion of a transposon into plasmid or chromosomal DNA is readily detectable because of the acquired resistance to an antibiotic. Transposition produces a short direct repeat of the target site on either side of the newly integrated transposon, just as for IS elements. Figure 6-3. General structure of bacterial transposons, such as Tn9 of E. coli. This transposon consists of a chloramphenicol-resistance gene (dark blue) flanked by two copies of IS1 (orange), one of the smallest IS elements. Other copies of IS1, without the drug-resistance gene, are located elsewhere in the E. coli chromosome. The internal inverted repeats of IS1 abutting the resistance gene are so mutated that transposase does not recognize them. During transposition, the IS-element transposase makes cuts at the positions indicated by small red arrows, so the entire transposon is moved from the donor DNA (e.g., a plasmid). The target-site sequence at the point of insertion becomes duplicated on either side of the transposon during transposition, which occurs via the replicative mechanism. Note that the 5-bp target-site direct repeat (light blue) is not to scale.Transposons are very valuable tools for the bacterial geneticist. They can be introduced into cells on plasmids or viral genomes. Once transferred into a cell, transposons can act as mutagens that affect only a single cellular gene. Although transposition is a rare event, mutagenized cells are readily isolated because of their newly acquired antibiotic resistance gene. The site of the transposon-generated mutation can be determined readily by restriction-enzyme mapping, which reveals the insertion of the large transposon DNA. The precise sequence of bacterial DNA at the site of insertion can then be determined by dideoxy DNA sequencing using a primer complementary to the known sequence of the inverted repeats at the ends of the transposon.Usually the ITRs that are internal (the right ITR of the left IS, the left ITR of the right IS) in the Transposon are heavily mutated and inactive for transposition.When internal ITRs ARE functional, and the Tn hops to a small genome, e.g. a plasmid, a second hop to the chromosome can use the initially internal ITRs, creating a new Inside-Out Transposon with the IS elements reversed and with the plasmid DNA substituted for the DNA found between the IS elements of the initial Tn, as shown in the following Figure:Figure6-4 Inside-Out Transposon1.3 DNA RearrangementsA) Reciprocal Recom between Direct Tn Repeats = Deletions between Tn Repeats. Voet and Voet, Fig. 31-6Figure 6-5B) Reciprocal Recom between Inverted Tn Repeats = Inversion between Tn Repeats:. Voet and Voet, Fig. 31-6Figure6-61.4 Transposition MechanismsGeneral Mechanism: Staggered nicks, insert and join Tn, fill in gaps, ligateBasic route: Replicon Fusion with (replicative) or without (nonreplicative) CoIntegrate formedThe following is slightly modified and clarified . Fall, 2002:1. 4.1 Replicative Transposition . CoIntegrate Intermediate: . Lod3: 9-18; Lod4: absent; Voet and Voet, Fig. 31-60Tn is duplicated during transposition, copy transposed . Copy number of Tn increasesNeed 2 enzymes: Transposase - acts on ends of parent TnResolvase - acts on ends of copied, daughter TnFour nicks, from Transposase, 2 in donor at ends of Tn, 2 in target, staggered at each 5 end of Target site .The latter defines the size of the resulting Host/Recipient DNA Tandem DuplicationTn 5 ends in Donor ligated to Host 3 ends of the Tn: Crossover StructureDNA Repair Synthesis by PolI THROUGH the Tn AND one Direct Repeat, to yield CoIntegrate with Reciprocal Direct RepeatsResolvase executes site-specific homologous recombination at the Res Site in the CoIntegrate, yielding a Tn with Direct Repeats at either end in BOTH the Donor DNA and the Target DNAResolution of the CoIntegrate, via Resolvase, releases daughter replicons, each with one copy of the Transposon.1.4.2. Non Replicative Transposition: Phage Mu; Tn5; Tn10 . Lod3: 9-17; Lod4: 9-12True Hopping, Tn moves to new site . original site is repaired . Tn no longer in Donor DNAOnly Transposase is neededSame as above, but 2 more nicks in Donor DNA in Crossover Structure, followed by DNA repair to repair the Tandem Repeats . sometimes called Cut and Paste Transposition1.4.3. Conservative TranspositionSimilar to NonReplicative Transposition, but uses a Site-Specific recombinase similar to the Integrase of Phage LambdaExamples1. TnA family: Tn3 and gd . Voet and Voet, Fig. 31-56; Lewin, Fig. 34.13Fall, 2004: Figure of Tn3Figure 6-7NOT composite Transposons, no IS elements . 38 bp Inverted Terminal Repeats target Direct Repeat of 5 bp . Ap-resistancetnpA gene - TransposasetnpR gene - Repressor and ResolvaseTnpR Resolvase: binds to promoter region of both tnpA and tnpR at 3 sitesThis region is called res, for resolution of the CoIntegrateIf only one site is bound, Resolvase is thought to act as the RepressorIf all 3 sites are bound and the DNA structure is correct, a large nucleoprotein complex is formed, Resolvase introduces nicks spaced 2 bp apart in a TTATAA sequence in the res site, rearranges DNA ends, and ligates appropriate ones together to yield the resolved DNA products2. Tn10: Composite Tn: 9300 bp; IS10R, IS10L (nonFunc) inverted; Tc-resisFall, 1999: Figure of Tn10Figure 6-8IS10: 1300 bp, 22 bp ITRs; Host Target: 9 bp, NGCTNAGCN hot spotsFrequency of Tn10 Transposition tightly regulated:IS10R: Pin - Pout overlapping Promoters: Pout stronger than Pin, mRNA more stable1. MultiCopy Inhibition: RNA from Pout of Tn10 on plasmid is an Antisense RNA, limits expression from Pin, which expresses the Transposase . Limits hopping2. Transposase cis-preference: functions best on Tn from which it is expressed3. GATC hemimethylated state needed at 2 sites: GATC in Pin; GATC in ITR . enhances Transposase transcription and binding . coupling to ReplicationDaughter Tn10 with Template Strand GATC methylated: 1000x more active . Daughter Tn10 with other Strand GATC methylated: 3x more active.2. Eukaryotic Transposons 2. 1 Corn Ac and Ds elements Ac - Activator element - like a bacterial Insertion Sequence - active TransposaseDs - Dissociative element - like Ac element but has no active Transposase due to internal deletions - requires presence of Ac element (and its Transposase) for transposition.McClintocks original discovery of mobile elements came from observation of certain spontaneous mutations that affect production of any of the several enzymes required to make anthocyanin, a purple pigment. Mutant kernels are white, and wild-type kernels are purple. One class of these mutations is revertible at high frequency, whereas a second class of mutations does not revert unless they occur in the presence of the first class of mutations. McClintock called the agent responsible for the first class of mutations the activator (Ac) element and those responsible for the second class dissociation (Ds) elements because they also tended to be associated with chromosome breaks.Many years after McClintocks pioneering discoveries, cloning and sequencing revealed that Ds elements are deleted forms of the Ac element in which a portion of the sequence encoding transposase is missing. Because it does not encode a functional transposase, a Ds element cannot move by itself. However, in plants that carry the Ac element and thus express a functional transposase, Ds elements can move. The structure of these eukaryotic elements are similar to bacterial IS elements, and they appear to move by the nonreplicative mechanism shown in Figure 9-12.2.2 Drosophila P Element the P element functions as a tra