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1.
Partial Revertants of the Transposable Element-Associated Suppressible Allele White-Apricot in Drosophila Melanogaster: Structures and Responsiveness to Genetic Modifiers 总被引:24,自引:13,他引:11
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The eye color phenotype of white-apricot (wa), a mutant allele of the white locus caused by the insertion of the transposable element copia into a small intron, is suppressed by the extragenic suppressor suppressor-of-white-apricot (su(wa] and enhanced by the extragenic enhancers suppressor-of-forked su(f] and Enhancer-of-white-apricot (E(wa]. Derivatives of wa have been analyzed molecularly and genetically in order to correlate the structure of these derivatives with their response to modifiers. Derivatives in which the copia element is replaced precisely by a solo long terminal repeat (sLTR) were generated in vitro and returned to the germline by P-element mediated transformation; flies carrying this allele within a P transposon show a nearly wild-type phenotype and no response to either su(f) or su(wa). In addition, eleven partial phenotypic revertants of wa were analyzed. Of these, one appears to be a duplication of a large region which includes wa, three are new alleles of su(wa), two are sLTR derivatives whose properties confirm results obtained using transformation, and five are secondary insertions into the copia element within wa. One of these, waR84h, differs from wa by the insertion of the most 3' 83 nucleotides of the I factor. The five insertion derivatives show a variety of phenotypes and modes of interaction with su[f) and su(wa). The eye pigmentation of waR84h is affected by su(f) and E(wa), but not su(wa). These results demonstrate that copia (as opposed to the interruption of white sequences) is essential for the wa phenotype and its response to genetic modifiers, and that there are multiple mechanisms for the alteration of the wa phenotype by modifiers. 相似文献
2.
The maize P gene conditions red phlobaphene pigmentation to the pericarp and cob. Starting from two unstable P alleles which carry insertions of the transposable element Ac, we have derived 51 P null alleles; 47 of the 51 null alleles have a 17-kb deletion which removes the 4.5-kb Ac element and 12.5 kb of P sequences flanking both sides of Ac. The deletion endpoints lie within two 5.2-kb homologous direct repeats which flank the P gene. A P allele which contains the direct repeats, but does not have an Ac insertion between the direct repeats, shows very little sporophytic or gametophytic instability. The apparent frequency of sporophytic mutations was not increased when Ac was introduced in trans. Southern analysis of DNA prepared from the pericarp tissue demonstrates that the deletions can occur premeiotically, in the somatic cells during development of the pericarp. Evidence is presented that the deletions occurred by homologous recombination between the two direct repeats, and that the presence of an Ac element at the P locus is associated with the recombination/deletion. These results add another aspect to the spectrum of activities of Ac: the destabilization of flanking direct repeat sequences. 相似文献
3.
Prevalence of Localized Rearrangements Vs. Transpositions among Events Induced by Drosophila P Element Transposase on a P Transgene
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We have studied P transposase-induced events on a P[w] transgene, P[w(d1)], harboring the whole white gene with a 3.44-kb direct duplication of its 5' regulatory sequences (containing the ZESTE-binding region, ZBR). We have recovered mutations leading to an increase or a decrease of zeste(1) repression, generally as the consequence of modifications of the number of ZBR in close physical proximity and/or jumps to other sites. We describe mutants displaying deletions of the original duplicated sequence or increases in the number of repeats from two to three or four. Internal deletions are more frequent than amplifications. Both require the integrity of P-element ends. We have also observed a high frequency of double P elements localized at the original P[w(d1)] insertion site. These double P elements are arranged in nonrandom configurations. We discuss the frequencies and the possible mechanisms leading to the various types of derivatives, in light of the current models for P excision and transposition. We propose that the P transposase induces mainly localized events. Some of these could result from frequent changes of template during gap-repair DNA synthesis, and/or from abortive transposition. 相似文献
4.
5.
Mobilization of a Minos Transposon in Drosophila Melanogaster Chromosomes and Chromatid Repair by Heteroduplex Formation 总被引:1,自引:0,他引:1
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Transposase-mediated mobilization of the element Minos has been studied in the Drosophila melanogaster genome. Excision and transposition of a nonautonomous Minos transposon in the presence of a Minos transposase gene was detected with a dominant eye color marker carried by the transposon. Frequencies of excision in somatic tissues and in the germ line were higher in flies heterozygous for the transposon than in homozygotes or hemizygotes. Transposition of a X chromosome-linked insertion of Minos into new autosomal sites occurred in 1-12% of males expressing transposase, suggesting that this system is usable for gene tagging and enhancer trapping in Drosophila. Sequence analysis of PCR-amplified donor sites after excision showed precise restoration of the original target sequence in ~75% of events in heterozygotes and the presence of footprints or partially deleted elements in the remaining events. Most footprints consisted of the four terminal bases of the transposon, flanked by the TA target duplication. Sequencing of a chromosomal donor site that was directly cloned after excision showed a characteristic two-base mismatch heteroduplex in the center of the 6-bp footprint. Circular extrachromosomal forms of the transposon, presumably representing excised Minos elements, could be detected only in the presence of transposase. A model for chromatid repair after Minos excision is discussed in which staggered cuts are first produced at the ends of the inverted repeats, the broken chromatid ends are joined, and the resulting heteroduplex is subsequently repaired. The model also suggests a simple mechanism for the production of the target site duplication and for regeneration of the transposon ends during reintegration. 相似文献
6.
Precise and nearly-precise excision of the symmetrical inverted repeats of Tn5; common features of recA-independent deletion events in Escherichia coli 总被引:23,自引:0,他引:23
The transposon Tn5 contains a unique central region bordered by 1.5-kb inverted repeats. The in vitro deletion of the centre of Tn5, with a restriction endonuclease (XhoI) which cuts within the inverted repeats leads to the production of a palindrome on subsequent ligation. This palindromic region is unstable on subsequent transformation into Escherichia coli (Collins, 1981). Precise excision of the Tn5 region plus one copy of the bracketing 9-bp direct repeat occurred in about one-third of the transformants. The rest of the transformants contain only remnants of the inverted repeat. Sequence analysis indicated that deletion had occurred between short direct repeats. The precise excision of these "nearly precise" excision products continued with high frequency and was found to be affected by mutations that interfere with the normal precise excision of transposons. In a recB, sbcB host precise excision was markedly reduced. A common mechanism is proposed for all recA-independent deletions occurring in E. coli. 相似文献
7.
We report the formation of deletions and inversions by intramolecular transposition of Tn5-derived mobile elements. The synthetic transposons used contained the IS50 O and I end segments and the transposase gene, a contraselectable gene encoding sucrose sensitivity (sacB), antibiotic resistance genes, and a plasmid replication origin. Both deletions and inversions were associated with loss of a 300-bp segment that is designated the vector because it is outside of the transposon. Deletions were severalfold more frequent than inversions, perhaps reflecting constraints on DNA twisting or abortive transposition. Restriction and DNA sequence analyses showed that both types of rearrangements extended from one transposon end to many different sites in target DNA. In the case of inversions, transposition generated 9-bp direct repeats of target sequences. 相似文献
8.
Isolation and Characterization of Escherichia Coli Mutants with Altered Rates of Deletion Formation 总被引:9,自引:4,他引:5
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Using site-specific mutagenesis in vitro we constructed a genetic system to detect mutants with altered rates of deletion formation between short repeated sequences in Escherichia coli. After in vivo mutagenesis with chemical mutagens and transposons, the system allowed the identification of mutants with either increased or decreased deletion frequencies. One mutational locus, termed mutR, that results in an increase in deletion formation, was studied in detail. The mutR gene maps at 38.5 min on the E. coli genetic map. Since the precise excision of many transposable elements is also mediated at short repeated sequences, we investigated the effects of the mutant alleles, as well as recA, on precise excision of the transposon Tn9. Neither mutR nor recA affect precise excision of the transposon Tn9, from three different insertions in lacI, whereas these alleles do affect other spontaneous deletions in the same system. These results indicate that deletion events leading to precise excision occur principally via a different pathway than other random spontaneous deletions. It is suggested that, whereas precise excision occurs predominantly via a pathway involving replication enzymes (for instance template strand slippage), deletions on an F'factor are stimulated by recombination enzymes. 相似文献
9.
10.
Plasmid-borne cadmium resistance genes in Listeria monocytogenes are present on Tn5422, a novel transposon closely related to Tn917. 总被引:1,自引:1,他引:0
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The complete (6,449-bp) nucleotide sequence of the first-described natural transposon of Listeria monocytogenes, designated Tn5422, was determined. Tn5422 is a transposon of the Tn3 family delineated by imperfect inverted repeats (IRs) of 40 bp. It contains two genes which confer cadmium resistance (M. Lebrun, A. Audurier, and P. Cossart, J. Bacteriol. 176:3040-3048, 1994) and two open reading frames that encode a transposase (TnpA) and a resolvase (TnpR) of 971 and 184 amino acids, respectively. The cadmium resistance genes and the transposition genes are transcribed in opposite directions and are separated by a putative recombination site (res). The structural elements presumed to be involved in transposition of Tn5422 (IRs, transposase, resolvase, and res) are very similar to those of Tn917, suggesting a common origin. The transposition genes were not induced by cadmium. Analysis of sequences surrounding Tn5422 in nine different plasmids of L. monocytogenes indicated that Tn5422 is a functional transposon, capable of intramolecular replicative transposition, generating deletions. This transposition process is probably the reason for the size diversity of the L. monocytogenes plasmids. Restriction analysis and Southern hybridization revealed the presence of Tn5422 in all the plasmid-mediated cadmium-resistant L. monocytogenes strains tested but not in strains encoding cadmium resistance on the chromosome. 相似文献
11.
Gilmore RC Baker J Dempsey S Marchan R Corprew RN Byrd G Maeda N Smithies O Bukoski RD Harewood KR Chatterjee PK 《Gene》2001,275(1):65-72
Highly polymorphic di- and tetranucleotide repeats in and around Npr3, a potential candidate gene for hypertension, have been identified using a novel approach. Because this chromosomal site is rich in repetitive DNA and difficult to sequence, P1 artificial chromosomes were retrofitted with a loxP transposon to map the gene sequence within a clone using a series of nested deletions. Sequences from ends of deletions 1-3 kb apart identified a (CA)(20) and a (TA)(18)-(CA)(8) repeat 8 kb upstream and within an intron of Npr3, respectively. DNA from 17 individuals was analyzed for length polymorphisms in these and eight additional repeats identified in 200 kb of working draft sequence from this region in GenBank. The sequence contigs and microsatellite repeats from GenBank were ordered using the P1-derived artificial chromosome deletion series. Several of these repeats were found to vary considerably in length in the set of genomic DNA tested. Since this site in chromosome 5p has recently been implicated in disease in studies with genetically hypertensive rats, the microsatellite markers reported here will be useful for genetic analysis and may even be implicated in the disease process in humans. We discuss how these types of data are useful for interpreting draft DNA sequence coming out of the genome projects, and the utility of deletion clones as a resource for ordering contigs and gap filling. 相似文献
12.
13.
Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates. 相似文献
14.
Mariner family transposons are perhaps the most widespread transposable elements of eukaryotes. While we are beginning to understand
the precise mechanism of transposition of these elements, the structure of their transposases are still poorly understood.
We undertook an extensive mutagenesis of the N-terminal third of the transposase of the Himar1 mariner transposon to begin the process of determining the structure and evolution of mariner transposases. N and C-terminal deletion analyses localized the DNA binding domain of Himar1 transposase to the first 115 amino acids. Alanine scanning of 23 selected sites within this region uncovered mutations that
not only affected DNA binding but DNA cleavage as well. The behavior of other mutations strongly suggested that the N-terminus
is also involved in multimerization of the transposase on a single inverted terminal repeat and in paired ends complex formation
which brings together the two ends of the transposon. Finally, two hyperactive mutations at conserved sites suggest that mariner transposases are under a pattern of stabilizing selection in nature with regard to how efficiently they mediate transposition,
resulting in a population of “average” transposons. 相似文献
15.
DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3. 总被引:138,自引:0,他引:138
The complete nucleotide sequence of the transposon Tn3 and of 20 mutations which affect its transposition are reported. The mutations, generated in vitro by random insertion of synthetic restriction sites, proved to contain small duplications or deletions immediately adjacent to the new restriction site. By determining the phenotype and DNA sequence of these mutations we were able to generate an overlapping phenotypic and nucleotide map. This 4957 bp transposon encodes three polypeptides which account for all but 350 bp of its total coding capacity. These proteins are the transposase, a high molecular weight polypeptide (1015 amino acids) encoded by the tnpA gene; the Tn3-specific repressor, a low molecular weight polypeptide (185 amino acids) encoded by the tnpR gene; and the 286 amino acid beta-lactamase. The 38 bp inverted repeats flanking Tn3 appear to be absolutely required in cis for Tn3 to transpose. Genetic data suggest that Tn3 contains a third site (Gill et al., 1978), designated IRS (internal resolution site), whose absence results in the insertion of two complete copies of Tn3 as direct repeats into the recipient DNA. We suggest that these direct repeats of complete copies of Tn3 are intermediates in transposition, and that the IRS site is required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 (Gill et al., 1978). A 23 nucleotide sequence within the amino terminus of the transposase which shares strong sequence homology with the inverted repeat may be the internal resolution site. 相似文献
16.
We report a transposase gene (tnpA) upstream of the opdA phosphotriesterase gene of Agrobacterium radiobacter P230, as well as inverted repeats indicative of insertion sequences, flanking the two genes. Both the tnpA gene and the inverted repeats resemble the Tn610 transposon from Mycobacterium fortuitum. Two additional putative open reading frames separate opdA and tnpA with inferred translation products with similarity to two proteins encoded on the Geobacillus stearothermophilus IS5376 transposon. To test the proposition that these genes were contained on a transposon, an artificial composite transposon was constructed. This artificial transposon was then delivered into Escherichia coli DH10beta cells. Transposition was demonstrated by the presence of opdA on the E. coli chromosome and confirmation of insertion by inverse polymerase chain reaction. The data presented suggest a possible role of transposition in the distribution of the opd/opdA genes across a wide range of soil bacteria. 相似文献
17.
Local Transposition of P Elements in Drosophila Melanogaster and Recombination between Duplicated Elements Using a Site-Specific Recombinase 总被引:2,自引:1,他引:1
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K. G. Golic 《Genetics》1994,137(2):551-563
The transposase source Δ2-3(99B) was used to mobilize a P element located at sites on chromosomes X, 2 and 3. The transposition event most frequently recovered was a chromosome with two copies of the P element at or near the original site of insertion. These were easily recognized because the P element carried a hypomorphic while gene with a dosage dependent phenotype; flies with two copies of the gene have darker eyes than flies with one copy. The P element also carried direct repeats of the recombination target (FRT) for the FLP site-specific recombinase. The synthesis of FLP in these flies caused excision of the FRT-flanked white gene. Because the two white copies excised independently, patches of eye tissue with different levels of pigmentation were produced. Thus, the presence of two copies of the FRT-flanked white gene could be verified. When the P elements lay in the same orientation, FLP-mediated recombination between the FRTs on separated elements produced deficiencies and duplications of the flanked region. When P elements were inverted, the predominant consequence of FLP-catalyzed recombination between the inverted elements was the formation of dicentric chromosomes and acentric fragments as a result of unequal sister chromatid exchange. 相似文献
18.
Genotype-environment interactions and the maintenance of polygenic variation 总被引:8,自引:6,他引:2
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The Enhancer of wa [E(wa)] mutation was shown to interact strongly with 4 of 41 tested alleles of the white (w) eye color locus. All four of the affected w alleles result from the insertion of a transposable element. E(wa) was further localized cytogenetically. The locus lies between the breakpoints of T(Y;2)L11 and T(Y;2)H137 (section 60) in 2R. The original mutation was shown to be antimorphic on the basis of its action in the presence of additional normal copies and the ability to revert the original allele to one that mimics the effect of a deficiency for the locus. The RNA transcribed from wa was analyzed from flies segregating for E(wa) and normal. The low level of normal functional messenger RNA present in white-apricot is reduced further in Enhancer homozygotes. Total copia RNA was also examined on Northern analyses from the segregating population but no quantitative change in the major copia RNA was produced by E(wa) homozygotes compared to normal. 相似文献
19.
Simple and efficient generation in vitro of nested deletions and inversions: Tn5 intramolecular transposition. 总被引:2,自引:0,他引:2
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We have exploited the intramolecular transposition preference of the Tn 5 in vitro transposition system to test its effectiveness as a tool for generation of nested families of deletions and inversions. A synthetic transposon was constructed containing an ori, an ampicillin resistance (Ampr) gene, a multi-cloning site (MCS) and two hyperactive end sequences. The donor DNA that adjoins the transposon contains a kanamycin resistance (Kanr) gene. Any Amprreplicating plasmid that has undergone a transposition event (Kans) will be targeted primarily to any insert in the MCS. Two different size targets were tested in the in vitro system. Synthetic transposon plasmids containing either target were incubated in the presence of purified transposase (Tnp) protein and transformed. Transposition frequencies (Ampr/Kans) for both targets were found to be 30-50%, of which >95% occur within the target sequence, in an apparently random manner. By a conservative estimate 10(5) or more deletions/inversions within a given segment of DNA can be expected from a single one-step 20 microl transposition reaction. These nested deletions can be used for structure-function analysis of proteins and for sequence analysis. The inversions provide nested sequencing templates of the opposite strand from the deletions. 相似文献
20.
We show that a single low-dose exposure of human epidermal keratinocytes (NHEK) to an FS20 light source in vitro can induce the formation of mitochondrial DNA deletions in a PCR detection assay. We used primer sets specifically designed to exclude amplification of segments containing the common deletion, but which could detect possibly lower abundance deletions generated within the same region of the mitochondrial genome. We characterized eight novel deletions of which six were generated from cut sites within, or adjacent to, short direct repeats. Two deletions involved cut sites in inverted tetrameric repeats; one of these also involved an insertion. 相似文献