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Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells. 总被引:29,自引:14,他引:15
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J R Stringer 《Molecular and cellular biology》1985,5(6):1247-1259
CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length. 相似文献
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Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site. 总被引:69,自引:32,他引:37
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The nucleotide sequence of the long terminal repeat (LTR) of three murine retroviral DNAs has been determined. The data indicate that the U5 region (sequences originating from the 5' end of the genome) of various LTRs is more conserved than the U3 region (sequences from the 3' end of the genome). The location and sequence of the control elements such as the 5' cap, "TATA-like" sequences, "CCAAT-box," and presumptive polyadenylic acid addition signal AATAAA in the various LTRs are nearly identical. Some murine retroviral DNAs contain a duplication of sequences within the LTR ranging in size from 58 to 100 base pairs. A variant of molecularly cloned Moloney murine sarcoma virus DNA in which one of the two LTRs integrated into the viral DNA was also analyzed. A 4-base-pair duplication was generated at the site of integration of LTR in the viral DNA. The host-viral junction of two molecularly cloned AKR-murine leukemia virus DNAs (clones 623 and 614) was determined. In the case of AKR-623 DNA, a 3- or 4-base-pair direct repeat of cellular sequences flanking the viral DNA was observed. However, AKR-614 DNA contained a 5-base-pair repeat of cellular sequences. The nucleotide sequence of the preintegration site of AKR-623 DNA revealed that the cellular sequences duplicated during integration are present only once. Finally, a striking homology between the sequences flanking the preintegration site and viral LTRs was observed. 相似文献
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We have developed an experimental approach to distinguish the 40-60 endogenous C-type proviruses of mice and to determine their association with well characterized developmental and physiological mutations. The hairless (hr) mutation causes a variety of pleiotropic effects. Using oligonucleotide probes specific for different classes of murine leukemia virus, we have identified and cloned a provirus present in HRS/J hr/hr animals but absent in HRS/J +/+. Genetic analyses showed perfect concordance between the hr phenotype and the presence of the provirus in a number of inbred and congenic strains of mice. Molecular analysis of a haired revertant established the causal relationship since it revealed the excision of most of the proviral genome leaving behind one long terminal repeat. These findings show that virus integration caused the hairless mutation and point to the utility of naturally occurring retroviral integrations for accessing the genome of the mouse. 相似文献
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Homologous recombination enhancement conferred by the Z-DNA motif d(TG)30 is abrogated by simian virus 40 T antigen binding to adjacent DNA sequences. 总被引:5,自引:0,他引:5
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The Z-DNA motif polydeoxythymidylic-guanylic [d(TG)].polydeoxyadenylic-cytidylic acid [d(AC)], present throughout eucaryotic genomes, is capable of readily forming left-handed Z-DNA in vitro and has been shown to promote homologous recombination. The effects of simian virus 40 T-antigen-dependent substrate replication upon the stimulation of recombination conferred by the Z-DNA motif d(TG)30 were analyzed. Presence of d(TG)30 adjacent to a T-antigen-binding site I can stimulate homologous recombination between nonreplicating plasmids, providing that T antigen is absent, in both simian CV-1 cells and human EJ cells (W. P. Wahls, L. J. Wallace, and P. D. Moore, Mol. Cell. Biol. 10:785-793). It has also been shown elsewhere that the presence of d(TG)n not adjacent to the T-antigen-binding site can stimulate homologous recombination in simian virus 40 molecules replicating in the presence of T antigen (P. Bullock, J. Miller, and M. Botchan, Mol. Cell. Biol. 6:3948-3953, 1986). However, it is demonstrated here that d(TG)30 nine base pairs distant from a T-antigen-binding site bound with T antigen does not stimulate recombination between either replicating or nonreplicating substrates in somatic cells. The bound T antigen either prevents the d(TG)30 sequence from acquiring a recombinogenic configuration (such as left-handed Z-DNA), or it prevents the interaction of recombinase proteins with the sequence by stearic hindrance. 相似文献
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Map locations of mouse hepatitis virus temperature-sensitive mutants: confirmation of variable rates of recombination. 总被引:6,自引:6,他引:0
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Using standard genetic recombination techniques, studies in our laboratory suggest that recombination rates are very high and vary in different portions of the mouse hepatitis virus (MHV) genome. To determine the actual recombination frequencies in the MHV genome and localize the nucleotide boundaries of individual viral genes, we have sequenced temperature-sensitive and revertant viruses to identify the location of specific mutant alleles. Complementation group F RNA+ ts mutants (LA7, NC6, and NC16) each contained a unique mutation which was tightly linked to the ts phenotype and resulted in a conservative or nonconservative amino acid change in the MHV S glycoprotein gene. In agreement with previous recombination mapping studies, the mutation in LA7 and NC6 mapped within the S1 domain while NC16 mapped within the S2 domain. To determine the map coordinates of the MHV polymerase genes, several RNA- mutants and their revertants belonging to complementation groups C (NC3 and LA9) and E (LA18 and NC4) were also sequenced. Mutations were identified in each virus that were tightly linked to the ts phenotype and resulted in either a conservative or nonconservative amino acid change. The group C allele spanned the ORF 1a/ORF 1b junction, while the group E mutants mapped at the C terminus of ORF 1b about 20 to 22 kb from the 5' end of the genome. Mutation rates, calculated from the reversion frequencies of plaque-purified ts viruses requiring a single nucleotide alteration for reversion, approached 1.32 (+/- 0.89) x 10(-4) substitutions per nucleotide site per round of template copying. Detailed recombination mapping studies across known distances between these different ts alleles has confirmed that homologous recombination rates approached 25% and varied within different portions of the MHV genome. 相似文献
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Molecular analysis of two mouse dilute locus deletion mutations: spontaneous dilute lethal20J and radiation-induced dilute prenatal lethal Aa2 alleles. 总被引:3,自引:0,他引:3
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M C Strobel P K Seperack N G Copeland N A Jenkins 《Molecular and cellular biology》1990,10(2):501-509
The dilute (d) coat color locus of mouse chromosome 9 has been identified by more than 200 spontaneous and mutagen-induced recessive mutations. With the advent of molecular probes for this locus, the molecular lesion associated with different dilute alleles can be recognized and precisely defined. In this study, two dilute mutations, dilute-lethal20J (dl20J) and dilute prenatal lethal Aa2, have been examined. Using a dilute locus genomic probe in Southern blot analysis, we detected unique restriction fragments in dl20J and Aa2 DNA. Subsequent analysis of these fragments showed that they represented deletion breakpoint fusion fragments. DNA sequence analysis of each mutation-associated deletion breakpoint fusion fragment suggests that both genomic deletions were generated by nonhomologous recombination events. The spontaneous dl20J mutation is caused by an interstitial deletion that removes a single coding exon of the dilute gene. The correlation between this discrete deletion and the expression of all dilute-associated phenotypes in dl20J homozygotes defines the dl20J mutation as a functional null allele of the dilute gene. The radiation-induced Aa2 allele is a multilocus deletion that, by complementation analysis, affects both the dilute locus and the proximal prenatal lethal-3 (pl-3) functional unit. Molecular analysis of the Aa2 deletion breakpoint fusion fragment has provided access to a previously undefined gene proximal to d. Initial characterization of this new gene suggests that it may represent the genetically defined pl-3 functional unit. 相似文献
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Cloning of the riboB locus of Aspergillus nidulans 总被引:9,自引:0,他引:9
We have complemented the riboB2 mutation of Aspergillus nidulans by transformation with a plasmid library of wild-type (wt) sequences. We have isolated, by marker rescue from a riboB+ transformant, a plasmid that complements riboB2 efficiently. From this plasmid we have subcloned an A. nidulans sequence that complements riboB2 efficiently and that integrates by homologous recombination at a site closely linked to the riboB locus. We conclude that this sequence contains the wt riboB+ allele. 相似文献
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The dispersion of four replication-defective endogenous proviruses, originally detected in 129 strain mice and shown to have extensive deletions of gag, pol, and env gene regions, was investigated in 13 inbred strains and substrains of mice. Using probes to sequences flanking the integration sites in 129 mice, unique genomic Eco RI fragments were assigned to each of the four endogenous proviral elements. Analyses revealed that certain of these proviral elements are present both in strains closely related to strain 129 (i.e., strains 101 and LP/J) and in more distantly related strains (i.e., strains BALB/cJ, A/J, and C3H/HeJ). In mouse strains lacking proviral integration at a particular locus, the size of the corresponding Eco RI genomic fragment and absence of a characteristic Kpn I site indicated the lack of a residual solitary long terminal repeat. Hybridization of oligonucleotide probes that distinguish the specific deletions present within these elements identified additional analogous proviral integrations at many different sites in all strains investigated. These data indicate that the diversification of these proviral elements found in inbred strains is generated by integration of new copies, rather than excision through homologous recombination. Moreover, the results are consistent with other endogenous retroviruses providing the trans-acting proteins necessary to package the defective viral RNA. 相似文献
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Molecular characterization of a nonautonomous transposable element (dTph1) of petunia. 总被引:7,自引:3,他引:4
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An insertion sequence of 283 base pairs has been isolated from the DFR-C gene (dihydroflavonol-4-reductase) of petunia. This insert was found only in a line unstable for the An1 locus (anthocyanin 1, located on chromosome VI) and not in fully pigmented progenitor and revertant lines or in stable white derivative lines. This implies that the An1 locus encodes the DFR-C gene. The unstable An1 system in the line W138 is known to be a two-element system, the autonomous element being located on chromosome I. In the presence of the autonomous element, W138 flowers exhibit a characteristic pattern of red revertant spots and sectors on a white background. In the absence of the autonomous element, the W138 allele gives rise to a stable recessive (white) phenotype. Sequence analysis of progenitor, unstable, and revertant alleles revealed dTph1 to contain perfect terminal inverted repeats of 12 base pairs. In DFR-C, it is flanked by an 8-base pair target site duplication. Sequences homologous to dTph1 are present in at least 50 copies in the line W138. Sequence analysis of An1 revertant alleles indicated that excision, including removal of the target site duplication, is required for reversion to the wild-type phenotype. Derivative stable recessive alleles showed excision of dTph1 and a rearrangement of the target site duplication. dTph1 is the smallest transposable element described to date that is still capable of transposition. The use of dTph1 in tagging experiments and subsequent gene isolation is discussed. 相似文献
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The bacterial lactose operator (lacO) was introduced into the PstI site of the long terminal repeat of the SL3-3 murine leukemia virus, generating a virus, SL3-3lacO, that can replicate in NIH3T3 cell cultures. DNA sequences harboring the lacO sequence might be recovered by molecular cloning in Escherichia coli lac+ lacZ+ using bacteriophage lambda or plasmid vectors. The high copy numbers of the lacO sequence titrate out the lac repressor, leading to the induction of the lac operon in the host. We show here that the lacO and the proviral sequences are carried stably together in the genomes of SL3-3lacO-infected cell cultures and in viral particles. This system is designed to facilitate studies on the provirus and the site of viral integration. 相似文献
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By screening a library of unintegrated, circular Moloney murine leukemia virus (M-MuLV) DNA cloned in lambda phage, we found that approximately 20% of the M-MuLV DNA inserts contained internal sequence deletions or inversions. Restriction enzyme mapping demonstrated tht the deleted segments frequently abutted a long terminal repeat (LTR) sequence, whereas the inverted segments were usually flanked by LTR sequences, suggesting that many of the variants arose as a consequence of M-MuLV DNA molecules integrating within their own DNA. Nucleotide sequencing also suggested that most of the variant inserts were generated by autointegration. One of the recombinant M-MuLV DNA inserts contained a large inverted repeat of a unique M-MuLV sequence abutting an LTR. This molecule was shown by nucleotide sequencing to have arisen by an M-MuLV DNA Molecule integrating within a second M-MuLV DNA molecule before cloning. The autointegrated M-MuLV DNA had generally lost two base pairs from the LTR sequence at each junction with target site DNA, whereas a four-base-pair direct repeat of target site DNA flanked the integrated viral DNA. Nucleotide sequencing of preintegration target site DNA showed that this four-base-pair direct repeat was present only once before integration and was thus reiterated by the integration event. The results obtained from the autointegrated clones were supported by nucleotide sequencing of the host-virus junction of two cloned M-MuLV integrated proviruses obtained from infected rat cells. Detailed analysis of the different unique target site sequences revealed no obvious common features. 相似文献
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RAD10, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination. 总被引:18,自引:5,他引:13
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The RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of UV-damaged DNA. We show that the RAD10 gene is also required for mitotic recombination. The rad10 delta mutation lowered the rate of intrachromosomal recombination of a his3 duplication in which one his3 allele has a deletion at the 3' end and the other his3 allele has a deletion at the 5' end (his3 delta 3' his3 delta 5'). The rate of formation of HIS3+ recombinants in the rad10 delta mutant was not affected by the rad1 delta mutation but decreased synergistically in the presence of the rad10 delta mutation in combination with the rad52 delta mutation. These observations indicate that the RAD1 and RAD10 genes function together in a mitotic recombination pathway that is distinct from the RAD52 recombination pathway. The rad10 delta mutation also lowered the efficiency of integration of linear DNA molecules and circular plasmids into homologous genomic sequences. We suggest that the RAD1 and RAD10 gene products act in recombination after the formation of the recombinogenic substrate. The rad1 delta and rad10 delta mutations did not affect meiotic intrachromosomal recombination of the his3 delta 3' his3 delta 5' duplication or mitotic and meiotic recombination of ade2 heteroalleles located on homologous chromosomes. 相似文献
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Recombination between a defective retrovirus and homologous sequences in host DNA: reversion by patch repair. 总被引:10,自引:9,他引:1
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The genomes of mammalian species contain multiple copies of sequences homologous to exogenous retroviruses. When a mutant retrovirus carrying a lethal deletion in an essential viral gene was introduced into mammalian cells, revertant viruses appeared and spread throughout the culture. Analysis of one such revertant showed that the mutation had been repaired by homologous recombination with endogenous sequences. Our results suggest that defective retroviruses can draw upon the genetic complement of the host cell to repair lesions in viral genes. 相似文献