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Avian Retrovirus pp32 DNA-Binding Protein I. Recognition of Specific Sequences on Retrovirus DNA Terminal Repeats 总被引:21,自引:10,他引:11
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The avian retrovirus pp32 protein possesses a DNA-nicking activity which prefers supercoiled DNA as substrate. We have investigated the binding of pp32 to avian retrovirus long terminal repeat (LTR) DNA present in both supercoiled and linear forms. The cloned viral DNA was derived from unintegrated Schmidt-Ruppin A (SRA) DNA. A subclone of the viral DNA in pBR322 (termed pPvuII-DG) contains some src sequences, tandem copies of LTR sequences, and partial gag sequences in the order src-U(3) U(5):U(3) U(5)-gag. Binding of pp32 to supercoiled pPvuII-DG DNA followed by digestion of this complex with a multicut restriction enzyme (28 fragments total) permitted pp32 to preferentially retain on nitrocellulose filters two viral DNA fragments containing only LTR DNA sequences. In addition, pp32 also preferentially retained four plasmid DNA fragments containing either potential promoters or Tn3 "left-end" inverted repeat sequences. Mapping of the pp32 binding sites on viral LTR DNA was accomplished by using the DNase I footprinting technique. The pp32 protein, but not the avian retrovirus alphabeta DNA polymerase, is able to form a unique protein-DNA complex with selected regions of either SRA or Prague A LTR DNAs. Partial DNase I digestion of a 275-base pair SRA DNA fragment complexed with pp32 gives upon electrophoresis in denaturing gels a unique ladder pattern, with regions of diminished DNase I susceptibility from 6 to 10 nucleotides in length, in comparison with control digests in the absence of protein. The binding of pp32 to this fragment also yields enhanced DNase I-susceptible sites that are spaced between the areas protected from DNase I digestion. The protected region of this unique complex was a stretch of 170 +/- 10 nucleotides that encompasses the presumed viral promoter site in U(3), which is adjacent to the src region, extends through U(5), and proceeds past the joint into U(3) for about 34 base pairs. No specific protection or DNase I enhancement by pp32 was observed in experiments with a 435-base pair SRA DNA fragment derived from a part of U(3) and the adjacent src region or a 55-base pair DNA fragment derived from another part of U(3). The DNA sequence of Prague A DNA at the fused LTRs differs from that of SRA DNA. The alteration in the sequence at the juncture of the LTRs prevented pp32 from forming a stable complex in this region of the LTR. Our results are relevant to two aspects of the interaction between pp32 and LTR DNA. First, the pp32 protein in the presence of selected viral DNA restriction fragments possibly forms a higher order oligomer analogous to Escherichia coli DNA gyrase-DNA complexes or eucaryotic nucleosome structures. Second, the specificity of the binding suggests a role for pp32 and the protected DNA sequences in the retrovirus life cycle. The preferred sequences to which pp32 binds include two adjacent 15-base pair inverted terminal repeats at the joint between U(5) and U(3) in SRA DNA. This region is involved in circularization of linear DNA and is perhaps the site that directs integration into cellular DNA. 相似文献
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Alterations in the U3 region of the long terminal repeat of an infectious thymotropic type B retrovirus. 总被引:19,自引:12,他引:7
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J K Ball H Diggelmann G A Dekaban G F Grossi R Semmler P A Waight R F Fletcher 《Journal of virology》1988,62(8):2985-2993
We isolated and characterized a type B thymotropic retrovirus (DMBA-LV) which is highly related to mouse mammary tumor virus (MMTV) isolates and which induces T-cell thymomas with a high incidence and a very short latent period. Regions of nonhomology between the DMBA-LV genome and the MMTV genome were identified by heteroduplex mapping and nucleotide sequence studies. In the electron microscope heteroduplex mapping studies the EcoRI-generated 5' and 3' fragments of the DMBA-LV genome were compared with the corresponding fragments of the MMTV (C3H and GR) genome isolated from mammary tumors. The results indicated that DMBA-LV contained a region of nonhomologous nucleotide sequences in the 3' half of the U3 region of the long terminal repeat (LTR). Nucleotide sequence studies confirmed these results and showed that in this region 440 nucleotides of the MMTV (C3H) sequences were deleted and substituted with a segment of 122 nucleotides. This substituted segment in the form of a tandem repeat structure contained nucleotide sequences derived exclusively from sequences which flanked the substitution loop. The distal glucocorticoid regulatory element was unaltered, and two additional copies of the distal glucocorticoid regulatory element-binding site were present in the substituted region. The restriction endonuclease map of the reconstructed molecular clone of DMBA-LV was identical to that corresponding to unintegrated linear DMBA-LV DNA present in DMBA-LV-induced tumor cell lines. Since the nucleotide sequences of the LTRs present in four different DMBA-LV proviral copies isolated from a single thymoma were identical, we concluded that they were derived from the same parental virus and that this type B retrovirus containing an alteration in the U3 region of its LTR could induce thymic lymphomas. Thus, DMBA-LV represents the first example of a productively replicating type B retrovirus that contains an LTR modified in the U3 region and that has target cell and disease specificity for T cells. 相似文献
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Activation of an endogenous retrovirus enhancer by insertion into a heterologous context. 总被引:1,自引:1,他引:0
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K F Conklin 《Journal of virology》1991,65(5):2525-2532
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Presence of a retroviral encapsidation sequence in nonretroviral RNA increases the efficiency of formation of cDNA genes. 总被引:6,自引:3,他引:3
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We showed previously that retrovirus vector particles can encapsidate RNAs without retroviral cis-acting sequences, that such RNAs are reverse transcribed in infected target cells, and that the cDNA copies are inserted into the host genome resulting in cDNA genes (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To provide further evidence that this retrovirus-mediated gene transfer occurred through an RNA intermediate, we constructed retroviral vectors containing an intron from a cellular gene. This intron was lost in a cDNA gene formed after infection with retroviral particles, establishing that an RNA intermediate had existed. Retroviral vectors with additional encapsidation sequences were constructed. The presence of a murine leukemia virus encapsidation sequence in an mRNA transcribed from the hygromycin B phosphotransferase gene increased the efficiency of encapsidation into spleen necrosis virus vector particles and the formation of cDNA genes by approximately 2 orders of magnitude. 相似文献
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Characterization of unintegrated retroviral DNA with long terminal repeat-associated cell-derived inserts.
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We have used a replication-competent shuttle vector based on the genome of Rous sarcoma virus to characterize genomic rearrangements that occur during retrovirus replication. The strategy involved cloning circular DNA that was generated during an acute infection. While analyzing a class of retroviral DNA clones that are greater than full length, we found several clones which had acquired nonviral inserts in positions adjacent to the long terminal repeats (LTRs). There appear to be two distinct mechanisms leading to the incorporation of cellular sequences into these clones. Three of the molecules contain a cell-derived insert at the circle junction site between two LTR units. Two of these molecules appear to be the results of abortive integration attempts, because of which, in each case, one of the LTRs is missing 2 bases at its junction with the cell-derived insert. In the third clone, pNO220, the cellular sequences are flanked by an inappropriately placed copy of the tRNA primer-binding site on one side and a partial copy of the U3 sequence as part of the LTR on the other side. A fourth molecule we characterized, pMD96, has a single LTR with a U5-bounded deletion of viral sequences spanning gag and pol, with cell-derived sequences inserted at the site of the deletion; its origin may be related mechanistically to pNO220. Sequence analysis indicates that all of the cellular inserts were derived from the cell line used for the acute infection rather than from sequences carried into the cell as part of the virus particle. Northern (RNA) analysis of cellular RNA demonstrated that the cell-derived sequences of two clones, pNO220 and pMD96, were expressed as polyadenylated RNA in uninfected cells. One mechanism for the joining of viral and cellular sequences suggested by the structures of pNO220 and pMD96 is recombination occurring during viral DNA synthesis, with cellular RNA serving as the template for the acquisition of cellular sequences. 相似文献
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Retrovirus-like sequences and their solitary (solo) long terminal repeats (LTRs) are common repetitive elements in eukaryotic
genomes. We reported previously that the tandemly arrayed genes encoding U2 snRNA (the RNU2 locus) in humans and apes contain a solo LTR (U2-LTR) which was presumably generated by homologous recombination between
the two LTRs of an ancestral provirus that is retained in the orthologous baboon RNU2 locus. We have now sequenced the orthologous U2-LTRs in human, chimpanzee, gorilla, orangutan, and baboon and examined numerous
homologs of the U2-LTR that are dispersed throughout the human genome. Although these U2-LTR homologs have been collectively
referred to as LTR13 in the literature, they do not display sequence similarity to any known retroviral LTRs; however, the
structure of LTR13 closely resembles that of other retroviral LTRs with a putative promoter, polyadenylation signal, and a
tandemly repeated 53-bp enhancer-like element. Genomic blotting indicates that LTR13 is primate-specific; based on sequence
analysis, we estimate there are about 2,500 LTR13 elements in the human genome. Comparison of the primate U2-LTR sequences
suggests that the homologous recombination event that gave rise to the solo U2-LTR occurred soon after insertion of the ancestral
provirus into the ancestral U2 tandem array. Phylogenetic analysis of the LTR13 family confirms that it is diverse, but the
orthologous U2-LTRs form a coherent group in which chimpanzee is closest to the humans; orangutan is a clear outgroup of human,
chimpanzee, and gorilla; and baboon is a distant relative of human, chimpanzee, gorilla, and orangutan. We compare the LTR13
family with other known LTRs and consider whether these LTRs might play a role in concerted evolution of the primate RNU2 locus.
Received: 29 September 1997 / Accepted: 16 January 1998 相似文献
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Diverse long terminal repeats are associated with murine retroviruslike (VL30) elements. 总被引:10,自引:3,他引:7
The VL30 family is a retroviruslike gene family with no apparent nucleic acid homology to any known retrovirus. Over 100 copies of VL30 DNA elements are dispersed throughout the mouse genome. Sequence analysis of the VL30 long terminal repeat (LTR) units showed that, whereas the LTR units of a given VL30 DNA element were almost identical, the LTR units associated with distinct members of the family were very different from one another. Comparison of the LTR sequences possessed by two particular VL30 DNA elements revealed a pattern of extensively homologous DNA segments adjacent to only distantly related DNA sequences. With the aid of sub-LTR probes, it was shown that a certain LTR is composed of both U5 sequences that are abundantly present in all species of the genus Mus and a U3 region detected only in Mus musculus. In addition, we isolated a VL30 DNA element in which the LTR units were replaced by the LTR units of an apparently novel retroviruslike family. These findings suggest that recombinations have played a role in generating the diverse population of VL30-associated LTRs. 相似文献
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Du Hyeong Lee Woo Hyeon Bae Hongseok Ha Eun Gyung Park Yun Ju Lee Woo Ryung Kim Heui-Soo Kim 《Molecules and cells》2022,45(8):522
Transposable elements (TEs) account for approximately 45% of the human genome. TEs have proliferated randomly and integrated into functional genes during hominoid radiation. They appear as right-handed B-DNA double helices and slightly elongated left-handed Z-DNAs. Human endogenous retrovirus (HERV) families are widely distributed in human chromosomes at a ratio of 8%. They contain a 5′-long terminal repeat (LTR)-gag-pol-env-3′-LTR structure. LTRs contain the U3 enhancer and promoter region, transcribed R region, and U5 region. LTRs can influence host gene expression by acting as regulatory elements. In this review, we describe the alternative promoters derived from LTR elements that overlap Z-DNA by comparing Z-hunt and DeepZ data for human functional genes. We also present evidence showing the regulatory activity of LTR elements containing Z-DNA in GSDML. Taken together, the regulatory activity of LTR elements with Z-DNA allows us to understand gene function in relation to various human diseases. 相似文献
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Integrated retroviral genomes are flanked by direct repeats of sequences derived from the termini of the viral RNA genome. These sequences are designated long terminal repeats (LTRs). We have determined and analyzed the nucleotide sequence of the LTRs from several exogenous and endogenous avian retroviruses. These LTRs possess several structural similarities with eukaryotic and prokaryotic transposable elements: 1) inverted complementary repeats at the termini, 2) deletions of sequences adjacent to the LTR, 3) small duplications of host sequences flanking the integrated provirus, and 4) sequence homologies with transposable and other genetic elements. These observations suggest that LTRs function in the integration and perhaps transposition of retrovirus genomes. Evidence exists for the presence of a strong promoter sequence within the LTR. The retroviral LTR also contains a "Hogness box" up-stream of the capping site and a poly(A) signal. These features suggest an additional role for the LTR in the regulation of gene expression. 相似文献
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在水稻第四号染色体的长臂上鉴定了一个结构完整的Ty3型逆转录转座子RIRE10。RIRE10两LTR间的中间区域在gag pol的上游还包含另一个开放阅读框。通过RT PCR与Northern印迹杂交检测到来自LTR区的转录产物 ;根据点杂交结果 ,鉴定出包含中间区域的RIRE10成员的个数以及LTR区的拷贝数。除了 6 5个完整的逆转录转座子所具备的两个LTR外 ,水稻基因组还含有近 90 0个RIRE10的solo LTR。LTR区的转录以及导致solo LTR产生的同源重组可能影响了RIRE10成员在水稻基因组中的转座活性 相似文献
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Kim S Lee K Kim MD Kang S Joo CW Kim JM Kim SH Yu SS Kim S 《Biochemical and biophysical research communications》2006,343(4):1017-1022
The long terminal repeat (LTR) of retrovirus contains the nucleotide sequences that control gene expression. Although several different LTRs have been used in the context of retroviral vector, the activity of the various LTRs has not yet been systematically compared for their level of gene expression. We evaluated the effect of four different LTRs on gene expression using luciferase, stem cell factor, and enhanced green fluorescence protein as reporter genes. LTRs tested in this study were derived from Moloney murine leukemia virus, myeloproliferative sarcoma virus, murine stem cell virus, and spleen focus-forming virus. It was found that the level of gene expression is affected by not only LTRs but also the transgenes and the cell types in which gene expression occurs. Furthermore, the presence of other nucleotide sequences such as the internal ribosome entry site (IRES)-neo cassette could also significantly affect gene expression. Our results suggested that the LTR should be chosen carefully, more or less on an empirical basis. 相似文献
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Development of minimal lentivirus vectors derived from simian immunodeficiency virus (SIVmac251) and their use for gene transfer into human dendritic cells 总被引:4,自引:0,他引:4
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Mangeot PE Nègre D Dubois B Winter AJ Leissner P Mehtali M Kaiserlian D Cosset FL Darlix JL 《Journal of virology》2000,74(18):8307-8315
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Retrovirus like sequences homologous to mouse IAP are present in Chinese hamster genome (Lueders K.K. and Kuff, E.L., 1981, 1983, Servenay et al., 1990). Murine IAP long terminal repeats (LTRs) can function as effective promoters in different cell types (Horowitz M. et al., 1984, Howe, C.C. et al., 1986). Thus CHO IAP sequences could act as retrotransposons in the cellular genome, and in this way affect the expression of other genes at the target sites. We had sequenced previously a Chinese hamster IAP genomic region corresponding mainly to the gag gene and including 57 nucleotides of U5 5' LTR (Servenay et al., 1988). In this paper, we present the 5' LTR complete nucleotide sequence of the Chinese hamster IAP element and its comparison with those of mouse and Syrian hamster. 相似文献