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PubMed1989

Characterization of the long terminal repeats of micropia elements microdissected from the Y-chromosomal lampbrush loops "threads" of Drosophila hydei.

Four micropia elements from Drosophila melanogaster and D. hydei have been analysed by sequencing. Two elements, from D. hydei, micropia-DhMiF8 and -DhMiF2, were recovered by cloning microdissected Y-chromosomal lampbrush loops "threads". This method allows isolation of repetitive sequences from defined chromosomal positions, but recovery of large and overlapping inserts is difficult. In case of the Y-chromosomal micropia elements it was not possible to define the endpoints of their long terminal repeat sequences precisely. Comparison of these locus-defined micropia elements to complete micropia elements isolated from D. melanogaster allowed identification of micropia-DhMiF8 and micropia-DhMiF2 long terminal repeats (LTRs). LTR sequences from the two Drosophila species are not conserved except for a few short sequences found at comparable positions that are believed to have functional significance. In contrast, the Leu-tRNA primer binding site and plus strand primer binding site are conserved between D. melanogaster and D. hydei.

Animals↗

PubMed1992

Interaction of SEWA sarcoma cell proteins with the intracisternal A-type particle long terminal repeat DNA sequence.

Intracisternal A-type particle (IAP) transcripts are endogenous retrovirus-like sequences expressed during specific stages of normal development and in a variety of murine tumors. In this study, we have analyzed two cell lines derived originally from the SEWA murine osteosarcoma and grown either as ascites or as solid tumors, for proteins that might regulate IAP expression. We found that subline AA7-NA, originally derived from the ascites tumor, expressed about five times more IAP RNA than the AS12-AD subline, which was derived from a solid tumor. In view of this finding, we examined the binding of cellular proteins from the two cell lines to the 5' end of an IAP long terminal repeat sequence. Gel retardation assays of DNA-protein complexes and DNase I footprinting assays identified several DNA sequences within the long terminal repeat fragment that were protected by protein extracts from both SEWA sublines. Gel retardation assays using specific synthetic oligonucleotide sequences that correspond to two of these protected regions revealed different patterns of DNA-protein complexes with extracts from the two SEWA sublines. These data suggest that expression of IAP sequences is regulated by complex mechanisms involving several proteins that appear to differ between the two sublines.

Animals↗

PubMed1998

The position and length of the steroid-dependent hypersensitive region in the mouse mammary tumor virus long terminal repeat are invariant despite multiple nucleosome B frames.

Stimulation of the mouse mammary tumor virus with steroids results in the generation of a DNase I-hypersensitive region (HSR) spanning the hormone responsive element (HRE) in the long terminal repeat. Restriction enzymes were used to characterize the accessibility of various sites within the HSR of mouse mammary tumor virus long terminal repeat-reporter constructions in four different cell lines. The glucocorticoid-dependent HSR was found to span minimally 187 bases, a stretch of DNA longer than that associated with histones in the core particle. Although the 5'-most receptor binding site within the HRE is downstream of -190, hypersensitive sites were found further upstream to at least -295. The relationship in the accessibility between pairs of sites in the vicinity of the HSR was further examined in one cell line by a two-enzyme restriction access assay. In the uninduced state, the accessibilities at these sites were found to be independent of each other. In contrast, when stimulated with hormone, the accessibilities at these sites were observed to become linked. That is, once a distinct promoter was activated, all of the sites within the HSR of that molecule became accessible. The HSR formed along an invariant stretch of DNA sequence despite the multiplicity of nucleosome frames in the nucleosome B region, where the HRE is located. The results indicate that the macroscopic length of the HSR does not arise from core length-remodeling events in molecules containing Nuc-B in alternative positions.

Animals↗

PubMed1984

Characterization of enhancer elements in the long terminal repeat of Moloney murine sarcoma virus.

A series of recombinant molecules were constructed which direct the expression of the easily assayed gene chloramphenicol acetyltransferase. We have used these recombinants to show that the 73/72-base-pair tandem repeat unit from the Moloney murine sarcoma virus long terminal repeat shares a number of properties with the prototypic enhancer element, the simian virus 40 72-base-pair repeat. Specifically, the Moloney murine sarcoma virus sequence significantly enhances the level of gene expression at both 5' and 3' locations and in either orientation relative to the test gene. It is able to enhance gene activity both from its own promoter and from a heterologous (simian virus 40) promoter. The 73/72-base-pair subunits of the Moloney murine sarcoma virus enhancer differ in sequence by four nucleotides and also in the strength of their enhancer function. The promoter distal A repeat is at least three times as active as the promoter proximal B repeat in enhancing chloramphenicol acetyltransferase expression. Results of these studies also show that the enhancer sequence alone is unable to induce gene activity but requires other promoter elements, including a proximal GC-rich sequence and the Goldberg-Hogness box.

DNA, Viral↗

PubMed1991

Involvement of long terminal repeat U3 sequences overlapping the transcription control region in human immunodeficiency virus type 1 mRNA 3' end formation.

In retroviral proviruses, the poly(A) site is present in both long terminal repeats (LTRs) but used only in the 3' position. One mechanism to account for this selective poly(A) site usage is that LTR U3 sequences, transcribed only from the 3' poly(A) site, are required in the RNA for efficient processing. To test this possibility, mutations were made in the human immunodeficiency virus type 1 (HIV-1) U3 region and the mutated LTRs were inserted into simple and complex transcription units. HIV-1 poly(A) site usage was then quantitated by S1 nuclease analysis following transfection of each construct into human 293 cells. The results showed that U3 sequences confined to the transcription control region were required for efficient usage of the HIV-1 poly(A) site, even when it was placed 1.5 kb from the promoter. Although the roles of U3 in processing and transcription activation were separable, optimal 3' end formation was partly dependent on HIV-1 enhancer and SP1 binding site sequences.

Cloning, Molecular↗

PubMed2000

Green fluorescence protein as a transcriptional reporter for the long terminal repeats of the human immunodeficiency virus type 1.

Using the enhanced green fluorescence protein (EGFP), a transient reporter expression system was established to assess the transcriptional activity of the long terminal repeats (LTR) of primary isolates of the human immunodeficiency virus type 1 (HIV-1). Consistent with the conventional chloramphenicol acetyl transferase (CAT) reporter, EGFP expression, under the direction of HIV-1 LTR, was readily detected in the transient transfection and was elevated by co-transfection of HIV-1 tat-expression vector. Comparing to CAT, however, EGFP expression system has two advantages: (i) Using a fluorescence activated cell sorter (FACS), it was possible to simultaneously measure transfection efficiency and fluorescence intensity of the transfected live cells without the necessity of co-transfection of a reference plasmid for comparing the transcriptional activity of two promoters; and (ii) EGFP expression was readily detected at a DNA concentration where CAT activity was not detectable possibly because the transfectants could be 'gated'. On the other hand, at a higher concentration of DNA, CAT signal became more prominent than that of EGFP, possibly because the enzymatic activity of CAT 'amplified' the signal. EGFP fluorescence detected by FACS was a direct measurement of the expressed chromophore. It is concluded that the system is rapid, reproducible, convenient and useful for quantitative analysis of transcription.

Chloramphenicol O-Acetyltransferase↗

PubMed1994

Naturally occurring genotypes of the human immunodeficiency virus type 1 long terminal repeat display a wide range of basal and Tat-induced transcriptional activities.

The primary body of information on the structure of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)/gag leader genotypes has been determined from the analysis of cocultivated isolates. Functional studies of this regulatory portion of the provirus have been derived from the study of in vitro-generated mutations of laboratory-adapted molecular clones of HIV-1. We have performed a longitudinal analysis of molecular clones from the LTR/gag leader region amplified directly from the peripheral blood of four patients over three years. We have found a remarkable number of point mutations and length polymorphisms in cis- and trans-acting regulatory elements within this cohort. Most of the length polymorphisms were associated with duplications of Sp1 and TCF-1 alpha sequences. These mutations were associated with a wide range of transcriptional activities for these genotypes in a reporter gene assay. Mutations in conserved Sp1 sequences correlated with a diminished capacity of such genotypes to bind purified Sp1 protein. Although no generalized trend in transcriptional activity was seen, a single patient accumulated mutations in NF-kappa B, Sp1, and TAR elements over this period. The analysis of naturally occurring mutations of LTR genotypes provides a means to study the molecular genetic consequences of virus-host interactions and to assess the functional impact of HIV therapeutics.

Base Sequence↗

PubMed1995

Distinct HIV-1 long terminal repeat quasispecies present in nervous tissues compared to that in lung, blood and lymphoid tissues of an AIDS patient.

OBJECTIVE: To investigate the phylogenetic relationship of HIV-1 proviral long terminal repeat (LTR) variants present in postmortem samples of lymph node, spleen, lung, dorsal root ganglion and spinal cord as well as in the peripheral blood of an HIV-1-infected patient dying with AIDS. DESIGN AND METHODS: Postmortem tissues were studied by a combination of histology, cell culture and molecular analyses. The patient had a stable CD4 count of 10 x 10(6)/I during the 12 months preceding death. A 540 base-pair fragment of the LTR including U3/R/U5 was amplified using polymerase chain reaction on proviral DNA from the five postmortem tissues and peripheral blood mononuclear cells obtained 2 months prior to death. The population of viral variants was determined by sequencing at least five plasmid clones of the amplicons. The relationship between the variants present in different body sites was investigated using molecular phylogeny methods. RESULTS: HIV-1 was present in all organs analysed and correlated with the presence of abnormal histology. Genetic variation leading to divergence from the consensus sequence was more frequently present in characterized transcription factor binding sites within the LTR (P < 0.0001) although the HIV-1 LTR quasispecies in the different body sites showed similar, relatively low levels of divergence (intra-organ median heterogeneity ranging from 0.0094 to 0.017). Phylogenetic analysis showed that the spinal cord and dorsal root ganglion harboured an LTR population genetically distinct from that present in other organs and more closely related to a previously characterized neurotropic strain of HIV (strain JRcsf). CONCLUSION: The independent clustering of HIV-1 LTR variants present in spinal cord and dorsal root ganglion shows that HIV-1 LTR evolution can occur in a compartmentalized fashion. The data show that the LTR is an important region to analyse in sequence variation studies of HIV since it may play a role in nervous tissue adaptation of HIV-1 and neuropathogenicity. Outgrowth of HIV-1 LTR variants that are most fit for the utilization of tissue-specific transcription factors can occur in the nervous tissue.

Acquired Immunodeficiency Syndrome↗

PubMed1987

5' long terminal repeats of myc-associated proviruses appear structurally intact but are functionally impaired in tumors induced by avian leukosis viruses.

B-cell lymphomas induced in chickens infected with avian leukosis viruses are characterized by integration of the virus within the cellular myc locus and alteration of c-myc expression. Although avian leukosis viruses are intact, replication-competent retroviruses, the structures of many myc-associated proviruses are altered by deletions, raising the possibility that proviral defectiveness plays an essential role in oncogenesis. We found that all myc-associated proviruses in 21 independent tumors had deletions, which were confined to the viral genome and did not extend into adjacent cellular sequences. Deletions were not random but, in at least 85% of the myc-associated proviruses, involved a region near the 5' end of the proviral genome where elements implicated in control of viral gene expression have been localized. A second class of deletions involved sequences in the 3' half of the viral genome and included the splice acceptor site used in generating viral env mRNA. Both the 5' and 3' long terminal repeats of myc-associated proviruses appeared to be structurally intact in most tumors, although the 5' long terminal repeats were not involved in expression of either U5-myc transcripts or detectable steady-state viral RNAs. A complex array of repeated sequence elements surrounded the junctions of the internal deletions in two myc-associated proviruses. The organization of the deleted proviruses was similar to that of deleted unintegrated viral molecules, consistent with a model in which deletions occurred prior to integration.

Avian Leukosis Virus↗

PubMed1990

Hepatitis B virus X protein transactivates the long terminal repeats of human immunodeficiency virus types 1 and 2.

The X gene product of the hepatitis B virus (HBV) has been expressed transiently in HepG2 cells, and the 17-kilodalton protein has been detected by Western (immuno-) blot analysis. Cotransfection of the X gene with the long terminal repeat of human immunodeficiency virus type 1 or 2 results in a stimulation of long terminal repeat-directed expression that is higher than the X-induced stimulation of the HBV enhancer linked to either autologous promoter or to the heterologous simian virus 40 promoter. A frameshift mutation abolished this transactivation. In vitro nuclear transcription assays revealed that HBV X acts at the transcriptional level. The carboxy terminus of the HBV X protein does not seem to be necessary for its transactivating activity, as demonstrated by using HBV X protein deletion mutants.

Blotting, Western↗

PubMed1997

Non-random deletions in human foamy virus long terminal repeat during viral infection.

We have characterized a new form of human foamy virus (HFV) non-random deleted long terminal repeat (LTR) sizing 1078-bp, deleted in its U3 region, sensitive to the viral transactivator and functional in an infectious proviral clone. Besides two known HFV LTRs of 1260-bp and 1123-bp, this LTR represents the smallest, designed S. Analysis of the LTR sequence shows the presence of short direct repeats surrounding the deletions, suggesting a mechanism generating deletion by misalignment of the growing strand during replication. Our data suggest that the deleted LTRs, preferentially associated with chronic viral infection, could be related with viral persistence.

Animals↗

PubMed1989

Reconstitution of nucleosomes in vitro with a plasmid carrying the long terminal repeat of Moloney murine leukemia virus.

The potential of nucleosome assembly along the sequence of a plasmid carrying the long terminal repeat (LTR) and its flanking region of Moloney murine leukemia virus was analyzed by in vitro reconstitution experiments with histones from chicken erythrocytes. The results of electrophoretic mobility-shift and micrococcal nuclease-digestion assays indicated that the plasmid DNA contained four preferred sites for nucleosome formation. However, all of these sites were mapped on the vector moiety but not on the LTR moiety. Computer analysis of the sequences in the four preferred sites, each spanning about 150 bp, indicated that short runs of (dA,dT) containing two kinds of triplets, AAA/TTT and AAT/ATT, occurred frequently. Furthermore, many of these triplets tended to occur in the same side of the DNA helix, suggesting that DNA curvature was involved in the preferred sites for nucleosome assembly. Consistent was the observation that DNA fragments carrying these preferred sites showed anomalous electrophoretic mobilities at a low temperature.

Base Sequence↗

PubMed1988

The long terminal repeat of feline endogenous RD-114 retroviral DNAs: analysis of transcription regulatory activity and nucleotide sequence.

Six cloned 5' long terminal repeat (LTR) and adjoining cellular DNA regions of partially deleted feline endogenous RD-114 proviral loci were linked to the chloramphenicol acetyltransferase (CAT) gene and assayed for their ability to promote transient CAT expression. One endogenous LTR (clone CRL-3) and the LTR from an infectious RD-114 provirus, EX-LTR, were capable of actively expressing the CAT gene. DNA sequence comparison of these LTRs with an inactive endogenous LTR (CR-1) revealed extensive homology in all regions except in the 5' half of U3. The homologous portion contained transcriptional regulatory sequences including CAT, TATA, polyadenylation signal boxes and an octamer enhancer, which is rarely seen in retroviruses. Variations in the 5' half of U3 were primarily due to insertions and deletions. A major difference was in number of copies and integrity of tandemly repeated sequences. EX-LTR contained two pairs of tandem direct repeats, while the two endogenous LTRs contained different deletions of repeated sequences. DNA sequence data also revealed that the primer binding site for RD-114 loci was complementary to a glycine tRNA isotype, the use of which is distinct from any other known retrovirus. An analysis of the steady state RNA levels in T-lymphoid cell lines showed that at least three different incomplete proviral transcripts and their spliced products made up the majority of expressed RD-114 mRNA, and further demonstrated that partially deleted proviral loci have the potential to be transcriptionally vigorous in certain feline cell types.

Acetyltransferases↗

PubMed1982

Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site.

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.

AKR murine leukemia virus↗

PubMed1992

Characterization of nuclear proteins that bind the EFII enhancer sequence in the Rous sarcoma virus long terminal repeat.

The EFII cis element is a 38-bp sequence at the 5' end of the Rous sarcoma virus long terminal repeat, extending from nucleotides -229 to -192 (with respect to the viral transcription start site), which is recognized by sequence-specific DNA-binding proteins in avian fibroblast nuclear extracts (L. Sealy and R. Chalkley, Mol. Cell. Biol. 7:787-798, 1987). We demonstrate that multiple copies of the EFII cis element strongly activate transcription of a reporter gene in vivo. We correlate the region of the EFII cis element which activates transcription in vivo with the in vitro binding site for three nuclear factors, EFIIa, EFIIb, and EFIIc. The sequence motif recognized by EFIIa, -b, and -c is also found in consensus binding sites for members of a rapidly growing family of transcription factors related to the CCAAT/enhancer-binding protein (C/EBP). EFIIa, -b, and -c are present in fibroblast and epithelial cell lines from various species but are much less abundant in differentiated rat liver and kidney cells. The EFIIa binding activity is particularly abundant in an avian B-cell lymphoma line. As judged from molecular weight analysis, cell type distribution, and sequence recognition properties, the EFII factors under study appear to differ from most of the previously described C/EBP-related factors and thus may expand the diversity of the C/EBP family.

Avian Sarcoma Viruses↗

PubMed1995

A redundant nuclear protein binding site contributes to negative regulation of the mouse mammary tumor virus long terminal repeat.

The tissue specificity of mouse mammary tumor virus (MMTV) expression is controlled by regulatory elements in the MMTV long terminal repeat (LTR). These regulatory elements include the hormone response element, located approximately between -200 and -75, as well as binding sites for NF-1, Oct-1 (OTF-1), and mammary gland enhancer factors. Naturally occurring MMTV deletion variants isolated from T-cell and kidney tumors, transgenic-mouse experiments with MMTV LTR deletions, and transient transfection assays with LTR constructs indicate that there are additional transcription regulatory elements, including a negative regulatory element (NRE), located upstream of the hormone response element. To further define this regulatory region, we have constructed a series of BAL 31 deletion mutants in the MMTV LTR for use in transient transfection assays. These assays indicated that deletion of two regions (referred to as promoter-distal and -proximal NREs) between -637 and -201 elevated basal MMTV promoter activity in the absence of glucocorticoids. The region between -637 and -264 was surveyed for the presence of nuclear protein binding sites by gel retardation assays. Only one type of protein complex (referred to as NRE-binding protein or NBP) bound exclusively to sites that mapped to the promoter-distal and -proximal NREs identified by BAL 31 mutations. The promoter-proximal binding site was mapped further by linker substitution mutations and transfection assays. Mutations that mapped to a region containing an inverted repeat beginning at -287 relative to the start of transcription elevated basal expression of a reporter gene driven by the MMTV LTR. A 59-bp DNA fragment from the distal NRE also bound the NBP complex. Gel retardation assays showed that mutations within both inverted repeats of the proximal NRE eliminated NBP binding and mutations within single repeats altered NBP binding. Intriguingly, the NBP complex was detected in extracts from T cells and lung cells but was absent from mammary gland cells. These results suggest that a factor contributing to high-level expression of MMTV in the mammary gland is the lack of negative regulation by NBP.

Animals↗

PubMed1999

Interaction of Gli2 with CREB protein on DNA elements in the long terminal repeat of human T-cell leukemia virus type 1 is responsible for transcriptional activation by tax protein.

The long terminal repeat (LTR) of human T-cell leukemia virus type 1 (HTLV-1) has two distinct DNA elements, one copy of TRE2S and three copies of a 21-bp sequence that respond to the viral trans-activator protein, Tax. Either multiple copies of the 21-bp sequence or a combination of one copy each of TRE2S and 21-bp sequence is required for efficient trans activation by Tax. In the trans activation of multiple copies of 21-bp sequence, CREB/ATF protein plays an essential role in forming a complex with Tax. To understand the role of TRE2S in trans activation of one copy of 21-bp sequence, we examined protein binding to the DNA elements by DNA affinity precipitation assay including Gli2 protein binding to TRE2S and CREB protein binding to 21-bp sequence. Binding of CREB to a DNA probe containing both elements, TRE2S-21bp probe, was dependent on Gli2 protein under restricted conditions and was enhanced in a dose-dependent fashion by the binding of Gli2 protein to the same probe. Mutation in either element abolished the efficient binding of CREB. A glutathione S-transferase fusion protein of a fragment of Gli2 was able to bind to CREB. Therefore, Gli2-CREB interaction on the DNA probe is proposed to stabilize CREB binding to DNA. Tax can bind to CREB protein on the DNA; therefore, stabilization of DNA binding of CREB results in more recruitment of Tax onto DNA. Conversely, Tax increased the DNA binding of CREB, although it had almost no effect on the binding of Gli2. These results suggest that Gli2 binds to the DNA element and interacts with CREB, resulting in more recruitment of Tax, which in turn stabilizes DNA binding of CREB. Similar cooperation of the protein binding to TRE2S-21bp probe was also observed in nuclear extract of an HTLV-1-infected T-cell line. Consistent with the Gli2-CREB interaction on the DNA elements, Tax-mediated trans activation was dependent on the size of the spacer between TRE2S and 21-bp sequence. The effective sizes of the spacer suggest that TRE2S in the LTR would cooperate with the second and third copies of the 21-bp sequence and contribute to trans activation of the viral gene transcription.

Cyclic AMP Response Element-Binding Protein↗

PubMed1988

Multiple regulatory domains in the mouse mammary tumor virus long terminal repeat revealed by analysis of fusion genes in transgenic mice.

Transcription initiated within the mouse mammary tumor virus (MTV) long terminal repeat (LTR) is regulated by glucocorticoids, androgens, and estrogen. However, expression of the virus in vivo and transcription of MTV LTR fusion genes in transgenic mice are not readily interpretable solely in terms of the influence of these hormones. To investigate whether there is a regulatory role for sequences within the LTR but outside the region known to be responsible for glucocorticoid induction, we have produced transgenic mice carrying genes in which various regions of the LTR have been linked to the human growth hormone gene. Analysis of expression of the fusion genes in these transgenic mice has demonstrated that the 5' end of the LTR can profoundly influence transcription initiated within the MTV LTR.

Animals↗