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PubMed1981

Intracisternal A-particle genes in Mus musculus: a conserved family of retrovirus-like elements.

The structural organization of intracisternal A-particle genes has been studied, using isolates from a mouse gene library in lambda phage Charon 4A. The predominant gene form among the isolates was 7.3 kilobases (kb) in length. R-loops between the 7-kb (35S) A-particle genomic ribonucleic acid and several of these genes were colinear, with no visible evidence of intervening deoxyribonucleic acid sequences. One recombinant was found with an A-particle gene that contained a 1.7-kb deletion. Using the deletion as a reference, the deoxyribonucleic acid and ribonucleic acid homology regions were localized with respect to one another and to the restriction map: the 5' terminus of the ribonucleic acid was several hundred base pairs within the 5' end of the deoxyribonucleic acid homology region. Restriction endonuclease fragments encompassing the 5' and 3' regions of one 7.3-kb gene were separately subcloned into pBR322. Heteroduplexes between the two subclones revealed an approximately 300-base pair segment of terminally redundant sequences. The cloned 3' fragment hybridized with restriction fragments from the 5' end of several other A-particle genes, demonstrating the presence of common (though not necessarily identical) terminally repeated sequences. A-particle genes varied in the occurrence of specific restriction sites at characteristic internal loci. However, heteroduplexes between several variant 7.3-kb genes showed continuous homology regions even when spread under stringent hybridization conditions. The relative abundance of restriction site variants was highly conserved in 12 laboratory strains of Mus musculus, in embryonic and adult tissues of a single inbred strain, and in the SC-1 cell line of feral mouse origin, but appeared to differ in a feral Japanese substrain, Mus musculus molossinus. Some evidence suggests that subsets of A-particle genes may have similar flanking sequences. The results are discussed in terms of the evolution of this multigene family.

Animals↗
PubMed1987

Human retroviral sequences on the Y chromosome.

Novel endogenous human retroviral sequences were cloned by low-stringency hybridization, using the pol gene of endogenous human retrovirus 51-1. One clone, lambda NP-2, contained gag, pol, env, and long terminal repeat sequences related to the corresponding portions of clone 51-1 and the closely related full-length endogenous human retrovirus 4-1. The sequence of the env gene of NP-2 was 73% homologous to that of 4-1. Genomic Southern blots of male and female DNAs showed that NP-2 is located on the Y chromosome and that the Y chromosome also contains one other sequence closely related to the env and 3' flanking regions of NP-2. Conservation of flanking DNA suggests that the second Y chromosome copy of the NP-2 env sequence arose by gene duplication rather than provirus insertion.

Base Sequence↗
PubMed1981

Hybrid plasmids carrying part of the Rous sarcoma virus-specific leader sequence.

'Strong-stop' DNA, complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus genomic RNA and subgenomic mRNAs can be used to isolate RSV-specific mRNA from infected cells. To analyse leader sequences of these mRNAs and to study the mechanism of their translation, relatively large quantities of strong-stop DNA are required. The construction of four plasmids carrying useful strong-stop DNA sequences for mRNA isolation is described. One such hybrid plasmid carries a DNA insertion containing sequences derived from a region starting 42 nucleotides from the 5'-end of RSV RNA, and ending at the tRNATry primer attachment site. Since this plasmid lacks the 20-nucleotide terminal repeat sequence it can be used for the specific isolation of 5'-end fragments of RSV-specific mRNAs and of complementary DNA transcripts of these fragments.

Animals↗
PubMed1993

Efficient insertion from an internal long terminal repeat (LTR)-LTR sequence on a reticuloendotheliosis virus vector is imprecise and cell specific.

To examine the fidelity and efficiency of integration from a covalently closed long terminal repeat (LTR)-LTR sequence in vivo, we isolated individual spleen necrosis virus proviruses that arose following infection of chicken embryo fibroblasts (CEFs) and sequenced the provirus-cell DNA junctions. Some but not all CEF preparations allowed efficient insertion from the internal sequence. Moreover, in contrast to integration from the normal ends of the viral DNA, which occurs with precision with respect to the viral DNA, insertion from the internal sequence was not precise. In particular, there were short deletions of variable size from the viral DNA and these proviruses were not flanked by short direct repeats. Although this imprecise insertion can be efficient in CEFs, such integration is very inefficient in two other cell types (D17 and QT47) that support the replication of reticuloendotheliosis viruses. Thus, it is possible that there is a cell-specific factor(s) in CEFs required for efficient but imprecise insertion or, alternatively, D17 and QT47 cells contain a factor that abrogates integration from an internal LTR-LTR junction. Virus particles released from CEFs do not efficiently use the LTR-LTR junction following infection of D17 cells. Therefore, if there is a CEF-specific factor required for insertion, it does not appear to be transferred through particles.

Animals↗
PubMed1988

B-cell lymphoproliferation and lymphomagenesis are associated with clonotypic intracellular terminal regions of the Epstein-Barr virus.

We analyzed 17 B-cell lineages cloned from two patients with infectious mononucleosis and found that different B-cell lineages exhibited notable variation in the length of the fused Epstein-Barr virus (EBV) terminal region on intracellular EBV episomes. EBV termini in different B-cell clones from the same person differed by as many as 15 to 20 reiterations of the ca. 500-base-pair terminal repeat sequence. In contrast, analysis of seven B-cell lineages cloned from a patient with a fatal, oligoclonal lymphoma revealed that three of the cell clones had the same-sized EBV terminal region. These three clones had previously been shown, by immunoglobulin gene analysis, to be metastatic daughter cells descended from a common progenitor. Similarity of the EBV terminal regions in the three daughter clones suggested that EBV infected the progenitor cell before proliferation and metastasis. Individual, EBV-infected cells from a single individual showed sufficient heterogeneity in their EBV termini to allow use of terminal fragment size as a clonal marker in studies addressing the contribution of EBV to the clonal pathogenesis of tumors with which this virus has been associated.

B-Lymphocytes↗
PubMed1983

Glucocorticoids and chromosomal position modulate murine mammary tumor virus transcription by affecting efficiency of promoter utilization.

The rate of transcription of murine mammary tumor virus (MTV) sequences in MTV-infected rat hepatoma tissue culture cells is strongly affected by both glucocorticoid hormones and the chromosomal position of provirus integration. We have characterized MTV RNAs produced in J2.17 and M1.54, independent isolates containing, respectively, 1 and 10 proviruses integrated at distinct chromosomal loci. M1.54, but not J2.17, synthesized MTV RNA in the absence of glucocorticoids; the rate of hormone-stimulated viral gene transcription in M1.54 was 50- to 100-fold higher than in J2.17. In each case in which MTV genes were expressed (J2.17 induced, M1.54 basal and induced), the viral RNAs produced were indistinguishable. RNA blotting revealed accumulation of two transcripts, 7.8 and 3.8 kilobases; the latter was likely produced from the former by RNA splicing. Sites used for transcription initiation, polyadenylation, and splicing have been identified from the sizes of end-labeled hybridization probes protected from digestion with mung bean nuclease; the unique initiation and polyadenylation sites were both encoded within the MTV long-terminal-repeat sequence. The efficiencies of splicing and of utilization of the polyadenylation signal did not appear to vary as functions of chromosomal position or hormonal stimulation. Differences in rates of viral gene transcription were reflected in the differential accumulation of the 5'-terminal 136 nucleotides of MTV RNA. Thus, glucocorticoids and chromosomal position appeared to affect solely the efficiency of utilization of the MTV promoter, leaving unchanged the sites of initiation, splicing, and polyadenylation, as well as the efficiencies of the latter two processes.

Animals↗
PubMed1997

CpG-rich sequences close to the site of duplication within the human IGH constant region.

Human immunoglobulin heavy chains are encoded by a highly complex locus, IGH, which encompasses many transcriptional units, including nine alternative constant region genes. Much of the constant region gene cluster was duplicated during hominoid evolution. In rodents, IGH is known to be regulated by multiple elements, including several enhancers 3' of the alpha chain-encoding A constant region gene, CA, the last transcribed region. Sequences downstream of the two human CA genes, possibly containing homologous enhancer elements, have not yet been reported. By long-range mapping of genomic DNA, a cluster of unmethylated rare restriction sites, representing a potential CpG island, was previously reported downstream of each CA gene, close to the 3' end of the duplicated region. Such potential CpG islands are candidates for additional IGH regulatory elements. We isolated bacteriophage clones containing these clusters of methylation-sensitive restriction sites, which lie within short CpG-rich stretches. Some of these sites showed tissue-specific methylation. 3' of the unmethylated CpG-rich sequences, clones derived from the 5' (telomeric) copy of the duplicated region, contained, in order, endogenous retroviral sequences, a processed ELK1 pseudogene, and the pseudogene IGHGP. Comparison with the presumed 3' (centromeric) copy of the duplicated region showed that similarity was lost exactly at the end of the retroviral long terminal repeat sequences. These results imply that an endogenous retroviral integration was present immediately 3' of IGH in the common hominoid ancestor and suggest models for the duplication of the region.

Base Sequence↗
PubMed2006

Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells.

We established a method for production of recombinant adeno-associated virus type 5 (rAAV5) in insect cells by use of baculovirus expression vectors. One baculovirus harbors a transgene between the inverted terminal repeat sequences of type 5, and the second expresses Rep78 and Rep52. Interestingly, the replacement of type 5 Rep52 with type 1 Rep52 generated four times more rAAV5 particles. We replaced the N-terminal portion of type 5 VP1 with the equivalent portion of type 2 to generate infectious AAV5 particles. The rAAV5 with the modified VP1 required alpha2-3 sialic acid for transduction, as revealed by a competition experiment with an analog of alpha2-3 sialic acid. rAAV5-GFP/Neo with a 4.4-kb vector genome produced in HEK293 cells or Sf9 cells transduced COS cells with similar efficiencies. Surprisingly, Sf9-produced humanized Renilla green fluorescent protein (hGFP) vector with a 2.4-kb vector genome induced stronger GFP expression than the 293-produced one. Transduction of murine skeletal muscles with Sf9-generated rAAV5 with a 3.4-kb vector genome carrying a human secreted alkaline phosphatase (SEAP) expression cassette induced levels of SEAP more than 30 times higher than those for 293-produced vector 1 week after injection. Analysis of virion DNA revealed that in addition to a 2.4- or 3.4-kb single-stranded vector genome, Sf9-rAAV5 had more-abundant forms of approximately 4.7 kb, which appeared to correspond to the monomer duplex form of hGFP vector or truncated monomer duplex SEAP vector DNA. These results indicated that rAAV5 can be generated in insect cells, although the difference in incorporated virion DNA may induce different expression patterns of the transgene.

Alkaline Phosphatase↗
PubMed1987

Mechanism of leukemogenesis by human T-cell leukemia virus types I and II: role of the lor gene.

The human T-cell leukemia (HTLV) family of viruses now include two groups of transforming viruses (HTLV-I and HTLV-II) and one group of viruses with immunosuppressive activity but no apparent transforming ability (HTLV-III). Nucleotide sequence analysis of an HTLV-I provirus by Seiki et al [Proc Natl Acad Sci USA 1982: 6899-902] has revealed a distinct region of the genome located between env and the long terminal repeat sequences (LTR). Analysis of a variant of HTLV-I, called HTLV-Ib, which retains transforming ability, revealed a deletion in the 5' portion of this sequence that indicates that this portion of the sequence probably does not play an essential role in transformation. Hybridizations at varying stringencies show that at least a portion of this additional region of HTLV-I and -II is more highly conserved than most other regions of the genome. The DNA sequence of this HTLV-II region localizes the area of homology within the 1,000 base pairs just upstream of the LTR; the remainder of the region is not conserved. The conserved region includes a long open reading frame, is denoted lor, and has the coding capacity for a protein of at least 38 kilodaltons (kd). Studies of the transcriptional activity of the LTR sequences show that the rate of LTR-directed transcription is greatly augmented in HTLV-infected cells, a phenomenon called trans-acting transcriptional regulation. It is suggested that transformation of lymphocytes by HTLV-I or -II is mediated by action of HTLV-encoded trans-acting transcription factors on cellular genes that control the replication of lymphocytes.

Deltaretrovirus Infections↗
PubMed1980

Molecular cloning of avian sarcoma virus closed circular DNA: structural and biological characterization of three recombinant clones.

Unintegrated, circular viral DNA, isolated from Prague A avian sarcoma virus (PrA-ASV)-infected quail cells (QT6), was cloned in the lambda vector lambda gtWES x lambda B. Three independent lambda-ASV recombinants were identified, and each contained a complete copy of the PrA-ASV genome. The arrangement of the ASV sequences within the recombinants was determined by restriction enzyme analysis and hybridization with labeled ASV-specific complementary DNA. One of the recombinants (lambda RPA101) resulted from cloning at the EcoRI site located within the terminally repeated sequence and therefore was virtually co-linear with PrA-ASV virion RNA. The other two recombinants (lambda RPA102 and 103) resulted from cloning at the EcoRI site located within the viral env gene. By restriction enzyme analysis and by measurement of R-loops formed between lambda RPA101 and PrA-ASV virion 35S RNA, the viral genome was estimated to be 9,100 bases in length. Genome length viral DNA purified from clones lambda RPA102 and 103 was biologically active. Transfection of chicken embryo cells with viral DNA, in the form of either circles or linear dimers, produced foci of transformed cells within 8 to 10 days. Linear DNA was much less efficient at inducing transformation. Viral DNA from the clone lambda RPA101 was unable to cause transformation; the basis for this defect is unknown.

Alpharetrovirus↗
PubMed1994

Molecular characterization of a newly recognized mouse parvovirus.

Mouse parvovirus (MPV), formerly known as orphan parvovirus, is a newly recognized rodent parvovirus distinct from both serotypes of minute virus of mice (MVM). Restriction analysis of the MPV genome indicated that many restriction sites in the capsid region were different from those of MVM, but most sites in the nonstructural (NS) region of the genome were conserved. MPV resembled MVM in genome size, replication intermediates, and NS proteins. Replication intermediates in infected cells were the same for MPV and MVM, including packaging of the 5-kb minus (V) strand. Furthermore, the MPV NS proteins were the same size as and present at the same ratio as the MVM(i) proteins in infected cells. Cloning and sequencing of the MPV genome revealed a genome organization closely resembling that of MVM, with conservation of open reading frames, promoter sequences, and splice sites. The left terminal hairpin was identical to that of MVM(i), but the right terminus was not conserved. Also, the MPV genome was unique in that it contained 1.8 copies of the terminal repeat sequence rather than the 1 or 2 copies found in other parvoviruses. The predicted amino acid sequence of the NS proteins of MPV and MVM(i) were nearly identical. In contrast, the predicted amino acid sequence of the capsid proteins of MPV was different from sequences of other parvoviruses. These results confirm that MPV is a distinct murine parvovirus and account for the antigenic differences between MPV and MVM.

Amino Acid Sequence↗
PubMed1987

Physical analysis of the conjugative shuttle transposon Tn1545.

The conjugative shuttle transposon Tn1545 from Streptococcus pneumoniae confers resistance to kanamycin (aphA-3), erythromycin (ermAM), and tetracycline (tetM). The 25.3-kb element is self-transferable to various gram-positive bacterial genera where it transposes. Tn1545 is also capable of transposition, but not of conjugation, after cloningoff Escherichia coli. Analysis of the element by restriction endonucleases, molecular cloning, electron microscopy of heteroduplexes, DNA hybridization, and sequencing allowed us to establish a physical map of Tn1545, localize the resistance genes, determine their direction of transcription, and compare them with other characterized resistance determinants, and show that Tn1545 is not flanked by large terminal repeated sequences in opposite orientation.

Chromosome Mapping↗
PubMed2004

Genome structure and thymic expression of an endogenous retrovirus in zebrafish.

In a search for previously unknown genes that are required for lymphocyte development in zebrafish, a retroviral sequence was identified in a subtracted thymus cDNA library and in genomic DNA libraries. The provirus is 11.2 kb and contains intact open reading frames for the gag, pol, and env genes, as well as nearly identical flanking long terminal repeat sequences. As determined by in situ hybridization, the thymus appears to be a major tissue for retroviral expression in both larval and adult fish. Several viral transcripts were found by Northern blotting in the adult thymus. The provirus was found at the same genomic locus in sperm from four fish, suggesting that it is an endogenous retrovirus. Phylogenetic analysis indicates that it is closest to, yet distinct from, the cluster of murine leukemia virus-related retroviruses, suggesting that this virus represents a new group of retroviruses.

Animals↗
PubMed1981

Characterization of coliphage lambda hybrids carrying DNA fragments from Herpes simplex virus type 1 defective interfering particles.

We describe the characterization of 34 hybrid lambda bacteriophages carrying EcoRI fragments obtained from DNA of defective interfering particles of the Patton strain of Herpes simplex virus type 1 (HSV-1). All cloned fragments contained S region terminal repeat sequences (TRs) fused to unique HSV-1 DNA. Several fragments contained deletions and rearrangements not described previously for DNA of HSV-1 defective interfering particles. A model describing the generation of defective interfering DNA based on recombination events involving the terminal "a" sequence as presented.

Bacteriophage lambda↗
PubMed2005

Global effects on gene expression in fission yeast by silencing and RNA interference machineries.

Histone modifications influence gene expression in complex ways. The RNA interference (RNAi) machinery can repress transcription by recruiting histone-modifying enzymes to chromatin, although it is not clear whether this is a general mechanism for gene silencing or whether it requires repeated sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr mutants derepressed similar subsets of genes, many of which also became transcriptionally activated in cells that were exposed to environmental stresses such as nitrogen starvation. Many genes that were repressed by the Clr proteins clustered in extended regions close to the telomeres. Surprisingly few genes were repressed by both the silencing and RNAi machineries, with transcripts from centromeric repeats and Tf2 retrotransposons being notable exceptions. We found no correlation between repression by RNAi and proximity to LTRs, and the wtf family of repeated sequences seems to be repressed by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing.

Cell Cycle Proteins↗
PubMed1997

Characterization of the early region 4 of porcine adenovirus type 3.

The nucleotide sequence of a 3028 bp DNA segment, located between map co-ordinates 100 and 92 in the genome of porcine adenovirus type 3 (PAV-3), was determined. The segment includes the entire early region 4 (E-4) and the right inverted terminal repeat sequences. There were two TATA boxes and one canonical polyadenylation signal on the 1 strand. Homology searches of the GenBank data base for the predicted amino acid sequences revealed that, of the eight open reading frames (ORFs) on the 1 strand, and four ORFs on the r strand, only ORF 8 on the 1 strand showed homology with the 34 kDa E-4 protein of human adenovirus types 2, 12 and 34. Northern blot analysis showed that transcription from the E-4 region of PAV-3 began 4 h after infection, peaked at 8 h and declined after 10 h, before DNA replication began 16 h after infection. The E-4 region of PAV-3 was further characterized by 5' and 3' end mapping of the transcription unit.

Adenoviridae↗
PubMed2003

Duplication of U3 sequences in the long terminal repeat of mink cell focus-inducing viruses generates redundancies of transcription factor binding sites important for the induction of thymomas.

The ability of mink cell focus-inducing (MCF) viruses to induce thymomas is determined, in part, by transcriptional enhancers in the U3 region of their long terminal repeats (LTRs). To elucidate sequence motifs important for enhancer function in vivo, we injected newborn mice with MCF 1dr (supF), a weakly pathogenic, molecularly tagged (supF) MCF virus containing only one copy of a sequence that is present as two copies (known as the directly repeated [DR] sequence) in the U3 region of MCF 247 and analyzed LTRs from supF-tagged proviruses in two resulting thymomas. Tagged proviruses integrated upstream and in the reverse transcriptional orientation relative to c-myc provided the focus of our studies. These proviruses are thought to contribute to thymoma induction by enhancer-mediated deregulation of c-myc expression. The U3 region in a tagged LTR in one thymoma was cloned and sequenced. Relative to MCF 1dr (supF), the cloned U3 region contained an insertion of 140 bp derived predominantly from the DR sequence of the injected virus. The inserted sequence contains predicted binding sites for transcription factors known to regulate the U3 regions of various murine leukemia viruses. Similar constellations of binding sites were duplicated in two proviral LTRs integrated upstream from c-myc in a second thymoma. We replaced the U3 sequences in an infectious molecular clone of MCF 247 with the cloned proviral U3 sequences from the first thymoma and generated an infectious chimeric virus, MCF ProEn. When injected into neonatal AKR mice, MCF ProEn was more pathogenic than the parental virus, MCF 1dr (supF), as evidenced by the more rapid onset and higher incidence of thymomas. Molecular analyses of the resultant thymomas indicated that the U3 region of MCF ProEn was genetically stable. These data suggest that the arrangement and/or redundancy of transcription factor binding sites generated by specific U3 sequence duplications are important to the biological events mediated by MCF proviruses integrated near c-myc that contribute to transformation.

Animals↗
PubMed1984

Homology of genome of AIDS-associated virus with genomes of human T-cell leukemia viruses.

A T lymphotropic virus found in patients with the acquired immune deficiency syndrome (AIDS) or lymphadenopathy syndrome has been postulated to be the cause of AIDS. Immunological analysis of this retrovirus and its biological properties suggest that it is a member of the family of human T-lymphotropic retroviruses known as HTLV. Accordingly, it has been named HTLV-III. In the present report it is shown by nucleic acid hybridization that sequences of the genome of HTLV-III are homologous to the structural genes (gag, pol, and env) of both HTLV-I and HTLV-II and to a potential coding region called pX located between the env gene and the long terminal repeating sequence that is unique to the HTLV family of retroviruses.

Acquired Immunodeficiency Syndrome↗