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[Fine structure of long terminal repeats and stages of reverse transcription of mobile dispersed genes in Drosophila].

In Drosophila melanogaster cultured cells, RNA reverse transcription intermediate forms connected with initiation of minus and plus DNA strand synthesis (minus and plus strong-stop DNA) are detected for mobile dispersed genetic elements MDG1, MDG3 and MDG4 (gypsy). A comparative analysis of intermediate forms has proved that mdg elements pass the same stages of reverse transcription as retroviruses, revealing a complete similarity between intermediate products. It has also been established that these three mdg elements possess a common mechanism of reverse transcription, despite their structural differences. The length of the minus strong-stop DNA, that gives the position of the RNA start site, coincides with the data obtained from SI nuclease analysis of transcription initiation. SI mapping has also revealed that mdg RNA carries a repeated sequence R on its ends, similar to retroviral RNA molecules, and that mdg LTRs have a U3-R-U5 structure analogous to that of proretroviral LTRs. Transcription of mdg1, mdg3 and mdg4 is initiated within or immediately after the same sequence TCAGTPy. Neither TATA box nor CAAT box can be found in their characteristic positions upstream of the 5' ends of mRNA.

Animals↗

Human immunodeficiency virus long terminal repeat responds to transformation by the mutant T24 H-ras1 oncogene and it contains multiple AP-1 binding TPA-inducible consensus sequence elements.

We have employed a short term transfection assay to examine the response of HIV-1 LTR to transformation by the human normal and mutant T24 H-ras1 genes. The plasmid pBC12HIVCAT which carries the HIV-1 LTR sequences linked to the reporter gene chloramphenicol acetyl transferase (CAT) was transfected into rat 208F fibroblasts and their derivatives RFHO6N1-1 and RFHO6T1-1 transfectants. RFHO6N1-1 and RFHO6T1-1 express an exogenous human normal or mutant T24 H-ras1 gene respectively. Expression of the mutant T24 but not the normal H-ras1 gene resulted in increased levels of HIV-1 LTR driven CAT activity. We have noted four motifs in the HIV-1 LTR region which resemble TPA-inducible and AP-1 binding consensus sequences. Since H-ras1 fos and jun/AP-1 respond to TPA and T24 H-ras1 is known to induce both fos and jun/AP-1 nuclear transcriptional factors, it is possible that the latter genes play a role in HIV-1 transcription. Moreover, H-ras1 oncogene activation may play an important role in HIV gene expression and in the activation of latent HIV.

Animals↗

[Study of conditions for cleaving bovine leukemia virus RNA in vitro using ribozymes directed against long terminal repeats].

Ribozyme has been constructed by the oligonucleotide synthesis technique that is capable of splitting bovine leukemia virus (BLV) RNA-substrate in the R-region (358th nucleotide). Ribozyme has been shown to efficiently split the RNA containing R-region of the virus. The efficiency of ribozyme function depends on conditions of the reaction reaching its maximum at 25 degrees C and pH 7.3-7.6. The efficiency of splitting is increased by multiple heating-cooling cycles of the substrate-ribozyme mixture.

Base Sequence↗

[Structural characteristics of four long terminal repeats (LTR) of human endogenous retroviruses and features of their integration sites].

Four LTR-containing regions of human chromosome 19 were sequenced by the primer walking technique using strings of short oligonucleotides tightly bound to the template. A comparative and evolutionary analysis of sequences homologous to human endogenous retroviruses (HERV) was performed, and the prototypes of the LTRs were determined. Analysis of the chromosome 19 sequences adjacent to LTR revealed that LTRs of HERV-K share a common location with other retroposons.

Base Sequence↗

The GT-rich sequence in the U5 region of Rous sarcoma virus long terminal repeat is required for transcription termination and 3' processing.

The sequences in the LTR of Rous sarcoma virus that are required for transcription termination and polyadenylation have been determined. A vector containing LTR-neo-LTR has been constructed and deletions in the U5 region of the downstream LTR have been made. The DNAs from wild-type and deletion mutant recombinant plasmids were introduced into QT6 cells and G418-resistant transformants were selected. Those transformants with neo sequences in the arrangement, LTR-neo-LTR, were analyzed for transcription termination and polyadenylation by Northern blot analysis and by S1 protection experiments. The results indicate that the polyadenylation signal, AATAAA, located in the U3 region alone, is not sufficient for 3' end processing and that the sequence between +20 and +44 in the U5 region is absolutely required for transcription termination or endonucleolytic cleavage and polyadenylation.

Animals↗

Circular DNA of human immunodeficiency virus: analysis of circle junction nucleotide sequences.

During infection of cells by retroviruses, some of the nonintegrated viral DNA can be found as a circular form containing two tandem, directly repeated long terminal repeats. The nucleotide sequence at the point where the long terminal repeats join (the circle junction) can be used to deduce the terminal nucleotides of the linear form of the viral DNA. Comparison of the termini of linear viral DNA with sequences at the junctions between the integrated provirus and the host chromosome has revealed that for most retroviruses 2 bp are removed from each end of the linear viral DNA during integration. For human immunodeficiency virus type 1 (HIV-1), however, sequence considerations involving primer-binding sites had suggested that only 1 bp is removed during integration. We obtained the nucleotide sequences at the ends of HIV-1 DNA by using the polymerase chain reaction to amplify fragments corresponding to the HIV-1 circle junction. Of 17 clones containing amplified sequences, 10 had identical circle junctions that contained an additional 4 bp (GTAC) relative to the integrated provirus. This indicates that, as for other retroviruses, 2 bp are removed from each end of the linear HIV-1 viral DNA during integration. The remaining seven isolates contained insertions or deletions at the circle junction.

Base Sequence↗

Retroviral insertions into a herpesvirus are clustered at the junctions of the short repeat and short unique sequences.

We previously described the integration of a nonacute retrovirus, reticuloendotheliosis virus (REV), into the genome of a herpesvirus, Marek disease virus (MDV), following both long-term and short-term coinfection in cultured fibroblasts. The long-term coinfection occurred in the course of attenuating the oncogenicity of the JM strain of MDV and was sustained for > 100 passages. The short-term coinfection, which spanned only 16 passages, was designed to recreate the insertion phenomenon under controlled conditions. We found that REV integrations into MDV were common and could occur within the first passage following coinfection. Now we have mapped the integration sites. After 5-16 passages in vitro, 17 out of 19 REV junction sites are clustered in two 1-kilobase regions at the junctions of the short unique and short repeat region of MDV. In the long-term cocultivation experiment, 6 out of 10 insertions also mapped in this region. In both cases, integrated proviruses are unstable and undergo subsequent recombinative deletion, often leaving a solitary long terminal repeat. The long terminal repeat sequences are, however, stably maintained for many rounds of passaging in vitro. This clustering of insertions presumably is influenced by selection for viable and fast-growing viruses, and occurs in a region of the MDV genome which shows significant size heterogeneity in several strains.

Animals↗

Graffi murine leukemia virus: molecular cloning and characterization of the myeloid leukemia-inducing agent.

The Graffi murine leukemia virus (MuLV) is a retroviral mixture that induces predominantly myeloid leukemia in several inbred strains of mice. To analyze the viral component responsible for the myeloid leukemogenesis, we cloned several proviruses from a Graffi MuLV-infected cell line. Several infectious molecular clones were obtained that could be classified into two distinct groups of infectious MuLV. Both types of MuLV were nondefective, ecotropic, and NB tropic and induced granulocytic leukemia in BALB/c and NFS mice. Restriction enzyme analysis and molecular hybridization with several MuLV probes on one molecular clone from each group revealed that both groups are closely related to each other but are clearly distinct from all known retroviruses. One component of MuLV, however, induced leukemia with a shorter latency period and harbored a lengthier long terminal repeat. The long terminal repeat of the more leukemogenic component of MuLV had acquired a 60-bp perfect duplication in the U3 region. Analysis of the tumor DNAs with probes for the mouse T-cell receptor and immunoglobulin heavy chain genes revealed frequent rearrangements with one or both probes. This concomitant expression by leukemic cells of markers of different lineages, observed in human leukemias, has been termed "lineage infidelity" and confirms that the latter rearrangements are not restricted to hematopoietic precursors committed to lymphoid differentiation.

Animals↗

Generation of a uniform 3' end RNA of murine leukemia virus.

Using the S1 nuclease mapping technique, we demonstrated that the majority of Moloney murine leukemia RNA molecules, isolated either from the nucleus or cytoplasm of infected mouse cells, share a uniform 3' end located at the border of the R and U-5 regions of the long terminal repeat. When the long terminal repeat sequences were inserted in the pSV plasmid downstream of the simian virus 40 late promoter, the 3' end of the viral RNA was also generated close to the R region of the long terminal repeat. These results demonstrate that the long terminal repeat signals the generation of an authentic 3' end when situated downstream of an actively transcribed region.

Animals↗

Prototypic sequences for human repetitive DNA.

We report a collection of 53 prototypic sequences representing known families of repetitive elements from the human genome. The prototypic sequences are either consensus sequences or selected examples of repetitive sequences. The collection includes: prototypes for high and medium reiteration frequency interspersed repeats, long terminal repeats of endogenous retroviruses, alphoid repeats, telomere-associated repeats, and some miscellaneous repeats. The collection is annotated and available electronically.

Animals↗

Nucleotide sequence analysis of the env gene and its flanking regions of the human spumaretrovirus reveals two novel genes.

Recombinant clones that represent the 3' part of the genome of the human spumaretrovirus (foamy virus) were established from viral DNA and from DNA complementary to viral RNA. The recombinant clones were characterized by blot hybridizations and nucleotide sequence analysis. The deduced protein sequence of the clones at their 5' ends was found to be homologous to the 3' domain of retroviral reverse transcriptases. Downstream of a small intergenic pol-env region a long open reading frame of 985 amino acid residues was identified that according to its genomic location, size, glycosylation signals, and hydrophobicity profile closely resembles the lentiviral env genes. The spumaretroviral env gene is followed by two open reading frames, termed bel-1 and bel-2 which are located between env and the long terminal repeat region. The long terminal repeat of 1259 nucleotides is preceded by a polypurine tract and contains the canonical signal sequences characteristic for transcriptional regulation of retroviruses. The provisional classification of the spumaretrovirus subfamily is discussed.

Amino Acid Sequence↗

An unusual transposon with long terminal inverted repeats in the sea urchin Strongylocentrotus purpuratus.

A 3-kilobase DNA segment characteristic of a transposable element was found within a histone H2B pseudogene in a higher eukaryote, the sea urchin Stronglyocentrotus purpuratus. The inserted segment (TU1) is flanked by 8-base pair (bp) direct repeats of the H2B sequence. TU1 has long terminal inverted repeats approximately 840 bp long with an outer domain of 15-bp tandem repeats and a non-repeating inner domain, and is a member of a heterogeneous family of transposable elements. TU1 differs from most previously characterized eukaryotic transposable elements with terminal direct repats, but resembles the foldback transposon family in Drosophila.

Animals↗

A new retrovirus packaging cell for gene transfer constructed from amplified long terminal repeat-free chimeric proviral genes.

The retroviral gene transfer system is a powerful tool for somatic gene therapy. A retroviral stock with a high viral titer and lacking replication-competent virus (RCV) is desirable for this type of gene transfer. To fulfill these requirements, we made a new packaging cell line, designated ampli-GPE. To reduce the homology between proviral DNA in the packaging cell and retroviral vector, the gag-pol and env genes of Moloney murine leukemia virus were separated onto two different plasmids, pGP-KV and pENV-KV, respectively, in which the 5' long terminal repeat and the 3' long terminal repeat had been replaced by the mouse metallothionein I promoter or the human beta-globin gene containing the polyadenylation site as control units for the gag-pol and env genes. In addition, these plasmids contained 69% of the bovine papillomavirus gene for gene amplification to obtain production of virus at a high titer. NIH 3T3 clones containing approximately 20 to 50 copies of the gag-pol and env genes were selected and designated ampli-GPE. When ampli-GPE was transfected with the N2 vector or pZipNeoSV(DHFR) derived from pZipNeoSV(X)1, we established clones producing titers of 5 x 10(6) and 1 x 10(6) CFU/ml, respectively. There was no sign of RCV generation in any virus-producing cells from ampli-GPE. However, virus-producing cells derived from psi 2 cells transfected with N2 did generate RCV. Thus, we showed that ampli-GPE, possessing the minimum complement of proviral genes, has potential for the development of a gene transfer system.

3T3 Cells↗

Demonstration of biological activity and nucleotide sequence of an in vitro synthesized clone of the Moloney murine sarcoma virus mos gene.

A clone of the Moloney murine sarcoma virus mos gene derived by in vitro reverse transcription was characterized. When assayed for focus formation by DNA transfection on NIH/3T3 cells, this clone was biologically inactive, presumably due to the absence of a long terminal repeat sequence. Therefore, a long terminal repeat was inserted into the clone by in vitro recombination, after which the most gene was able to transform NIH/3T3 cells efficiently. The nucleotide sequence encompassing the transforming region of this clone was determined. A single long open reading frame was observed, which potentially encodes a polypeptide of 41,000 daltons. This open reading frame initiates with the first five amino acids of the murine leukemia virus env gene, after which it enters the mos sequence, where it terminates. The nucleotide sequence described in this paper was compared with other sequences of mos in an effort to resolve discrepancies in the position of the long open reading frame. Although Moloney murine sarcoma virus retains the 3' splicing site of the murine leukemia virus env gene, a mos-specific mRNA which corresponds structurally to the murine leukemia virus env mRNA was not identified. The sequence described here revealed a single nucleotide change in the proposed env gene 3' splicing site which was retained in Moloney murine sarcoma virus. This deviation from the consensus 3' splicing sequence may underlie the observed absence of mos expression via the env gene splicing pathway.

Animals↗