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O Danos

Publications and source records attributed to O Danos.

18 recordsLinked to original sources
PubMed1992

Clonal segregation of oligodendrocytes and astrocytes during in vitro differentiation of glial progenitor cells.

To study the clonal lineage of the glial progenitor population, isolated from newborn rat brain (Lubetzki et al. J Neurochem 56:671, 1991), we combined somatic transgenesis using a retroviral vector encoding a modified bacterial beta-galactosidase with nuclear localization, and triple immunofluorescence labeling with A2B5, anti-galactosylceramide, and anti-glial acidic fibrillary protein antibodies. This allowed clonal analysis of the postnatal glial lineage with precise phenotypic identification of each cell within the lacZ-positive clones. When infected cells were cultivated under constant conditions, in the presence of either 1% or 10% fetal calf serum (FCS)-containing medium, all the 250 lacZ-positive clusters examined were homogeneous, i.e., either oligodendroglial or astroglial. Mixed astrocyte-oligodendroglial clones were observed when cells cultivated in the presence of 1% FCS were switched to a 10% FCS-containing medium, confirming the bipotentiality of glial progenitor cells (Temple and Raff Nature 313:223, 1985). However, even under the switch culture conditions, segregation into homogeneous clones of either oligodendrocytes or astrocytes still predominated, and the percentage of mixed clones dropped from 25 to 8 or to 3, when the switch took place at 8, 16, or 22 days in vitro, respectively. Two additional observations lead us to suggest that microenvironmental factors are responsible for the clonal segregation of glial progenitor cells: 1) the uneven distribution of oligodendrocyte and astrocyte clusters, the latter being seen mostly on the edge of the coverslips; and 2) the presence, in the vicinity of an homogeneous lacZ-positive clone, of some lacZ-negative cells expressing the same phenotype.

Animals↗
PubMed1992

Activation pathways and human immunodeficiency virus type 1 replication are not altered in CD4+ T cells expressing the nef protein.

While recent studies in Rhesus monkeys have pointed out the importance of an intact nef gene for the development of acquired immunodeficiency syndrome (AIDS), no biological function has been so far unambiguously attributed to its product. Since Nef has been described to possess GTP-binding properties and to down-regulate CD4 cell surface expression, we looked for evidences of Nef interfering with the transduction of activating signals in human CD4+ T cells. We used a murine leukemia retroviral vector to express the HIV-1BRU nef gene in two permanent tumoral T-cell lines (CEM and Jurkat) and in two nonimmortalized, interleukin-2 (IL2)-dependent, T-cell clones. The single copy recombinant provirus integrated in the genome of these cells directed the synthesis of a 27-kD protein with a half-life greater than 5 h. The levels of expression of cell surface molecules involved in T-cell functions (CD4, CD3, CD28, CD29, IL-2 receptor) were not modified in cell populations expressing Nef. In immunocompetent T-cell clones, cell proliferation and lymphokine production in response to activating stimuli (IL-2, alloantigens, phorbol esters, or antibodies directed against CD2, CD3, CD4, CD28) remained unmodified. Moreover, the presence of Nef did not change the kinetics of human immunodeficiency virus (HIV) infection.

3T3 Cells↗
PubMed1992

Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses.

The envelope glycoproteins (SU) of mammalian type C retroviruses possess an amino-terminal domain of about 200 residues, which is involved in binding a cell surface receptor. In this domain, highly conserved amino acid sequences are interrupted by two segments of variable length and sequence, VRA and VRB. We have studied the role of these variable regions in receptor recognition and binding by constructing chimeric molecules in which portions of the amino-terminal domains from amphotropic (4070A), xenotropic (NZB), and polytropic (MCF 247) murine leukemia virus SU proteins were permuted. These chimeras, which exchanged either one or two variable regions, were expressed at the surface of replication-defective viral particles by a pseudotyping assay. Wild-type or recombinant env genes were transfected into a cell line producing Moloney murine leukemia virus particles devoid of envelope glycoproteins in which a retrovirus vector genome carrying an Escherichia coli lacZ gene was packaged. The host range and sensitivity to interference of pseudotyped virions were assayed, and we observed which permutations resulted in receptor switch or loss of function. Our results indicate that the determinants of receptor choice are found within the just 120 amino acids of SU proteins. Downstream sequences contribute to the stabilization of the receptor-specific structure.

3T3 Cells↗
PubMed1991

Retroviral-mediated gene transfer into hepatocytes in vivo.

Stable gene transfer into hepatocytes might be used to compensate for a genetic deficiency affecting liver function or to deliver diffusible factors into the blood stream. In rats, we have combined retroviral-mediated gene transfer with a surgical procedure in which the liver is temporarily excluded from the circulation and infected in vivo. Partial hepatectomy was performed 24-48 hr before perfusion with virus to induce hepatocyte division and facilitate viral integration. A helper-free recombinant retrovirus coding for beta-galactosidase with nuclear localization was used to score cells that expressed the transgene. For at least 3 months after gene transfer, up to 5% of hepatocytes expressed nuclear beta-galactosidase. Whereas in vitro reimplantation of genetically modified hepatocytes has proved to be inefficient in stably transferring genes into the liver, our approach provides a feasible alternative.

Animals↗
PubMed1991

An amino-terminal fragment of the Friend murine leukemia virus envelope glycoprotein binds the ecotropic receptor.

Retrovirus entry into cells is mediated by specific binding of the envelope glycoprotein to a cell membrane receptor. Constitutive envelope gene expression prevents infection by interfering with the binding of viruses which recognize the same receptor. We have used this property to investigate the receptor binding capacities of deleted or truncated murine leukemia virus ecotropic envelope glycoproteins. Friend murine leukemia virus envelope glycoproteins bearing internal amino-terminal deletions, or a soluble 245-amino-acid gp70 amino-terminal fragment, were expressed in NIH 3T3 cells. The susceptibility of these cells to ecotropic and amphotropic virus infection was determined. We observed that both membrane-bound and soluble forms of the gp70 245-amino-acid amino-terminal domain induced resistance to ecotropic virus, indicating that this fragment binds the ecotropic receptor. Binding occurs both at the cell surface and in the endoplasmic reticulum, as shown by the use of soluble envelope fragments either secreted in the culture supernatants or retained in the endoplasmic reticulum lumen by a KDEL sequence. These results suggest that the gp70 amino-terminal domain folds into a structure which recognizes the ecotropic receptor regardless of the carboxy-terminal part of the molecule.

Amino Acid Sequence↗
PubMed1991

Gene transfer to somatic tissues using retroviral vectors.

Retroviral vectors have successfully been used for stable gene transfer in a number of in vivo situation. Before this approach can reallistically be applied in the treatment of human genetic diseases, the correction of pathological symptoms have to be documented in an animal model. In this perspective, we have chosen to use a strain of mice carrying the gusmps mutation, where homozygotes are deficient for beta-glucuronidase and develop a mucopolysaccharidosis. Two methods which result in the stable introduction of the human beta-glucuronidase cDNA into skin fibroblasts or hematopoietic stem cells are presented. A third method, whereby genes can be stably transferred to the liver is also discussed.

Animals↗
PubMed1990

Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells.

Recombinant retroviruses encoding human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans.

Adenosine Deaminase↗
PubMed1988

Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.

We have constructed a set of packaging cell lines useful for the generation of helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. To eliminate the problems of transfer of packaging functions and helper virus formation encountered with the previously available packaging systems, two mutant Moloney murine leukemia virus-derived proviral genomes carrying complementary mutations in the gag-pol or env regions were sequentially introduced into NIH 3T3 cells by cotransformation. Both genomes contained a deletion of the psi sequence necessary for the efficient encapsidation of retroviral genomes into virus particles and additional alterations at the 3' end of the provirus. We show that the resulting packaging cell lines psi CRIP and psi CRE can be used to isolate clones that stably produce high titers (10(6) colony-forming units/ml) of recombinant retroviruses with amphotropic and ecotropic host ranges, respectively. More importantly, we demonstrate that viral producers derived from the packaging cell lines do not transfer the packaging functions, or yield helper virus, even under conditions where existing packaging cell lines can be shown to yield transfer of packaging functions and/or helper virus. These properties of the psi CRIP and psi CRE packaging lines make them particularly valuable reagents for in vivo gene transfer studies aimed at cell lineage analysis and the development of human gene replacement therapies.

Animals↗
PubMed1988

Use of retroviral vectors for mapping of splice sites in cottontail rabbit papillomavirus.

Cottontail rabbit papillomavirus (CRPV) genomic sequences coding for virus early functions were introduced into a retroviral vector in order to produce cDNAs of the viral early region. Two constructs differing in the length of control sequences preceding the E6 open reading frame were transfected into Psi-2 cells and the released retroviral stock was used to infect NIH3T3 cells. The proviral sequences were rescued from antibiotic G418-resistant virus-infected cells after fusion with Cos cells, amplified as plasmids in Escherichia coli and analysed. Nucleotide sequencing showed that the splicing signals used in the construct containing only the early coding region are the same as in CRPV-expressing tumours, linking the beginning of E1 to the middle of E2. On the other hand, in a construct including most of the long control region a splice donor site located in the 5' end of this region, at position 7810, was very efficiently used, totally excluding the use of the donor site at position 1371. None of the constructs containing CRPV sequences transcribed from Moloney murine leukaemia virus promoter was able to transform mouse fibroblasts after DNA transfection.

Base Sequence↗
PubMed1987

Nucleotide sequence and comparative analysis of the human papillomavirus type 18 genome. Phylogeny of papillomaviruses and repeated structure of the E6 and E7 gene products.

The complete nucleotide sequence and genomic organization of human papillomavirus type 18, associated with cervical cancer, has been established. A detailed comparative analysis was undertaken leading to the identification of a number of features specific for genital papillomaviruses and the construction of a phylogenetic tree. Genital papillomaviruses differ from other human and animal papillomaviruses as they possess a longer E1 open reading frame (ORF) and have a characteristic control region. Phylogenetically, HPV 18 is located between the benign genital viruses, HPV 6 and HPV 11, and the malignant isolates, HPV 16 and HPV 33, and may represent an evolutionary intermediate among oncogenic papillomaviruses. Viral gene products known to be involved in cellular transformation are those of ORFs E5, E6 and E7. Significant sequence variation was found between the E6 to E7 regions of different integrated forms of HPV 18. On re-examination of the E6 primary structures we noticed that the gene has evolved by successive duplications of a unit encoding 33 amino acids, which include a Cys-X-X-Cys motif. Furthermore, the E7 gene product has apparently evolved in the same manner and is related to E6. Both gene products bear a striking resemblance to the transcriptional factor IIIA of Xenopus laevis, the prototype of a new class of nucleic acid binding proteins.

Amino Acid Sequence↗
PubMed1986

Production of spliced DNA copies of the cottontail rabbit papillomavirus genome in a retroviral vector.

The early region of the cottontail rabbit papillomavirus (CRPV) genome has been introduced into a retroviral vector and recombinant retroviruses, produced upon transfection of the psi 2 packaging cell line, have been used to infect NIH 3T3 cells. Spliced derivatives of the CRPV early region can be rescued from the infected cells. Sequence analysis demonstrates that the major splicing event observed in RNA in tumours is faithfully reproduced in this system. This splice generates a polycistronic mRNA that contains in its 5' portion the E7 open reading frame, or both E6 and E7, and at its 3' end a reading frame with codons for three amino acids from the N-terminus of E1 linked to codons for 100 amino acids from the C-terminus of the E4 region. Recombinant retroviruses containing intact or spliced CRPV sequences can now be used to introduce the viral genes efficiently into a variety of cell lines.

Animals↗
PubMed1985

Nucleotide sequence of the AIDS virus, LAV.

The complete 9193-nucleotide sequence of the probable causative agent of AIDS, lymphadenopathy-associated virus (LAV), has been determined. The deduced genetic structure is unique: it shows, in addition to the retroviral gag, pol, and env genes, two novel open reading frames we call Q and F. Remarkably, Q is located between pol and env and F is half-encoded by the U3 element of the LTR. These data place LAV apart from the previously characterized family of human T cell leukemia/lymphoma viruses.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Nucleotide sequence of the visna lentivirus: relationship to the AIDS virus.

We have determined the complete 9202 nucleotide sequence of the visna lentivirus. The deduced genetic organization most closely resembles that of the AIDS retrovirus in that there is a novel central region separating pol and env. Moreover, there is a close phylogenetic relationship between the conserved reverse transcriptase and endonuclease/integrase domains of the visna and AIDS viruses. These findings support the inclusion of the AIDS virus in the retroviral subfamily Lentivirinae.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Genomic structure of the cottontail rabbit (Shope) papillomavirus.

The nucleotide sequence of the cottontail rabbit papillomavirus (CRPV or Shope papillomavirus) genome has been determined. The overall organization of the genome is similar to that of the other three papillomaviruses already sequenced. The amino acid sequences of the putative viral proteins were compared to the available protein data banks. Of particular interest is the homology found between the COOH terminus of the E2 putative protein of the different papillomaviruses and the viral or cellular mos oncogene product. This analysis also reveals a specific feature of the rabbit virus that may be related to its natural oncogenic potential. One of its open reading frames (E6) shows significant homologies with the beta subunit of a family of ATP synthases from mitochondria, chloroplasts, and bacteria, including the conservation of amino acids residues involved in nucleotide binding. The viral noncoding region includes a highly A+T-rich segment and shows a complex array of repetitions and inverted repeats that may act as control elements for gene expression and genome replication.

Base Sequence↗
PubMed1985

Fine structure of the cottontail rabbit papillomavirus mRNAs expressed in the transplantable VX2 carcinoma.

We analyzed the polyadenylated mRNAs transcribed from the cottontail rabbit papillomavirus genomes present in the domestic rabbit transplantable carcinoma line VX2, employing a combination of S1 nuclease mapping and primer extension techniques with vector M13-based single-stranded DNA probes. Each of the two major mRNA species (1,860 and 1,110 bases long) contained two exons which corresponded to the E6-E7 and E2-E4 open reading frames. The splice donor site for these transcripts was located at position 1371 at the beginning of the E1 open reading frame. Consequently, the splicing event did not lead to the fusion of the E region proximal (E6 and E7) and distal (E2, E4, or E5) open reading frames. The translation of the polycistronic RNA could result in the production of E6 and E7 proteins alone or of an additional E1-E4 fusion product if translation reinitiation can occur after the E7 stop codon. A heterogeneity was detected in the 5' ends of the longer transcript; E6 transcripts could thus yield a full-length or a truncated E6 protein. We also detected a minor subset of mRNAs covering the E2-E4 coding region and including at least three species with estimated sizes of 4.2, 2.8, and 1.8 kilobases. All the viral transcripts detected in the VX2 tumor cells were polyadenylated at the same site (position 4367) 20 base pairs beyond the first AATAAA signal which borders the E region.

Animals↗