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O Brison

Publications and source records attributed to O Brison.

At least 55 records · Page 3Linked to original sources
PubMed1989

Methylation of ribosomal cistrons in diploid and tetraploid Odontophrynus americanus (Amphibia, Anura).

Odontophyrynus americanus (Amphibia, Anura) genomic DNA from diploid and tetraploid specimens was treated with restriction enzymes sensitive to cytosine and adenine methylation (5 meC and 6 meA). In both diploids and tetraploids a high proportion of the total DNA was not cleaved by 5 meC-sensitive enzymes as observed on agarose gels stained with ethidium bromide. The DNAs were transferred to nitrocellulose filters and hybridized with cloned fragments containing sequences of Xenopus laevis 28S and 18S ribosomal DNA (rDNA). A high level of methylation of the ribosomal repeat units was revealed by 5 meC-sensitive enzymes in blood, liver, kidney and testis tissues. Adenine was methylated to a lesser degree and similarly in the rDNA from both germinative and somatic tissues. Comparison of the results obtained with DNA of diploids and tetraploids showed that methylation of ribosomal genes was increased in tetraploid genomes of adult frogs, but exact quantitative determinations could not be performed by this methodology. Cloning of the 28S region of the rDNA repeat unit was performed in the lambda gtWES lambda C vector. Restriction patterns obtained with methylation-sensitive enzymes using diploid and tetraploid derived clones confirmed the high level of methylation of the corresponding region of the ribosomal repeat unit in genomic DNAs. The implications of these results in the regulation of expression of the ribosomal genes in diploids and tetraploids are discussed.

Adenine↗
PubMed1989

Selection of cells with different chromosomal localizations of the amplified c-myc gene during in vivo and in vitro growth of the breast carcinoma cell line SW 613-S.

The c-myc gene is amplified in the human breast carcinoma cell line SW 613-S. At early in vitro passages, the extra copies of the gene were mainly localized in double minute chromosomes (DMs), as shown by in situ hybridization with a biotinylated c-myc probe. However, cells without DMs were also present in which the c-myc genes were found integrated into any of several distinct chromosomes (mainly 7q+, 4 and 4q+, and 1). When this cell line was propagated in vitro, the level of c-myc amplification decreased because cells with DMs and a high amplification level were lost and replaced by cells without DMs and having a low amplification level. On the contrary, when early passage SW 613-S cells were grown in vivo, as subcutaneous tumours in nude mice, cells with numerous DMs and a high level of c-myc amplification were selected for. In one cell line (SW 613-Tu1) established from such a tumour, the DM-containing cells were substituted at late passages for cells with a high number of c-myc copies integrated within an abnormally banded region, at band 17q24 of a 17q+ chromosome. When only cells with integrated genes were present, this cell line was still highly tumorigenic indicating that the localization of the c-myc genes in DMs was not required for these cells to be tumorigenic in nude mice. Furthermore, cells of the secondary tumours induced by SW 613-Tu1 did not contain any DMs showing that in vivo growth did not promote the release of integrated c-myc copies into DMs.

Blotting, Southern↗
PubMed1989

Structure of four amplified DNA novel joints.

The structures of four novel joints present in the amplified DNA of a Syrian hamster cell line highly resistant to N-(phosphonacetyl)-L-aspartate were analyzed. Novel joints J1, J2, and J4 were formed by recombination between two regions of wild-type DNA, whereas joint J3 is the end point of an inverted duplication. A fraction of the J3 copies displays a cruciform structure in the purified genomic DNA. The formation of J1 and J2 apparently involved a simple breakage and joining of the two wild-type sequences, whereas extra nucleotides are present at the junction point of J3 and J4. The two regions of the wild-type DNA which have recombined to form J1, J2, and J4 show few sequence similarities, indicating that these joints probably resulted from nonhomologous recombination. AT-rich regions are present in the vicinity of the breakpoint for the four joints and eight of 10 crossover points could be associated with putative topoisomerase I cleavage sites. Our results indicate that different types of novel joints are present in the amplified DNA of this cell line, which was isolated after several steps of selection.

Animals↗
PubMed1989

Long-term study of chimaerism in bone marrow transplantation recipients for severe aplastic anaemia.

We used minisatellite probes to analyse by DNA fingerprints the long-term engraftment (median 4.3 years, range 1-2) of 21 bone marrow transplantation recipients for severe aplastic anaemia. Patients received their graft from histocompatible siblings. They were conditioned with cyclophosphamide (150 mg/kg) and a 6GY thoracoabdominal irradiation and did not have ex-vivo T cell depletion of marrow donor. DNA was extracted peripheral mononuclear cells and analysed by Southern blotting with 32P-labelled single-stranded RNA probes. Seven out of 21 donor-recipient pairs were sex-mismatched and additionally studied with a probe detecting a male specific repeated sequence on the Y chromosome. Red cell surface phenotype was also used as marker of engraftment in most cases. Long-term engraftment appeared complete for all patients studied with respect to the three methods.

Adolescent↗
PubMed1988

Plasmid vectors with multiple cloning sites and cat-reporter gene for promoter cloning and analysis in animal cells.

We have constructed two vectors, pGCAT-A and pGCAT-C, designed to facilitate the construction of recombinant plasmids containing the bacterial gene (cat) coding for chloramphenicol acetyltransferase (CAT) under the control of eukaryotic promoter and/or enhancer elements. The cat gene was inserted downstream from a multiple cloning site (MCS) region with eleven unique restriction sites. The MCS region is in opposite orientation in the two vectors. The CAT activity of control extracts from cells transfected with pGCAT-A or pGCAT-C is very low. Insertion of the viral SV40 early promoter into one of the sites of the MCS region of pGCAT-A or pGCAT-C results in a 30- to 400-fold stimulation of the CAT activity.

Acetyltransferases↗
PubMed1988

Both N-ras and c-myc are activated in the SHAC human stomach fibrosarcoma cell line.

A transforming N-ras gene was isolated from the SHAC human stomach fibrosarcoma cell line. A single-point mutation resulting in the substitution of histidine for glutamine at codon 61 was found in the SHAC transforming allele. The N-ras gene is overexpressed in the tumor cells and transformant cells. The N-ras p21 product was studied by immunoprecipitation and showed no alteration in mobility as compared to the normal p21 protein. The c-myc gene is amplified and overexpressed in these cells. This report gives evidence that an amplified c-myc and a mutated N-ras gene are both present in this tumor cell line and provides support for the idea that co-operation of at least 2 activated cellular oncogenes is required for carcinogenesis.

Animals↗
PubMed1988

cis activation of the c-myc gene in bovine papilloma virus type 1/human c-myc hybrid plasmids.

The c-myc gene amplification observed in human tumors is likely to represent an activation mechanism aiming at an increased transcription level. In order to evaluate the biological significance of this amplification in the malignant transformation we have designed an experimental model that could possibly mimic this situation in vitro. We have constructed a series of plasmids which physically link the human c-myc gene to the bovine papilloma virus type 1 genome (BPV1) and therefore should be maintained as amplified episomes upon transformation of rodent cells. Anticipating that the high copy number will bring about the immortalizing capacity of the c-myc gene, the constructions were introduced into primary rat embryo cells. Immortal cell lines were established by transfection of the hybrid plasmids carrying either the complete BPV1 genome or the transforming region of the viral genome. The BPV1 DNA alone or the c-myc gene alone has no activity in this assay. The analysis of the established cell lines demonstrates that the transfected plasmids are present not as free copies as anticipated but rather integrated as tandem repeats. We present data which strongly suggest that the immortalization capacity of the hybrid plasmids reflects the activation of the c-myc gene by the transactivable BPV1 enhancer. Although both the BPV1 early genes and the c-myc gene are actively transcribed, most of the cell lines do not display a transformed phenotype.

Animals↗
PubMed1988

High c-myc amplification level contributes to the tumorigenic phenotype of the human breast carcinoma cell line SW 613-S.

The c-myc gene is amplified in the SW 613-S cell line which was established from a human breast carcinoma. This line is heterogeneous: it contains cells with a high level of amplification and carrying the extra copies of the c-myc gene in double minute chromosomes (DMs) and cells with few c-myc genes integrated into chromosomes. Clones with different levels of amplification and different cytological localization of the c-myc copies were isolated from the SW 613-S cell population. Those with a high level of amplification and expression of the c-myc gene were highly tumorigenic in nude mice whereas those with a low level were not. Introduction of c-myc gene copies by transfection into the cells of several non-tumorigenic clones restored the tumorigenic phenotype. Our results indicate that a high level of amplification of the c-myc gene is a requirement for the tumorigenicity of SW 613-S cells in animals.

Animals↗
PubMed1986

An Epstein-Barr virus transcription unit is at least 84 kilobases long.

We have studied the structure of the Epstein-Barr virus mRNAs expressed in B95-8, a productively-infected Marmoset cell line established from in vitro-infected B-lymphocytes. We constructed a cDNA library from the cytoplasmic polyadenylated RNAs of B95-8 in the lambda gt10 bacteriophage. We present here the analysis of a 3.5 kbp cDNA containing exons transcribed from the US, IR and UL regions of the viral genome. The corresponding transcription unit is at least 84 kbp long. Two exons are transcribed from the US region, five from the IR region and two from the UL region. The exons from the IR region consist of two tandem repeats of a unit containing two exons, 66 and 132 nucleotides, and of a third copy of the 66 nucleotide exon. The exons from the UL region contain an open reading frame coding for a 944 amino acid polypeptide. The C-terminal end of this polypeptide harbors three types of repeated sequences. The corresponding mRNA is the second described of a family of mRNAs produced by alternative splicing of exons transcribed from the US, IR and UL regions.

Animals↗
PubMed1986

New RNA species is produced by alternate polyadenylation following rearrangement associated with CAD gene amplification.

Mammalian cells selected to resist N-(phosphonacetyl)-L-aspartate (PALA) contain amplified copies of the CAD gene. While a single 7.9-kb mRNA species is detected in PALA-sensitive and most PALA-resistant cell lines, two RNA species (7.9 and 10.2 kb) are detected in two related drug-resistant mutants presumably derived from the same parental cell. In this report we show that the 10.2-kb RNA is produced as a direct consequence of a sequence rearrangement adjacent to the 3' end of the CAD gene in these cell lines. A CAD gene containing the sequence rearrangement was cloned from one of these lines and found to produce both RNA species when transfected into CAD-deficient cells. DNA sequencing and S1 analysis demonstrate that the 10.2-kb RNA is produced by alternative polyadenylation rather than by alternative splicing. Sequence analysis also reveals that several consensus poly(A) addition signals (AATAAA) were brought into close proximity to the CAD gene by virtue of the rearrangement. While sequences adjacent to each of the polyadenylation signals contain additional features postulated to be important for the selection of the site of poly(A) addition, S1 mapping analysis indicates that only one of the polyadenylation signals is used. A comparison of all of these sites suggests that multiple sequence motifs are required to form a functional polyadenylation and cleavage signal.

Animals↗
PubMed1986

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.

Cell Line↗
PubMed1985

Increased level of amplification of the c-myc oncogene in tumors induced in nude mice by a human breast carcinoma cell line.

Cell line SW 613-S, derived from a human breast carcinoma, contained double minute chromosomes (DMs) but lost them progressively upon in vitro cultivation. These cells were tumorigenic in nude mice. Cell lines were derived from the tumors and were found to have a high DM content. In three such cell lines, DMs were stably maintained upon in vitro cultivation, whereas in another they were progressively lost. We found that the c-myc oncogene is amplified 5- to 10-fold in SW 613-S and 20- to 90-fold in the different cell lines derived from the tumors. At least part of the additional c-myc copies were found associated with a purified DM fraction. In cell lines which lost the DMs during in vitro passages, the level of amplification was maintained. In situ hybridization experiments indicated that this loss was compensated by the acquisition of copies of the c-myc gene integrated into a chromosome. No major rearrangement of the amplified c-myc gene was detected. The amount of c-myc messenger RNAs is roughly proportional to the level of amplification. Our results indicate that growth of SW 613-S cells as tumors in nude mice selected cells with an increased level of amplification and expression of the c-myc oncogene.

Animals↗
PubMed1983

Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.

Animals↗
PubMed1982

General method for cloning amplified DNA by differential screening with genomic probes.

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite. This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.

Animals↗
PubMed1980

Specific in vitro initiation of transcription on conalbumin and ovalbumin genes and comparison with adenovirus-2 early and late genes.

Specific initiation of transcription of cloned conalbumin and ovalbumin genes has been obtained in a cell-free system in the presence of purified calf thymus RNA polymerase B. Comparison with the adenovirus-2 early (E1A) and major late genes reveals marked variations in transcriptional efficiencies. Specific fragmentation of the conalbumin gene DNA indicates that a short DNA segment 5' to the initiation site is involved in specific transcription.

Adenoviruses, Human↗
PubMed1979

Adenovirus DNA template for late transcription is not a replicative intermediate.

The relationship between adenovirus replication and late transcription has been investigated using viral replication and transcription complexes isolated from infected HeLa cell nuclei. These two types of complexes extracted from adenovirus type 2-infected cell nuclei did not sediment at the same rate on sucrose gradients. Viral replicative intermediates were quantitatively precipitated by immunoglobulins raised against purified 72,000-dalton DNA-binding protein, whereas viral transcription complexes remained in the supernatant. These results show that late transcription does not occur on active replication complexes or on 72,000-dalton DNA-binding protein-containing replicative intermediates inactive in DNA synthesis. Additional evidence is presented indicating that it is very unlikely that replicative intermediates lacking the 72,000-dalton DNA-binding protein could be the template for late transcription.

Adenoviruses, Human↗
PubMed1978

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.

Adenoviruses, Human↗