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G Buttin

Publications and source records attributed to G Buttin.

At least 37 records · Page 2Linked to original sources
PubMed1993

The origin of chromosome rearrangements at early stages of AMPD2 gene amplification in Chinese hamster cells.

BACKGROUND: Gene amplification and chromosomal rearrangements are frequent properties of cancer cells, provoking considerable interest in the mechanism of gene amplification and its consequences - particularly its relationship to chromosomal rearrangements. We recently studied the amplification of the gene for adenylate deaminase 2 (AMPD2) in Chinese hamster cells. Using fluorescent in situ hybridization (FISH), we found that early amplification of the AMPD2 gene is based on unequal gene segregation at mitosis, rather than local over-replication. We observed large inverted repeats of the amplified sequences, consistent with an amplification mechanism involving cycles of chromatid breakage, followed by fusion after replication and, in mitosis, the formation of bridges between the fused sister chromatids that leads to further breaks - a process we refer to as chromatid breakage-fusion-bridge (BFB) cycles. Our previous work left open the question of how this mechanism of gene amplification is related, if at all, to the chromosomal rearrangements that generate the dicentric, ring and double-minute (DM) chromosomes observed in some AMPD2-amplified metaphase cells, which are not predicted intermediates of chromatid BFB cycles, although they could be generated by related chromosome BFB cycles. RESULTS: We have addressed this question using FISH with probes for the AMPD2 gene and other markers on the same chromosome. Our results are not consistent with the chromosome BFB cycle mechanism, in which two chromatids break simultaneously and fuse to generate, after replication, a dicentric chromosome. Rather, they suggest that dicentric chromosomes are generated by secondary events that occur during chromatid BFB cycles. Our results also suggest that DM chromosomes are generated by the 'looping-out' of a chromosomal region, generating a circular DNA molecule lacking a centromere; in this case, gene amplification would result from the unequal segregation of DM chromosomes at mitosis. CONCLUSION: We conclude that, at early stages of AMPD2 gene amplification, chromatid BFB cycles are a major source of both 'intrachromosomal' gene amplification and genomic rearrangement, which are first limited to a single chromosome but which can then potentially spread to any additional chromosome. It also seems that, occasionally, a DNA sequence including the AMPD2 gene can be excised, generating a DM chromosome and thus initiating an independent process of 'extrachromosomal' amplification.

Journal Article↗
PubMed1993

DNA puff C4 of Bradysia hygida (Diptera: Sciaridae) contains genes unequally amplified and differentially expressed during development.

We report here the isolation and characterization of a 2.3 kb genomic EcoRI fragment that co-localizes in the DNA puff C4 of Bradysia hygida with a 4 kb EcoRI fragment previously characterized as containing part of a gene amplified and expressed in the salivary gland at the time when puff C4 expands. Verification of the relative amount of DNA complementary to these two genomic fragments shows that they are unequally amplified in the salivary gland. The fragment containing part of the gene expressed when puff C4 expands amplifies about eight times more than the 2.3 kb fragment. This 2.3 kb fragment also carries sequences complementary to RNA species present in the gland in a period when puff C4 has already receded. Based on these data we discuss the nature of the DNA puff and the possible way in which amplification is occurring at these sites.

Animals↗
PubMed1992

Co-amplified markers alternate in megabase long chromosomal inverted repeats and cluster independently in interphase nuclei at early steps of mammalian gene amplification.

Two-colour in situ hybridization with probes for two co-amplified markers located several megabases apart on chromosome 1 has been used to analyse early stages of adenylate deaminase 2 (AMPD2) gene amplification in Chinese hamster cells. In the amplified chromosomal structures, the distribution of hybridization spots identifies megabase-long inverted repeats. Their organization is remarkably well accounted for if breakage-fusion-bridge cycles involving sister chromatids drive the amplification process at these early stages. During interphase the markers often segregate into distinct nuclear domains. Many nuclei have bulges or release micronuclei, carrying several copies of one or both markers. These observations indicate that the amplified units destabilize the nuclear organization and eventually lead to DNA breakage during interphase. We propose a model in which interphase breakage has a role in the progression of gene amplification.

AMP Deaminase↗
PubMed1992

The evolution of the amplified adenylate deaminase 2 domains in Chinese hamster cells suggests the sequential operation of different mechanisms of DNA amplification.

Fluorescent in situ hybridization was used to localize the adenylate deaminase 2 (AMPD2) genes and flanking sequences on the chromosomes of the Chinese hamster line GMA32 and to study the distribution of additional copies of these genetic sequences in amplified mutants selected at several early stages of the amplification process. The synteny of AMPD2 genes and MDR1 genes, located on chromosomes 1, was demonstrated; in GMA32 the existence of a rearrangement positioning the two AMPD2 genes at different distances from the telomeres was disclosed. Using this structural marker, we showed that the amplified copies distribute along only one of the chromosomes 1. Their organization in different cells of clonal mutant populations at a very early stage of amplification was extremely heterogeneous; classes of organization could be recognized however. Their quantitative distribution at this stage and in cells which went through 10 more division cycles suggests an evolution pathway common to the mutant clones under study: as a rule, tandems of few units of identical and very large size (47 Mb) appear to be the first detected product of amplification; this organization is progressively overtaken by structures with more units of reduced and irregular size, while, in a growing number of cells, clusters of much shorter units can be observed. The nature of segregative amplification mechanisms operating in these processes and the possible involvement of replicative ones are discussed.

AMP Deaminase↗
PubMed1991

Activity of the kappa B enhancer of the interleukin-2 receptor alpha chain in somatic cell hybrids is accompanied by the nuclear localization of NF-kappa B.

The two nuclear proteins NF-kappa B (consisting of subunits p50 an dp65) and the DNA-binding subunit of NF-kappa B (p50) by itself, also called KBF1, are constitutively expressed and localized in the nucleus of the human T-cell line IARC 301.5. In order to define the roles of these two factors, which bind to the same kappa B enhancers, in transcription activation we have prepared somatic cell hybrids between IARC 301.5 and a murine myeloma. Most hybrids express both KBF1 and NF-kappa B in their nuclei, but one hybrid expresses only KBF1. The kappa B enhancer of the gene encoding the interleukin-2 (IL-2) receptor alpha chain (IL-2R alpha) is functional only in the hybrids expressing nuclear NF-kappa B. These findings show that nuclear NF-kappa B is necessary to activate the kappa B enhancer, while KBF1 by itself is not sufficient. We propose that KBF1 is a competitive inhibitor of NF-kappa B and discuss how these factors may be involved in the transient expression of IL-2 and IL-2R alpha genes during the immune response.

Animals↗
PubMed1990

Coamplification of mu class glutathione S-transferase genes and an adenylate deaminase gene in coformycin-resistant Chinese hamster fibroblasts.

In Chinese hamster fibroblasts, we previously detected an expressed gene located near the AMP deaminase gene. This gene was named Y1. Upon selection for resistance to coformycin, an inhibitor of AMP deaminase activity, both genes were amplified in several mutants. We have determined the complete nucleotide sequence of Y1 cDNA and identified the Y1 gene as a mu class glutathione S-transferase gene by comparison with sequences present in a data bank. Accordingly, Y1-amplified mutants express an increased glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene; this activity, as well as the abundance of the corresponding RNA, appears, however, to reach a limit despite further increase in the Y1 gene copy number during successive amplification steps. Southern blot experiments showed that Y1 belongs to a multigene family, all or part of which has been amplified in mutant lines. These data provide a method to amplify and to overexpress the mu class of the glutathione S-transferase gene family on the basis of its linkage with the AMP deaminase gene.

AMP Deaminase↗
PubMed1990

The splenic pool of mouse stem cells: in vitro differentiation and expression of the BP-1 alloantigen on cells of the lymphoid and myeloid lineages.

The relative paucity of data about the development of the stem cell pool present in the spleen prompted this study. During in vitro cultures of B-enriched lymphocytes from mouse spleens and in the presence of a culture supernatant of WEHI-3 cells (WEHI-SUP), a population of cells expressing the BP-1 antigen appears progressively, reaches an optimal size 8 days after initiation of the culture, and disappears on day 28. In 8-day-old cultures, a minor population of cells bearing both BP-1 and B220 can be detected. The growth of this cell population, with characteristics of the B lymphoid lineage (pro-B), is strictly dependent on the presence of WEHI-SUP in the medium. After 2 weeks of culture, the BP-1 antigen is expressed on a cell population, which is essentially constituted of B220-, polynuclear cells. The BP-1 antigen, which is considered as characteristic of early cells of the B lymphoid lineage, can therefore also be expressed on cells of the myeloid lineage. The injection of BP-1+ or B220+ cells in irradiated mice can hardly reconstitute their B cell pool, whereas BP-1- and B220- cells are much more efficient in vivo progenitors of this cell lineage.

Animals↗
PubMed1988

The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification.

The amplified DNA of HC50474, a Chinese hamster fibroblast cell line selected in three steps for high resistance to coformycin, consists chiefly of 150 copies of a large inverted duplication including the adenylate deaminase gene. Most if not all of these units are more than 2 x 120 kb long. The inverted duplication was first detected in the cells recovered from the second selection step, at the same chromosomal location as the first step amplified units. Its formation and amplification appear to be coupled since the second step cell line already contained 40 copies of this novel structure. Reamplification of the inverted duplication occurred at the third step of selection concomitant with the loss of amplified DNA acquired during the first step. The head-to-head junction has been formed by recombination within a recombinational hotspot described previously [Hyrien, O., Debatisse, M., Buttin, G. and Robert de Saint Vincent, B. (1987) EMBO J., 6, 2401-2408]. Sequences at the joint and in the corresponding wild-type region reveal that the crossover sites, one of which occurs in the putative promoter region of B2 repeat, are located at the top of significant stem-loop structures and that patchy homologies between the parental molecules on one side of the breakpoints allow alignment of these crossover sites. We present a model which explains the formation and amplification of this and other large inverted duplications by errors in DNA replication.

Animals↗
PubMed1988

Antibody-mediated binding of a murine ecotropic Moloney retroviral vector to human cells allows internalization but not the establishment of the proviral state.

We have attempted to introduce a neomycin-resistance gene, recombined in a murine (ecotropic) Moloney retrovirus, into HEp2 human cells which are resistant to infection by this retrovirus. To allow the binding of the retroviral vector on HEp2 cells, we have tested several antibody constructions between two monoclonal antibodies, one (mAb 5E9) directed against the human transferrin receptor and the other (mAb 615) directed against the gp70 envelope viral protein. The crosslinking of mAbs 5E9 and 615 by a sheep anti-murine kappa light chain antibody allowed the binding of virus on HEp2 cells. After incubation at 37 degrees, virus was internalized by HEp2 cells. However, no neomycin-resistant colonies of HEp2 cells have been obtained under various conditions. Our results suggest that binding and internalization of a retrovirus are not sufficient for establishment of the proviral state in cells which do not express a specific receptor.

Antibodies, Monoclonal↗
PubMed1988

Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

In a previous study of three independent families of mutants selected for overproduction of adenylate deaminase (AMPD), we were not able to isolate a cDNA probe for the gene and so could not demonstrate its amplification directly. In addition to overproduction of AMPD, four proteins of unknown function, designated W, X, Y1, and Y2, accumulated, and by using the corresponding cDNA probes, we demonstrated amplification of all four genes. In independent mutant clones, sometimes all and sometimes only a subset of these genes were amplified. Assuming that all five genes are linked, the pattern of their coamplification suggested a genetic map in which AMPD lies between W and Y1. We show here that a two-step chromosome walk joins the W and Y1 genes, that the AMPD gene is the only expressed sequence between them, and that its amplification is indeed responsible for overproduction of the AMPD protein. In the course of this work, we cloned and studied two novel joints which mark rearrangements on either side of the AMPD gene. Each joint was generated independently in a single first-step mutant at single or low copy number. Remarkably, each joint was amplified preferentially in every second- and third-step mutant derived from the first-step line in which it was originally present, suggesting that the two independent rearrangements each generated amplification-prone structures.

AMP Deaminase↗
PubMed1987

A hotspot for novel amplification joints in a mosaic of Alu-like repeats and palindromic A + T-rich DNA.

We have identified, in the amplified domain of adenylate deaminase (AMPD) overproducing Chinese hamster fibroblasts, a 2.6 kb recombinogenic DNA region which is frequently involved in amplification-associated rearrangements. The nucleotide sequence reveals a mosaic organization of four Alu-equivalent repeats of the B1 and B2 families and eight long A + T-rich DNA segments. Part of this region is enriched with long imperfect palindromes. The center of one palindrome contains a putative topoisomerase I cleavage site and this site defines the position of a novel junction which was formed by illegitimate recombination with anther A + T-rich DNA sequence located far apart on the amplified DNA. These findings and their significance are discussed in the context of related data from other systems and in the light of current models for eukaryotic DNA recombination, replication and organization.

AMP Deaminase↗
PubMed1986

Increase of retroviral infection in vitro by the binding of antiretroviral antibodies.

Monoclonal antiretrovirus antibodies were assayed for their ability to influence retrovirus infection in vitro. Some antibodies increased murine cell infection by an ecotropic virus and both murine and human cell infection by an amphotropic virus. The stability of these viruses was not modified, suggesting that antibody-virus complexes may be more infectious than free virus particles.

Animals↗
PubMed1986

Segregation and rearrangement of coamplified genes in different lineages of mutant cells that overproduce adenylate deaminase.

Four genes encoding proteins designated as W, X, Y1, and Y2 were found previously to be amplified at different levels in a Chinese hamster fibroblast mutant line selected for overproduction of adenylate deaminase. To gain information on the molecular mechanisms responsible, we studied the levels of amplification and the structures of these four genes in several lineages of mutant cells with comparable activities of adenylate deaminase, the selected enzyme. Only the W gene was amplified in all the lines. In one line, the X, Y1, and Y2 genes were coamplified, while in others either the Y1 gene or the pair X and Y2 were coamplified. The results were consistent with linkage of all the genes--in a particular order--in an amplifiable sequence with variable endpoints. Novel joints with a nonrandom distribution were observed. We frequently detected rearranged copies of the W gene, but very few novel joints were present in the other three genes in the six highly amplified lines examined. Some of the novel joints in gene W were highly amplified; they were generated by reamplification of a rearrangement that appeared at an early selection step. In some lines, reamplification was accompanied by deletion or mass correction of preexisting units. We discuss mechanisms which might account for these observations.

AMP Deaminase↗
PubMed1985

A single VH-gene associated with a variety of D- and J-segments encodes for a large family of ABPC48-related antibodies induced by antiidiotypic immunization.

Two series of monoclonal antibodies have been obtained from BALB/c mice immunized against two antiABPC48 antiidiotypic antibodies. They are divided into two serologically different classes. Class I antibodies bind only the immunizing antibody; class II antibodies display a broad binding capacity to various antiidiotypic antibodies, and some bind levan, as does ABPC48. Northern blot analyses and partial mRNA sequencing show that all class II antibodies express the VH-gene coding for ABPC48 and UPC10 antilevan antibodies associated with a variety of D- and J-segments. The third hypervariable region of the sequenced antibody with antilevan activity is structurally related to that of ABPC48 and UPC10 antibodies but has a different genetic origin. This study indicates that the identification of idiotype-related antibodies arising from antiidiotypic immunization may be misleading, if based on their antigen-binding properties; and it stresses the importance of structural approaches for the analysis of regulatory mechanisms ruling immune responses.

Amino Acids↗
PubMed1985

Immune response induced by a single or several syngeneic monoclonal antiABPC48 antiidiotypic antibodies: no predominant coexpression of ABPC48 idiotopes.

BALB/c mice were immunized with monoclonal BALB/c antibodies IDA10, IDA16 and IDA17 raised against the BALB/c ABPC48 myeloma protein. Several procedures of immunization--copolymers with lipopolysaccharide or keyhole limpet hemocyanin, simultaneous or sequential injections of different IDAs--were performed in an attempt to orient the immune response towards the production of ABPC48-like idiotypes. We used a binding assay which identifies two idiotopes on the same molecule to measure the population of antibodies induced in these responses. The expression of ABPC48 cross-reactive idiotypes in immune sera was analyzed. The results show that, with all immunization protocols, immune responses to different monoclonal antiidiotypic antibodies are mostly independent of each other: the coexpression of ABPC48 idiotopes, either private or recurrent on the induced antibodies, is rarely found; it makes it difficult to discriminate by a serological approach between cross-reactive idiotypes and anti-antiidiotypic antibodies. We discuss the interest of combining molecular and serological approaches to identify these two populations of antibodies.

Animals↗
PubMed1985

The VK gene expressed by BALB/c ABPC48 cross-reactive idiotypes induced by anti-idiotypic immunization is identical to that of BALB/c anti-oxazolone and A/J anti-arsonate antibodies.

Anti-idiotypic immunization triggers the production of antibodies that are structurally related to the idiotype. We have shown that the heavy chain variable regions of antibodies produced after anti-ABPC48 (A48) anti-idiotypic immunization of BALB/c mice are homologous to that of A48, except for the third hypervariable region. We present here partial light chain sequences of A48 and of antibodies induced by anti-idiotypic immunization. Nearly perfect homology is found, suggesting that these chains are the products of genes derived from a unique VK germ-line gene. These observations indicate that the H and L hypervariable regions contribute to define the structure of A48 idiotopes. Remarkably, the VK sequence we identify is the same as that described for anti-arsonate and anti-oxazolone antibodies. We discuss the relative importance of particular amino acids for idiotype expression and antigen-binding activity.

Animals↗
PubMed1984

Expression of several amplified genes in an adenylate-deaminase overproducing variant of Chinese hamster fibroblasts.

Unstable variants with increasing amounts of adenylate-deaminase (AMPD) have been stepwise recovered from Chinese hamster fibroblasts plated in selective medium containing increasing coformycin concentrations; several polypeptides accumulate in the variants in parallel to AMPD: they are no longer detectable in cells which reverted to the wild-type enzyme level. We report here the molecular cloning of cDNA sequences complementary to mRNAs coding for four such polypeptides. The plasmidic probes have been exploited to characterize their complementary mRNAs and to quantify the copies of these cognate genes in a variant and in two revertant clones. The results show that different mRNAs code for the four polypeptides; their accumulation is accounted for by amplification of their specific genes; these observations suggest that cells overproducing AMPD are characterized by the presence of amplification units comprising several expressed genes.

AMP Deaminase↗