Gérard Buttin

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.

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.

Gene Amplification Mechanisms: The Role of Fragile Sites

We studied the early stages of gene amplification in a Chinese hamster cell line and identified two distinct amplification mechanisms, both relying on an unequal segregation of gene copies at mitosis. In some cases, a sequence containing the selected gene is looped out, generating an acentric circular molecule, and amplification proceeds through unequal segregation of such extrachromosomal elements in successive cell cycles. In other cases, the accumulation of intrachromosomally amplified copies is driven by cycles of chromatid breakage, followed by fusion of sister chromatids devoid of a telomere, which leads to bridge formation and further break in mitosis (BFB cycles). We showed that some clastogenic drugs specifically trigger the intrachromosomal amplification pathway and strictly correlated this induction of BFB cycles to the ability of these drugs to activate fragile sites. In three model systems, we also established, that the location of centromeric and telomeric fragile sites relative to the selected genes determines the size and sequence content of the early amplicons.

Molecular characterization of an 18 kb segment of DNA puff C4 of Bradysia hygida (Diptera, Sciaridae)

The data presented here are an extension of the molecular characterization of DNA puff C4 of Bradysia hygida. A cDNA related to a gene amplified in this puff and expressed when puff C4 expands was cloned and sequenced. Analysis of the amino acid sequence deduced from the open reading frame present in the cDNA indicate that the encoded protein is secreted and comprises mostly α-helical coiled-coil. An 18 kb genomic segment containing the transcription unit of this gene was also cloned and the structure and expression of the 1.4 kb mRNA was determined. Quantitative slot blot hybridization of DNA complementary to the transcription unit shows that this gene is amplified about 21 times in the salivary gland, confirming data previously obtained. Fragments upstream of the 5′ end, and beyond the 3′ end, of the gene transcription unit were also analysed and shown to be amplified at least eight and five times, respectively. Based on these data we discuss how amplification could occur at DNA puffs.

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.

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.

Matrix attachment regions and transcription units in a polygenic mammalian locus overlapping two isochores

Eukaryotic chromosomes are ponctuated by specialized DNA sequences (MARs) characterized by their ability to bind the network of nonhistone proteins that form the nuclear matrix or scaffold. We previously described an amplifiable cluster of genes with different tissue‐specific expression patterns, located on Chinese hamster chromosome 1q. This model is especially appropriate to study the relationships between MARs and transcription units. We show here that four attachment regions, with sequences exhibiting motifs specific to MARs, are present within the 100 kb of screened DNA. Three of them are relatively short sequences localized in intergenic regions. The last one extends over one of the transcription units and contains a region previously identified as a recombination hot spot. Moreover, the analysis of a DNA sequence extending over some 50 Kb of this region and spanning at least four genes, disclosed a strikingly sharp change in G + C content. This strongly suggests that the studied region contains the boundary of two isochores. We propose that the frequency and the size of MARs are correlated to their localization in G + C rich or poor domains. J. Cell. Biochem. 67:541–551, 1997.

[14] Rosette-forming cell assay for detection of antibody-synthesizing hybridomas

Publisher Summary This chapter describes rosette-forming cell assay for the detection of antibody-synthesizing hybridomas. Detection of antibody-synthesizing hybridomas represents a difficult problem and is always a tedious work. The assay required for that screening should be highly specific, fast, and sensitive enough to allow an extremely selective screening of several hundred clones growing simultaneously. The rosette-forming cell micromethod is the method of choice when the antigen can be coupled to erythrocytes. This assay can be successfully used for the screening of many different hybridomas, such as antiDNP, antidextran, antilevan, anti-idiotype, and antienzymes clones. The rosette assay may also be useful for the precise analysis of the specificity of the monoclonal antibody secreted by hybridoma cells. The chapter presents an example of the analysis of cross-reactive idiotypes by means of monoclonal antibodies and the rosette assay.

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.

Specificity and idiotypic analysis of monoclonal antibodies directed against the MOPC460 idiotype

Eight syngeneic anti-idiotypic hybridomas (IDMs) have been obtained against the BALB/c myeloma protein MOPC460 which displays anti-TNP activity. The study of their anti-idiotype specificity allowed us to distinguish them into two groups which define the presence of at least two idiotypic determinants or idiotopes in the MOPC460 idiotype. The biochemical analysis of the monoclonal antibodies is consistent with this dichotomy. This analysis, in fact, showed a striking correlation between anti-idiotypic specificity and biochemical characteristics of the monoclonal antibodies. Consequently, the idiotypic specificities of three of these hybridomas were studied. In accordance with what is expected, our results clearly indicate a strong idiotypic similarity for hybridomas belonging to the same group and a lack of idiotypic cross-reactivity.