The mouse Kin-17 gene codes for a new protein involved in DNA transactions and is akin to the bacterial RecA protein.

We have sought to characterize the molecular basis of the sensitivity to ionising radiation and to identify the genes involved in the cellular response of mammalian cells to such radiation. Using the Escherichia coli model, we tested the hypothesis that functional domains of RecA protein are represented in proteins of mammalian cells. We review here the results obtained in the detection of nuclear proteins of mammalian cells that are recognized by anti-RecA antibodies. We have called them kin proteins. Kin proteins likely play a role in DNA metabolism. We summarize the cloning of the mouse Kin-17 cDNA and our work on the identification and preliminary characterisation of the biochemical properties of mouse kin17 protein, a new nuclear protein able to recognize bent DNA and suspected to be involved in illegitimate recombination. We briefly describe our latest experiments on the molecular characterisation of the mouse Kin-17 gene. Finally, we discuss the properties of kin17 protein and the possible participation of kin17 protein in DNA transactions like transcription or recombination.

Nucleotide sequence and structure analysis of cDNAs encoding short-chain neurotoxins from venom glands of a sea snake (Aipysurus laevis).

Two cDNAs from the sea snake Aipysurus laevis have been cloned and sequenced. They encode isoforms of short chain neurotoxins. One of them is toxin b, previously isolated from the venom of Aipysurus laevis and sequenced by Maeda and Tamiya (Biochem. J. 153, 79, 1976), whereas the other corresponds to an isoform which was not hitherto described. The two toxin sequences differ from each other by three amino-acid residues. Both cDNA structures were comparable with that previously determined in our laboratory for erabutoxin a from Laticauda semifasciata.

Detection of ionizing radiations with the SOS Chromotest, a bacterial short-term test for genotoxic agents.

The effects of 3 types of ionizing radiation, gamma-rays, neutrons and accelerated alpha-particles, were examined using the SOS Chromotest, a bacterial colorimetric assay for genotoxic agents based on the measurement of the SOS response in Escherichia coli. The SOS Chromotest appeared to be a sensitive and simple assay to detect quantitatively these radiations as well as their biological effects. The range of adsorbed doses for which induction was observed was similar for the 3 types of radiation, the minimum inducing doses being in the order of 2.5-5 Gy. We discuss the possible use of these observations to study the molecular action of radiations and to compare their genotoxic effects with those of chemicals.

[A joint study of mutation of the Ki-ras oncogene and overexpression of the Tp53 oncogene in colorectal cancer]. / Etude conjointe des mutations de l'oncogène Ki-ras et de la surexpression de l'oncogène Tp53 dans des cancers colorectaux.

Twenty-five colorectal tumors (rectum 6, left colon 13, right colon 6) were studied with respect to the overexpression of p53 and the activation by point mutation of the Ki-ras oncogene. Single point mutations on codon 12 and codon 13 were analyzed after PCR amplivication, dotblotting and sequential hybridization with 12 different oligonucleotides. The intranuclear concentration of p53 protein was measured by flow cytometry after immunofluorescence staining with monoclonal antibody Pab 421. Twelve tumors were found to significantly overexpress p53 and 6 of them had an activated Ki-ras (5 on codon 12, 1 on codon 13). Of 13 tumors which failed to demonstrate over expression of p53, 8 had an activated Ki-ras (5 on codon 12, 3 on codon 13). In our series, p53 overexpression and ki-ras activation appeared to be independent.

Levallorphan-tolerant mutants of Excherichia coli with altered morphologies.

The penetration of levallorphan, a synthetic morphinan known to interact with the cytoplasmic membrane of E. coli, is seriously limited by the outer membrane. To select target-resistant mutants rather than outer membrane mutants, a two-step procedure was developed, which involved the selection by penicillin of an "intermediate" parental strain with a decreased penetration barrier and a subsequent positive selection of levallorphan-tolerant pseudo-revertant clones. Unlike the direct selection, this technique yielded various types of mutants in which the morphology, the septation ability, and the growth rate were greatly affected.

Analysis and sorting of chromosomes by flow cytometry: new trends.

Flow cytogenetic is widely used since 1975, and essentially contributes to karyotype analysis and chromosome sorting. The principles of experimentation and its possibilities and limitations are now well known. Recently several new technologies have appeared. What attitude should the cytometrist adopt regarding PCR, microdissection of chromosomes, in situ hybridization, slit-scan flow cytometry or image analysis?

Fluorescence response and sensitivity determination for ATC 3000 flow cytometer.

The fluorescence emitted by labeled particles after interaction with exciting light is conditioned by laser beam geometry and by the mode of fluorescence collection and filtration. A laser elliptic focusing mode is described, and the fluorescence characteristics of the sample cell flow are calculated. Fluorescence collection and detection through optical filters were analyzed, and efficiency was calculated for the ATC 3000 flow cytometer (Odam-Bruker, Wissembourg, France). A mathematical model is proposed for calculation of the fluorescence signal and its fluctuations. The background noise for the ATC 3000 was quantified experimentally using fluorescent microspheres of a known number of bound equivalent fluorescein isothiocyanate (FITC) molecules. These experimental measurements were found to fit the theoretical predictions, thus validating the proposed model.

Identification and mapping of swine cyclin-dependent kinase inhibitor CDKN2A and CDKN2B exon 2 sequences.

We have isolated the swine homologs of human CDKN2A and CDKN2B exon 2 sequences. As in the human and mouse genomes, the exon 2 sequences of these two genes present a high level of sequence homology and are tightly linked. Using fluorescence in situ hybridization, we have mapped swine CDKN2A and CDKN2B to chromosome 1q25. This confirms the comparative mapping data among man, mouse, and swine, showing a conserved synteny among chromosome segments 9p21, 4C3-C6, and 1q25, respectively.

[Sulfonamide-dependent growth of a strain of Escherichia coli K12 Thy- on Mueller-Hinton medium]. / Croissance sulfamido-dépendante d'une souche d'Escherichia coli K12 Thy- sur milieu de Mueller-Hinton.

The Mueller Hinton medium does not allow the growth of Escherichia coli Thy- strains. This effect is due to the presence of uridine (or a derived compound) which interferes with the function of thymidine phosphorylase. The presence of sulphonamides, in some E. coli K12 Thy- mutants, overcomes this inhibition. The same phenomenon of rescue by sulphonamides has been reproduced in a minimal medium containing thymine and uridine. The biochemical and clinical implications of this observation are discussed.

[Analysis of human chromosomes by the flow microcytometer]. / Analyse de chromosomes humains par microcytophotomètre de flux.

Human peripheral lymphocytes metaphase chromosomes, prepared for flow analysis were used to evaluate the resolving power of a new microscope-based flow cytometer "Leitz MPV flow". We were able to resolve the human karyotype into about 15 peaks after simple ethidium bromide staining and excitation with mercury arc lamp. These results showed that a simple flow cytometer with a common relatively inexpensive source of fluorescence excitation light, can compete with more sophisticated laser-illuminated flow cytometers for flow cytogenetic studies.

Recurrent t(11;22) breakpoint mapping by chromosome flow sorting and spot-blot hybridization.

The breakpoint of the recurrent t(11;22) translocation, one of the most frequent chromosome anomalies encountered in human population, always involves bands 11q23.2 and 22q11.2. The involvement of the C lambda locus of the immunoglobulin lambda gene cluster on chromosome 22 has been suggested: however, in situ hybridization experiments have yielded conflicting results. In order to solve these discrepancies by another approach, we have used bivariate flow sorting to separate the chromosomes of interest and to map the specific breakpoints by direct spot-blot hybridization with the gene-specific radiolabelled DNA probes, Alu, V lambda, ets. The results showed unambiguously that in the t(11;22) patient analysed, a set of C lambda and V lambda genes was translocated to the der(11) chromosome. Since V lambda genes are situated proximally to C lambda genes, we demonstrate that, in the case studied here, the chromosome 22 breakpoint is not located within or even immediately close to the C lambda region.

Simultaneous monitoring of P53 protein and DNA content of colorectal adenocarcinomas by flow cytometry.

The p53 transformation-related gene is located on 17p, a chromosomal segment frequently under-represented in colorectal adenocarcinoma karyotypes. We have developed a flow cytometric method for detection of its gene product on isolated nuclei by indirect immunofluorescence. Criteria for defining the presence of p53 were established, using parameters related to the difference of fluorescence obtained by incubating nuclei with a specific monoclonal antibody (MAB) versus isotypic control. This method allowed simultaneous quantitation of p53 and DNA in tumor nuclei to be made. Twenty-two of the 41 tumors analyzed (54%) were found to overexpress p53 when compared to 13 normal mucosa specimens. Repeated preparations from different fragments of the same tumor gave reproducible results for the 21 cases tested. p53 was detected in a higher proportion of disseminated tumors (64%) compared to localized disease (39%), but the difference did not reach statistical significance (chi 2 = 2.4, p greater than 0.10). Tumors containing aneuploid cell subpopulations were also more frequently positive (65%) than diploid ones (20%), the difference being significant (Fisher's exact test, p less than 0.03). This dual parameter flow cytometric method, evaluating both DNA ploidy and p53 expression, may prove useful in identifying different biological subgroups of colorectal cancer.

Comparative karyotype of pig and cattle using whole chromosome painting probes.

The extent and distribution of conserved chromosomal segments between pig and cattle chromosomes were established using hybridization of porcine chromosome painting probes on bovine metaphases. A total of 44 segments of conserved synteny were identified, resulting in a nearly complete coverage of the bovine karyotype. This study provides new data on chromosome evolution of mammals.

Swine chromosomes: flow sorting and spot blot hybridization.

Flow cytometry analysis was applied to swine chromosomes prepared from phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes. Flow karyotypes from both sexes and from t(3;7) translocation carrier females were obtained. A certain number of chromosome pairs could be assigned to various peaks. In fact, 13 peaks were observed for 18 autosomal pairs plus X and Y. Moreover, abnormalities owing to the t(3;7) translocation were readily observable. The number of base pairs for chromosomes associated with the various peaks was estimated by comparison with human flow karyotypes. The following four peaks were thus sorted: the peak assumed to represent the translocated chromosome 7 plus the normals associated with it; the corresponding peak from a normal swine; the peak assumed to contain among others the normal chromosome 7; and finally the peak corresponding to swine chromosome 1. Chromosomes of each peak were collected on Pall Biodyne membrane. Following appropriate denaturation and prehybridization, the four samples were hybridized with a human leucocyte antigen (HLA) class I 32P-labelled cDNA probe, representing most of the coding sequence of the HLA B7 gene. The results confirmed previous data from other techniques that assigned the swine MHC(SLA) to chromosome 7. Subsequently, sorted samples were hybridized with a porcine genomic Interferon alpha probe in order to confirm the mapping of this gene family on porcine chromosome 1.

Swine chromosomal DNA quantification by bivariate flow karyotyping and karyotype interpretation.

Human and swine chromosomes were analyzed separately and as a mix to obtain bivariate flow karyotypes. They were normalized to each other in order to use the human chromosomal DNA content as standard. Our results led to the characterization of the "DNA line" in swine identical to the human "DNA line." Estimation of the DNA content in mega-base pairs of the swine chromosomes is proposed. Chromosomal assignment to the various resolved peaks on the bivariate swine flow karyotype is suggested from the relation between DNA content quantified by flow cytometry and chromosomal size. Swine chromosomes 1, 13, 6, 5, 10, 16, 11, 18, and Y were assigned to peaks A, B, C, K, L, N, O, Q, and Y, respectively. Peaks D and E were assumed to contain chromosomes 2 and 14, but without specific assignment. Similarly, P and M peaks were expected to correspond to chromosomes 12 and 17. Of the remaining chromosomes (3, 7, X, 8, 15, 9, and 4), chromosomes 3, 7, and X, which were assigned previously to peaks F, G, and H, respectively, led us to deduce that chromosomes 15 and 8 belonged to peaks I and J, and chromosomes 9, 4, and X to peak H.

Recent advances of flow cytometry in fundamental and applied microbiology.

This review focuses on the recent applications of flow cytometry (FCM) in microbiological research (1987-mid 1992). It tries to give a scope of the important breakthroughs which occurred in this field during this period. The technical difficulties of microorganism analysis by flow cytometry is briefly appraised. The significance and the limits of the different microbial cell parameters attainable by flow analyses are systematically evaluated: light scatter for cell size and structure, fluorescence measurements for quantification of cellular components, microbial antigen detection and cell physiological activity estimation. Emphasis is given on the new technological advances which appeared in the last two years. The second part of the review is devoted to the analysis of the usefulness of flow cytometric approach in the different fields of microbiology: fundamental studies in microbial physiology, differentiation, microbial ecology and aquatic sciences, medical microbiology, parasitology, microbial pharmacology and biotechnology.

A PCR-based method to amplify DNA with random primers: determining the chromosomal content of porcine flow-karyotype peaks by chromosome painting.

We present here a new PCR-based technique that allows the production of several micrograms of DNA from only 300 flow-sorted chromosomes. During the first two PCR cycles, the annealing temperature is decreased to 30 degrees C, and numerous random loci are amplified under nonspecific conditions. As demonstrated here for pig chromosomes 1 and 18, the PCR products may be used to identify the chromosomal content of the flow-karyotype peaks of any species by fluorescence in situ hybridization.

A linkage map with microsatellites isolated from swine flow-sorted chromosome 11.

We have developed a simple and efficient method to construct partial libraries of swine Chromosome (Chr) 11, starting with only 300 flow-sorted copies. DNA is amplified by PARM-PCR with primer containing at the 5'-end the sequence AGCU-. After amplification, digestion of PCR products with uracil DNA glycosylase generates cohesive ends corresponding to the SstI site. The amplified fragments can then be ligated in vector linearized with the SstI enzyme. Using five different primers, we PARM-PCR amplified and cloned swine Chr 11 DNA. These chromosome-specific libraries have been used to develop 14 different (TG)n microsatellites. Ten of these markers were assigned to Chr 11 by PCR analysis of a panel of Pig-Rodent somatic hybrids and by linkage analysis of the 171 individuals of the PiGMaP reference families. A complete linkage map of 147 cM of this chromosome was then realized by integrating existing markers.

Flow cytometry studies of recombinant Escherichia coli in batch and continuous cultures: DNA and RNA contents; light-scatter parameters.

Flow cytometry has been used to study the contents of macromolecular compounds and light-scatter parameters in batch and continuous cultures of a recombinant Escherichia coli strain that forms protein inclusion bodies. Changes in relative DNA and RNA contents and cell mass as estimated by forward-angle light scatter were detected and tightly correlated in batch culture. In addition, heterogeneity of wide-angle light scatter (WALS), which we related to the presence of cellular inclusion bodies, was observed. In contrast, the relative RNA content and cell mass did not change during continuous culture, and homogeneity of WALS was found. In addition, unexpected changes in relative DNA content were observed after 67 h of culture, indicating a change in bacterial physiology.

Characterization of microsatellites from flow-sorted porcine chromosome 13.

Porcine flow-sorted Chromosome (Chr) 13 was PCR amplified with primers based on porcine short interspersed element (SINE) sequences. The product was cloned, gridded in microtiter plates, and screened with a [GT]10 oligonucleotide which gave 45 positive clones. Sequencing of these clones showed that 36 were unique, and 26 [GT]n microsatellites were characterized. Six other simple repeat sequences, the majority of which were associated with the 3' end of the SINE sequence, were also detected. Twenty-one primers sets were selected, and 13 of these detected useful polymorphisms in the grandparents (n = 26) of the European porcine mapping collaboration (PiGMaP) reference families. These 13 markers were mapped in the "PiGMaP" reference families, and a two-point linkage analysis was performed. The Lod scores indicated that three of the markers were not linked and the remaining 11 formed two linkage groups of two and nine markers respectively. The larger linkage group was also linked to the transferrin locus, permitting assignment of nine markers to porcine Chr 13.