Annette Schmitz

Allele-specific relative telomere lengths are inherited

Previous studies have indicated that single relative telomere lengths are defined in the zygote. In order to explore the possibility that single telomere lengths segregate in families, we compared relative telomere lengths obtained from allelic chromosome extremities transmitted from parent to child, representing a total of 31 independent meiotic events. We find a significant positive correlation of 0.65 (P=0.0004) between these telomere lengths, whereas the correlation between the non-transmitted parental homologue and the transmitted homologue in the child is not statistically significant (r=0.16; P=0.195). This study indicates that, even though there is a telomerase-mediated maintenance/elongation of telomeres in germ cells, allele-specific relative telomere lengths are preserved in the next generation.

Fine deletion mapping of chromosome 8p in non‐small‐cell lung carcinoma

Several somatic genetic alterations have been described in non‐small‐cell lung carcinomas (NSCLC). Recurrent chromosomal deletions have suggested the presence of tumor‐suppressor genes specifically involved in lung carcinogenesis. For one of these, 2 non‐overlapping regions have been proposed on the short arm of chromosome 8, encompassing the LPL and NEFL genes. The LPL region has been extensively studied in NSCLC and other cancer types. Two genes, N33 and PRLTS, have been identified, but the small number of mutations excludes their involvement in the vast majority of tumors. In order to delineate a reliable region of deletional overlap on chromosome 8p in NSCLC, a series of 77 NSCLC was studied for 34 microsatellite polymorphisms distributed on chromosome 8p, using multiplex‐PCR amplification. After purification of tumor nuclei by flow cytometry based on either the abnormal DNA index or the presence of a high expression of cytokeratin, allelic losses on chromosome 8p were observed in 39% of cases. Measurement of DNA index showed that 62% of tumors were hyperploid; allelic losses were more frequent in hyperploid than in diploid tumors (54% vs. 14%; p < 10−4). Deletions of part of the short arm were observed in 7 instances. Our data allow definition of an interval of common deletion, flanked by the loci D8S511 and D8S1992, where the putative tumor‐suppressor gene might be localized. Int. J. Cancer 81:854–858, 1999.

Comparative karyotype of rat and mouse using bidirectional chromosome painting.

A comparative karyotype of rat (Rattus norvegicus) and mouse (Mus musculus) based on chromosome G-banding morphology, heterologous chromosome painting results and available gene mapping data is proposed. Whole chromosome painting probes from both species were generated by PARM-PCR amplification of flow sorted chromosomes. Bidirectional chromosome painting identifies 36 segments of syntenic homology and allows us to propose a nearly complete comparative karyotype of mouse and rat (except for RNO 13 p and RNO 19 p12-13). Seven segments completely covered the RNO chromosomes 3, 5, 8, 11, 12, 15 and 18. Eight segments completely covered the MMU chromosomes 3, 4, 6, 7, 9, 12, 18 and 19. The RNO chromosomes 5, 8, 18 show complete homology with the MMU chromosomes 4, 9 and 18, respectively. Bidirectional hybridization results clearly assign 16 segments to subchromosomal regions in both species. Interpretation of the results allows subchromosomal assignment of all the remaining segments apart from seven distributed on chromosomes MMU 15, MMU 10 B2-D3 and MMU 17 E3-E5. The proposed comparative karyotype shows overall agreement with available comparative mapping data. The proposed repartition of syntenic homologous segments between the two species provides useful data for gene-mapping studies. In addition, these results will enable the reconstruction of the karyotype of the Cricetidae and Muridae common ancestor and the definition of the precise rearrangements which have occurred in both mouse and rat lineages during evolution.

Deletion Mapping of the Tumor Suppressor Locus Involved in Colorectal Cancer on Chromosome Band 8p21

Several somatic genetic alterations have been described in colorectal carcinoma (CRC). Recurrent chromosomal deletions have suggested the presence of tumor suppressor genes (TSG) specifically involved in colorectal carcinogenesis. For one of them, two non‐overlapping regions have been proposed on the short arm of chromosome 8, encompassing the LPL and NEFL genes. The short arm of chromosome 8 has been extensively studied in colorectal cancer and in other cancer types. Both regions have been reported as candidate loci for a TSG. In order to delineate a reliable region of deletional overlap on chromosome arm 8p in CRC, a series of 365 CRC samples was selected for the absence of microsatellite instability (RER, replication error); tumor and normal matched DNAs were studied for 54 microsatellite polymorphisms distributed on 8p using multiplex‐PCR amplification. After purification of tumor nuclei by flow cytometry based on either the abnormal DNA index or the presence of a high expression of cytokeratin, complete allelic losses on 8p were observed in 48% of cases. Measurement of the DNA index showed that 88% of RER tumors were hyperploid. Complete allelic losses of only part of the short arm were observed on 26 occasions. These data allowed us to define a 1 cM interval of common deletion, flanked by the loci D8S1771 and NEFL, where a putative TSG may be localized. Genes Chromosomes Cancer 25:147–153, 1999.

Pig standard bivariate flow karyotype and peak assignment for chromosomes X, Y, 3, and 7

A standard pig flow karyotype (2N = 38 chromosomes) was defined by standardization of several flow karyotypes obtained from stimulated peripheral blood lymphocytes of normal male and female pigs. Depending on the animals under study, the flow analysis of their chromosome suspensions gave rise to bivariate flow karyotypes comprising from 15 to 17 peaks, of which 11 to 15 represented single chromosomes. The results were used to propose a peak nomenclature. In addition, a male miniature pig lymphoblastoid cell line was characterized by flow cytogenetics. A very high-resolution flow karyotype, in which all peaks but one superimposed on those of the standard karyotype, was obtained. Peaks were assigned for chromosomes X and Y. Analysis of flow karyotypes obtained from translocated t(3,7)(p1.3;q2.1) pigs combined with polymerase chain reaction (PCR) studies of major histocompatibility complex (MHC)-linked sequences on flow-sorted chromosomes allowed identification of peaks 3 and 7 of normal pig chromosomes and of the derivative chromosomes associated with the t(3,7)(p1.3;q2.1) translocation.

Characterization of Reciprocal Translocations in Pigs using Dual-colour Chromosome Painting and Primed in situ DNA Labelling

We report the use of dual-colour chromosome painting to determine the exact nature of certain chromosome rearrangements observed in the pig (Sus scrofa domestica). The chromosomal abnormalities were detected by GTG- and RBG-banding techniques. The initially proposed interpretations were: (1) rcp(6;13)(p1.5;q4.1); (2) rcp(11;16)(p1.4;q1.4); (3) rcp(6;16)(p1.1;q1.1); (4) rcp(13;17)(q4.1;q1.1); (5) rcp(6;14)(q2.7;q2.1); (6) rcp(3;5)(p1.3;q2.3); (7) rcp(2; 14)(q1.3;q2.7); (8) rcp(15;17)(q1.3;q2.1). Hybridizations were carried out with biotin- and digoxigenin-labelled probes obtained by priming authorizing random mismatches polymerase chain reaction (PARM-PCR) amplification of porcine flow-sorted chromosomes. In some cases, i.e. (1), (4), (5), (6), (7) and (8), the fluorescence in situ hybridization (FISH) results allowed confirmation of the interpretations proposed with classical cytogenetic methods. Chromosome painting proved the reciprocity of the translocation in cases (1), (6) and (8), whereas modifications of the formula were proposed for case (2). Primed in situ DNA labelling (PRINS) experiments have also been carried out in case (3) using a primer specific for the centromeres of acrocentric chromosomes (first experiment) or a primer specific for the centromeres of a subset of meta- and submetacentric chromosomes including chromosome 6 (second experiment). It allowed us to demonstrate that the breakpoints occurred in the centromeric region of chromosome 16 and in the p arm of chromosome 6, just above the centromere.

Analysis and sorting of chromosomes by flow cytometry: new trends

Summary— Flow cytogenetic is widely used since 1975, and essentially contributes to caryotype 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?

New flow cytogenetics and large genomes analysis: from man to swine

Since the launching of the human genome research programme, the construction of detailed genetic maps of animal species of scientific or economic value, such as swine, caUle, sheep, dog, rat and mouse have also been in progress. For both genetic and physical mapping it is necessary to reduce the complexity of the genome. Its Inst natural division is the chromosome which represents a source of genomic DNA subunit which can be isolated by various methods. Among them flow cytometry is the technique of choice since it can sort out pure chromosomes at a speed of 15-20 chromosomes per second (9,13).