Florence Richard

Reconstruction of the ancestral karyotype of eutherian mammals

Applying the parsimony principle, i.e. that chromosomes identical in species belonging to different taxa were likely to be present in their common ancestor, the ancestral karyotype of eutherian mammals (about 100 million years old) was tentatively reconstructed. Comparing chromosome banding with all ZOO-FISH data from literature or studied by us, this reconstruction can be proposed with only limited uncertainties. This karyotype comprised 50 chromosomes of which 40–42 were acrocentrics. Ten ancestral pairs of chromosomes were homologous to a single human chromosome: 5, 6, 9, 11, 13, 17, 18, 20, X and Y (human nomenclature). Nine others were homologous to a part of a human chromosome: 1p+q (proximal), 1q, 2p+q (proximal), 2q, part of 7, 8q, 10p, 10q and 19p (human nomenclature). Finally, seven pairs of chromosomes, homologs to human chromosomes 3 + 21, 4 + 8p, part of 7 + 16p, part of 12 + part of 22 (twice), 14+15, 16q+19q, formed syntenies disrupted in man.

Aneuploidy in human lymphocytes: an extensive study of eight individuals of various ages.

Data on aneuploidy from a prospective study on a large number of lymphocyte metaphases (over 1000 in 72-h and 100 in 48-h cultures) per individual from eight healthy donors of various ages are reported. Chromosome losses were dependent on culture time, being significantly more frequent in 72-h than in 48-h cultures. All donors exhibited various degrees of aneuploidy which increased with age in women. This increase resulted essentially from X chromosome losses, as previously reported. Although the rate of aneuploidy limited to autosomes was similar in newborns and in adults, the distributions of the missing autosomes were different. In the two newborns studied, autosome aneuploidy was random. In the adults, a significant inverse correlation with autosome lengths was observed. The inverse correlation between chromosome lengths and losses may be explained by selective pressure against monosomic cells in the adults.

The frequency of micronuclei with X chromosome increases with age in human females

The rate of micronuclei counted on lymphocyte cultures from five healthy female donors, 27-80 years old, increased with age. Using pXBR1 probe, specific for the alphoid DNA of the X chromosome, the presence of this chromosome was investigated by FISH (fluorescence in situ hybridization) in both micronuclei and metaphases. Both X aneuploidy and frequency of X chromosome per micronuclei increased with age. However, this overinvolvement of X chromosome was not sufficient to explain the overall increase of micronuclei with age, suggesting that autosomes are also involved. Thus, the higher increase of X than autosome aneuploidy in lymphocytes may result from both an excess of X chromosome losses and a better survival of cells with a monosomy X.

Highly conserved chromosomes in an Asian squirrel (Menetes berdmorei, Rodentia: Sciuridae) as demonstrated by ZOO-FISH with human probes.

The chromosomes of Menetes berdmorei (Rodentia, Sciuridae, Sciurinae) were studied by ZOO-FISH using whole human chromosome probes. All homoeologies between M. berdmorei and human chromosomes were determined, except for two small chromosome segments. Twelve human chromosomes are conserved in a unique block of synteny; ten are split into two and one into three blocks. Thus, a small number of interchromosomal rearrangements, about twenty, separates human from this squirrel karyotype. Homoeologies between human and the presumed ancestral chromosomes of Sciurinae could also be deduced, as well as those with the presumed ancestral chromosomes of eutherian mammals. Sciurinae chromosomes appear to be much closer to those of non-rodent mammals than those of Muridae and Cricetidae species studied so far. Thus, they provide an interesting tool to link the rodent genome to those of other mammals.

Increased FISH efficiency using APC probes generated by direct incorporation of labeled nucleotides by PCR

Probes of various sizes from the adenomatous polyposis coli gene (APC) were directly biotinylated by polymerase chain reaction (PCR) from genomic DNA. PCR labeling gave high efficiency in detection of fluorescence in situ hybridization (FISH) signals. Probes as small as 250 base pairs could be visualized through a fluorescence microscope without any image processing.

Chromosome instability in lymphocytes from patients affected by or genetically predisposed to colorectal cancer

To determine whether there are chromosomal clues to inherited forms of colorectal cancer, we studied chromosome instability in lymphocytes cultured from persons with sporadic colorectal cancer before treatment and from persons with a genetic predisposition to colorectal cancer due to adenomatous polyposis coli, Peutz-Jeghers syndrome, or juvenile polyposis. Spontaneous aberrations of chromosome number and structure were scored and compared to control studies by the same methods. Sex chromosome aneuploidy was found increased in patients. Autosomal aneuploidy was not increased. Chromosome breakage was elevated in young persons genetically predisposed to colorectal cancer compared to young patients with sporadic colorectal cancer. Chromosome rearrangements, other than those of chromosomes 7 and/or 14, rose with age, particularly in patients. Despite considerable interindividual variations, it would seem that increased chromosome breakage and rearrangement in addition to sex chromosome aneuploidy may be signs of chromosome instability in the predisposition to colorectal cancer.

Chromosome painting comparison of Leontopithecus chrysomelas (Callitrichine, Platyrrhini) with man and its phylogenetic position

Using human probes of whole chromosomes, the homoeologies between human and Leontopithecus chrysomelas (Platyrrhini) karyotypes were established. Thirty-three conserved segments were observed between the two species. Intrachromosomal rearrangements between the two species were identified using hybridization of chromosome arm probes of human chromosomes 1 and 3. We also used chromosomal data to investigate phylogenetic relationships of Callitrichines. These data were encoded using Cebus capucinus, a species which kept fairly ancestral chromosomes, as reference. Two equi-parsimonious trees, including reversion or convergence events, were obtained. The monophyly of Callitrichines is confirmed. They share nine chromosomal rearrangements at least. The Cebuella-Callithrix group forms a clade sharing five rearrangements at least. According to the tree considered, the Tamarins, Leontopithecus and Saguinus share two chromosomal rearrangements restricted to these two taxa or none. Callimico accumulated seven chromosomal rearrangements unshared with other taxa, at least. To avoid convergence and reversion events, we propose the hypothesis of a network (or populational) evolution. Six chromosomal rearrangements would have occurred during the period of this network evolution. Finally, the karyotype of the last common ancestor to all Callitrichines has been reconstructed. It possessed 48 chromosomes.

Constitutional balanced translocations in patients with solid tumors.

In a sample of 329 patients with a solid tumor (colon and breast adenocarcinoma, cervical carcinoma, and meningioma), four balanced constitutional translocations were observed. Two were t(13q14q), and two were reciprocal translocations. Comparison with surveys of newborns showed a significant excess of translocations in our sample.

Simultaneous obtention of an intense G-banding and chromosome painting

A method for simultaneously obtaining brilliant G-banding and chromosome painting is described. It is based on a modification of methods enabling the detection of incorporated BrdU by anti-BrdU antibodies conjugated to a fluorochrome. It induces a banding of both painted and non-painted chromosomes. The method was adapted for in situ hybridizations on human and non human (ZOO-FISH) chromosomes.

Chromosome mapping of 11 human probes in the region 5q2→q3 by fluorescence in situ hybridization

Using single- and double-color fluorescence in situ hybridization (FISH), 11 probes from the human chromosome region 5q2-->q3 are mapped. The following map order is proposed, from proximal to distal: 5q21.3-->q22: 15A6 (D5S136), YN5.64, CRI-L372 (D5S49), YN5.48 (D5S81): 5q22: EF5.44 (D5S135), APC; 5q22-->q23.1; CI5.23, L5.69 (D5S137), CRI-T39 (D5S64), MC5.61 (D5S84); 5q31.1-->q31.2: CRI-L1265 (D5S52).