Marcello Siniscalco

High rate of sister chromatid exchanges of Bloom's syndrome chromosomes is corrected in rodent human somatic cell hybrids.

The high rate of sister chromatid exchange (SCE) characteristic of cultured somatic cells from patients with Blooms syndrome (BS) was found to be fully corrected in BS chromosomes retained by somatic cell hybrids between Chinese hamster cells (CHO-YH 21) and BS fibroblasts (GM 1492), independent of the type and the number of human chromosomes retained. On the contrary, the average rate of SCE per Chinese hamster chromosome remained unaffected by hybridization with both BS and normal human cells. A partial suppression of SCE of about 30% was observed in the BS cells themselves when these were co-cultivated with Chinese hamster/Blooms syndrome hybrid cells. In these hybrids, the rate of SCE per chromosome (Chinese hamster or human) was unaffected by co-cultivation. The data reported indicate that the high rate of SCE in BS cells must be considered to be the consequence of a lost normal function, rather than the acquisition of a new abnormal one, and that several independent genetic systems may be involved in the control of SCE during the replication of mammalian cells. Accordingly, the high rate of SCE in a cultured cell line or an individual should be looked upon as the common phenotype resulting from mutation(s) at any one of these systems. The occurrence of genetic complementation for SCE across the species barrier suggests that at least some of these genetic systems are homologous in different mammalian species and emphasizes the potential(s) of somatic cell hybridization for studying the biology of SCE, in general, and the genetics of Blooms syndrome, in particular.

Temperature-sensitive mutants of balb/3t3 cells. Iii. Hybrids between ts2 and other mouse mutant cells affected in dna synthesis and correction of ts2 defect by human x chromosome.

Complementation studies were performed withts 2, a mouse 3T3 cell mutant temperature sensitive (ts) for cell and viral DNA synthesis. Thets phenotype is corrected by non -ts mouse or human cells and a non-DNA−ts mutant. This gene had been localized to a region on the human X chromosome near the HPRT locus based on isozyme and karyotype analysis of hybrids. Unusually rapid loss and fragmentation of human chromosomes occurs in hybrids withts 2. Hybrids betweents 2 and other DNA−ts mutants of mouse cells did not show complementation of the growth phenotype.

Further linkage evidence for localization of mutational sites for nonsyndromic types of X‐linked mental retardation at the pericentromeric region

We used several microsatellite markers scattered along the X chromosome to search for linkage relationships in a large Sardinian pedigree segregating for nonspecific X-linked mental retardation (MRX). Markers DXS573 and AR, located at chromosomal subregions Xp11.4-p11.22 and Xq11.2-q12, respectively, were found to segregate in full concordance with the disease, leading to a LOD score of 4.21 at zero recombination value. Recombination with the disease was found with markers MAOB and DXS454 located at Xp11.4-p11.3 and Xq21.1-q22, respectively; accordingly, markers distal to Xp11.4 and Xq22 also segregated independently of the disease. These findings provide strong linkage evidence in favor of the localization of one MRX mutational site in the pericentromeric region of the human X chromosome, justifying the assignment of a new symbol (MRX26) to our pedigree. Finally, on the basis of the recombinational events observed in the Xq21-q22 region, we have been able to refine the assignment of marker DXS456 to Xq21.33-q22.

Human RNA transcripts in man-mouse somatic cell hybrids. II. Thermal denaturation studies and Cot analysis.

The present report confirms an earlier finding that human RNA transcripts from a man-mouse somatic cell hybrid that has regularly retained only the human X chromosome can be identified by molecular hybridization on nitrocellulose filters. From thermal denaturation studies it is concluded that molecular hybrids between hybrid cell and human nucleic acids have higher Tms, and thus greater specificity, than those between mouse and human nucleic acids. Additional data, utilizing the kinetics of molecular hybridization in solution (Cot analysis), demonstrate the presence of “few gene copy” DNA sequences in the above-mentioned hybrid cells that are complementary to human (HeLa) HnRNA. Also shown is a fraction of hybrid cell HnRNA that is complementary to “few gene copy” DNA sequences of human DNA. Thus, the experiments reported may represent a specific assay for identifying reiterated and “few gene copy” DNA sequences of the human X chromosome and their complementary RNA. Theoretically this experimental approach may be extended to any chromosome. Some of the short-term and long-term perspectives of these types of studies are discussed.

Immune Senescence Contributes to the Slow Growth of Tumors in Elderly Subjects

Cancer occurs more frequently in old than in young humans. Epidemiological studies reveal that the prevalence of cancer increases with age-from youth until 85 years of age. In fact, increasing age is the greatest single risk factor with respect to the development of cancer. The explanation for this direct correlation between age and the risk of cancer is not clear. In part, the explanation may be related to the fact that biological existence of an untransformed cell is finite while that of a cancer cell is infinite. Thus, as normal cells begin to reach the end of their biological life span the random chance of observing an immortal cell increases on purely statistical grounds as untransformed cells disappear. However, there is also experimental evidence that cells from old animals are more susceptible to the intracellular processes that lead to neoplastic transformation than are cells from young animals. Thus, with increasing age the chance of a cell undergoing neoplastic transformation increases. One of the clearest demonstrations of this thesis is the experiment reported by Ebbesen? In these studies, skin from young and old mice were transplanted to young, syngeneic animals in order to eliminate an effect of age of the host. This permitted a direct comparison of the susceptibility of old and young skin cells to neoplastic transformation. The transplanted skin was then exposed to a carcinogen and the numbers of papillomas and carcinomas in transplanted young and old skin was compared. Benign and malignant tumors arose significantly more frequently in the skin from the old as compared to the young donors. The biological basis for the increased susceptibility of cells from old animals to

Old and new genetics help ordering loci at the telomere of the human X-chromosome long arm.

SummaryA Sardinian pedigree described in 1964 for having been found to segregate at the X-linked loci for the Xga antigen, G6PD deficiency, Protan and Deutan color blindness, with an instance of recombination between the last two loci, was re-examined with respect to four common X-linked DNA polymorphisms detected by molecular probes homologous to critical subregions of the human X chromosome. Two branches of this pedigree-including the one with the Protan-Deutan recombinant-were found to segregate also for the common BamHI polymorphism identified with the cDNA probe pHPT-2 of the HPRT gene (Xq26). The analysis of the chromosome haplotypes in the male offspring of the phase known penta-heterozygous mother suggests that the probable order of the relevant loci is HPRT, Deutan, G6PD, Protan, Xq telomere. Though we are fully aware of the risks of generalizing the significance of observations made on a single exceptional pedigree, we believe that this report outlines the potential of families of the type described as reasearch tools to resolve the linear order of tightly X-linked loci and to investigate the biology of genetic recombination in humans.

Complementation Studies in Murine/Human Hybrids Suggest Multiple Etiology for Increased Rate of Sister Chromatid Exchange in Mammalian Cells

Two mutational changes which occurred in culture and are associated with a high rate of sister chromatid exchange (SCE) phenotype have been identified in the L-A9 murine cell genome by means of complementation studies with somatic cell hybrids. Preliminary cytogenetical evidence suggests that the retention of human autosome 6 (namely the region comprised between Xq12 and Xqter) or human autosome 19 is required in the hybrid metaphases for complementation to occur, independently of their being derived from normal human or Bloom syndrome (BS) cells. These data and other complementation studies previously reported by our group and by other investigators suggest that mammalian cells may possess several independent systems involved in the control of SCEs during chromatid replication. Thus, the high rate of SCE can be regarded as the common phenotype resulting from a variety of qualitative or quantitative changes affecting the mammalian cell genome. Bloom syndrome is evidently an example of homozygosity for a recessive mutation occurring in nature. The high SCE mutants found among rodent cells (as those seen in unstable rodent-human hybrid cells) are more likely the result of chromosomal loss or rearrangement occurring in culture at one or more of the genetic systems hypothesized above. The occurrence of complementation within or between the species barrier, following cell hybridization or cocultivation, indicates the recessive nature of the corresponding mutations and the possible homology of the relevant genetic systems in different mammalian species. The isolation of rodent clonal cell lines with a stable high rate of SCEs and the production of somatic cell hybrids between them and BS cells offer a promising experimental tool for studying the biology of SCEs in general and the genetics of BS in particular.

+2.71 LOD score at zero recombination is not sufficient for establishing linkage between X-linked mental retardation and X-chromosome markers.

Nonspecific X-linked mental retardation (MRX) is the denomination attributed to the familial type of mental retardation compatible with X-linked inheritance but lacking specific phenotypic manifestations. It is thus to be expected that families falling under such broad definition are genetically heterogeneous in the sense that they may be due to different types of mutations occurring, most probably, at distinct X-chromosome loci. To facilitate a genetic classification of these conditions, the Nomenclature Committee of the Eleventh Human Gene Mapping Workshop proposed to assign a unique MRX-serial number to each family where evidence of linkage with one or more X-chromosome markers had been established with a LOD score of at least +2 at zero recombination. This letter is meant to emphasize the inadequacy of this criterion for a large pedigree where the segregation of the disease has been evaluated against the haplotype constitution of the entire X-chromosome carrying the mutation in question. 12 refs., 2 figs., 1 tab.

Premutation for the Martin-Bell Syndrome Analyzed in a Large Sardinian Family: III. Molecular Analysis with the StB12.3 Probe

This report complements a series of clinical, cytogenetical, and psychological studies previously reported on a large Sardinian pedigree segregating for premutations and full mutations associated with the Martin-Bell syndrome (MBS). Using the StB12.3 probe, we report now the molecular classification of all of the critical members of the pedigree. These molecular findings are evaluated against the variable phenotypic manifestations of the disease in the course of a six-generation segregation of an MBS premutation allegedly present in a common female progenitor of 14 MBS male patients and 9 female MBS heterozygotes seen in the last two generations. The nature and stepwise progression of MBS-premutations toward the fully manifested Martin-Bell syndrome and the possibility of reverse mutational events toward the normal allele are discussed with respect to the application of the presently available diagnostic tools in genetic counselling.

Hybridization properties of human X-chromosomal RNA transcripts from murine-human hybrids

A human-mouse hybrid cell line which has retained the human X chromosome and a fragment of chromosome 2 contains RNA sequences transcribed from human DNA. Nuclear RNA enriched for human sequences was prepared from this hybrid cell line by a multistep hybridization procedure to mouse and human DNA immobilized on nitrocellulose filters. The properties of this RNA were analyzed by RNA-DNA hybridization techniques. The results indicate that we have pre pared a RNA fraction enriched for sequences homologous to repeated DNA sequences of the human X chromosome.