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M Siniscalco

Publications and source records attributed to M Siniscalco.

At least 37 records · Page 2Linked to original sources
PubMed1990

Site-specific integration by adeno-associated virus.

Cellular sequences flanking integrated copies of the adeno-associated virus (AAV) genome were isolated from a latently infected clonal human cell line and used to probe genomic blots derived from an additional 21 independently derived clones of human cells latently infected with AAV. In genomic blots of uninfected human cell lines and of primary human tissue, each flanking-sequence probe hybridized to unique bands, but in 15 of the 22 latently infected clones the flanking sequences hybridized not only to the original fragments but also to a total of 36 additional species. AAV probes also hybridized to 22 of these new bands, representing 11 of the 15 positive clones, but never to the fragment characteristic of uninfected cell DNA. From these data we conclude that the AAV genome preferentially integrates into a specific region of the cellular genome. We have determined that the integration site is unique to chromosome 19 by somatic cell hybrid mapping, and this sequence has been isolated from uninfected human DNA.

Cell Line↗
PubMed1990

Mental retardation in heterozygotes for the fragile-X mutation: evidence in favor of an X inactivation-dependent effect.

The still debated question of whether the expression of mental retardation in heterozygous carriers of the Martin-Bell syndrome is influenced by X inactivation has been investigated in a group of phase-known double heterozygotes for the FRA-X mutant and the G6PD Mediterranean variant. In these individuals, the number of somatic cells (fibroblasts or red cells) with an active FRA-X chromosome could be assessed through the G6PD phenotype at the single-cell level. The data reported indicate a significant inverse correlation between the IQ level (as measured by the Wechsler-Bellevue test) and the percentage of fibroblast cells with an FRA-X active chromosome. In contrast, no significant correlation was found when the IQ level and red cell data were compared, thus suggesting the occurrence of somatic selection against hematopoietic stem cells with an active FRA-X chromosome.

Dosage Compensation, Genetic↗
PubMed1989

Chromosomes of older humans are more prone to aminopterine-induced breakage.

We have adopted a simplified version of the "cell hybrid cotransfer method" to test the hypothesis that human lymphocytes derived from elderly individuals have a higher chromosome instability. Peripheral blood lymphocytes from "old" male individuals and "young" controls were fused with a Chinese hamster cell line (CHO-YH21), yielding 10 HAT-resistant rodent-human clones from the old propositi and 22 from the young controls (HAT = hypoxanthine/aminopterin/thymidine). Both series of hybrid clones were analyzed with respect to the retention of the enzyme glucose-6-phosphate dehydrogenase and the surface antigen MIC2 identified by monoclonal antibody 12E7, two human X chromosome-linked markers located at opposite ends of the X chromosome. Cell hybrid clones with an X chromosome from a young control retained both markers in about 70% of the cells. In contrast, cell hybrid clones with an X chromosome from an old donor retained the MIC2 marker in only 30% of their cells. Slot-blot hybridization studies have established that the observed loss of the MIC2 marker is due to loss of the coding gene, not to suppression of its expression. Similar hybridization studies with molecular probes specific for other regions of the X chromosome suggest preferential chromosomal breakage sites. T lymphocytes from old donors were also found to have an LD50 for aminopterine significantly lower than the concentration of this drug in the HAT medium used to grow the hybrids, suggesting that the higher level of gene loss observed in the X chromosomes from old donors may be directly related to their increased sensitivity to the clastogenic effect of aminopterine. We speculate that the higher rate of chromosomal breakage and of marker loss observed along the "old-age" X chromosomes could be the result of "molecular scars" accumulated with aging at sites of constitutive chromosomal fragility.

Adult↗
PubMed1987

Biological performance in beta-thal heterozygotes and normals: results of a longitudinal comparison in a former malarial environment.

Families of beta-thal heterozygotes and of normals studied in 1956 by Silvestroni and Bianco in the small town of Berra near Ferrara were studied again in 1981-85. It was possible to compare mortality, fertility, and migration, in the two groups of heterozygotes and normals after the lapse of one generation. At the power of resolution of the sample available and of the methods used, no difference was visible. However, it was possible to establish the upper limits of the differences in average age at death and in average fertility at the 5% level of significance.

Age Factors↗
PubMed1987

Comparison of cytologic and genetic distances between long arm subtelomeric markers of human autosome 14 suggests uneven distribution of crossing-over.

The analysis of two rodent X human somatic cell hybrids, carrying different inborn translocations of the human chromosome 14 long arm, has permitted us to narrow down the localization of the structural locus for alpha-1-antitrypsin (PI) to band 14q32.1, proximally to the highly polymorphic DNA locus D14S1 which has been localized by previous studies between 14q32.1 and 14q32.2. These data, evaluated in conjunction with other published information, suggest that the D14S1 locus is cytologically equidistant from both the PI locus and the complex locus for the immunoglobulin heavy chains (IGH) but, genetically, it appears much closer to the latter since the recombination frequency reported between the IGH complex and PI is six times greater than that between the IGH complex and D14S1 (lod score peaks respectively at 26% and 4% with narrow fiducial limits). The present report adds further strength to the frequently proposed hypothesis of a nonlinear relationship between cytologic and genetic distances of human genes. The possibility that this phenomenon may be a feature of frequent occurrence throughout the entire human genome is discussed.

Animals↗
PubMed1986

The myoblast defect identified in Duchenne muscular dystrophy is not a primary expression of the DMD mutation. Clonal analysis of myoblasts from five double heterozygotes for two X-linked loci: DMD and G6PD.

We previously proposed the hypothesis that the primary expression of the defect in X-linked Duchenne muscular dystrophy (DMD) occurred in the myoblast, or muscle precursor cell. This was based on the observation that the number of viable myoblasts obtained per gram DMD muscle tissue was greatly reduced and those that grew in culture had decreased proliferative capacity and an aberrant distended flat morphology. Here we test that hypothesis by determining whether the expression of the myoblast defect is X-linked. Muscle cells were obtained from five doubly heterozygous carriers of two X-linked loci, DMD and glucose-6-phosphate dehydrogenase (G6PD), and compared with those from five sex- and age-matched controls heterozygous for G6PD only. A total of 1,355 individual clones were determined to be muscle and evaluated at the single cell level for proliferative capacity, morphology, and G6PD isozyme expression. The results demonstrate that the proportion of defective myoblast clones is significantly increased in DMD carriers. However, since this cellular defect does not consistently segregate with a single G6PD phenotype in the myoblast clones derived from any of the carriers, it is unlikely to be the primary expression of the DMD mutant allele.

Adult↗
PubMed1985

A human Y-linked DNA polymorphism and its potential for estimating genetic and evolutionary distance.

A human DNA sequence (p12f2), derived from a partial Y-chromosome genomic library and showing homology with the X and Y chromosomes and with an undetermined number of autosomes, detected two Y-specific restriction fragment length variants on male DNA that had been digested with Taq I and Eco RI. These variants may have been generated through a deletion-insertion mechanism and their pattern of holoandric transmission indicates that they represent a two-allele Y-linked polymorphism (RFLP). By means of DNA from patients with inborn deletions in chromosome Y, this polymorphic DNA site was mapped to the interval Yq11.1-Yq11.22. The frequency of the rarest allele was about 35 percent in Algerian and Sardinian human males, whereas it was only 4 percent among Northern Europeans. The p12f2 probe also detected Y-specific DNA fragments in the gorilla and chimpanzee. In view of the monosomy of the Y chromosome in mammalian species, Y-linked RFLP's may prove to be more useful than autosomal or X-linked markers in estimating genetic distances within and between species.

Base Sequence↗
PubMed1985

The human genes for hemophilia A and hemophilia B flank the X chromosome fragile site at Xq27.3.

Two DNA recombinant clones, shown by separate studies to contain DNA sequences homologous to the genes coding for the human blood coagulation Factors VIII and IX, were hybridized in situ to metaphases or prometaphases derived from patients with the fragile-X syndrome and from a normal control. The results of these experiments indicate that (i) both genes are located in the subtelomeric region of the long arm of the human X chromosome flanking the fragile site at Xq27.3, (ii) the resolution of this localization is approximately 0.5% the length of the human haploid genome, i.e., 1.8 X 10(7) bp, (iii) the linear order of loci within the above region is Factor IX-fragile site-Factor VIII-Xqter. Both the localization and the linear order of these loci have been confirmed by Southern blotting studies using the same molecular probes and a panel of rodent-human somatic cell hybrids known to have retained different segments of the human X chromosome. The findings described herein and the knowledge that Factor IX deficiency recombines freely with at least two loci of the G6PD cluster support our hypothesis that the chromosomal region which includes the fragile-X site is normally a region of high meiotic recombination.

Chromosome Mapping↗
PubMed1985

Genes controlling gp25/30 cell-surface molecules map to chromosomes X and Y and escape X-inactivation.

The monoclonal antibody AbO13 defines a cell-surface antigen that is expressed on most cultured human cells, but not on rodent cells. AbO13 precipitates glycoproteins of 25,000 and 30,000 mol. wt. from lysates of [3H]glucosamine-labeled human cells. Results of the serological typing of a panel of 25 rodent-human somatic cell hybrid clones show that reactivity with AbO13 segregates with the human X and Y chromosomes. The presence of either of these chromosomes is sufficient for O13 expression on the hybrid cell surface. Analysis of hybrid clones containing human X chromosomes with karyotypically defined deletions permitted the regional assignment of the X-linked gene locus controlling the expression of O13 to Xp22-pter. In addition, AbO13 is reactive with Chinese hamster-human hybrids derived from fibroblasts of a 49,XXXXX individual that contained only inactivated copies of the human X chromosome. These results suggest that the X-linked locus determining the expression of O13 is not subject to X-inactivation.

ABO Blood-Group System↗
PubMed1984

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.

Animals↗
PubMed1984

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

A 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 or 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 research tools to resolve the linear order of tightly X-linked loci and to investigate the biology of genetic recombination in humans.

Blood Group Antigens↗
PubMed1984

Isolation and characterization of human random cDNA clones homologous to DNA from the X chromosome.

To search for human X-chromosome-specific probes useful for molecular mapping, we studied recombinant clones isolated from a human cDNA library. DNA preparations from 150 randomly selected clones were labeled and annealed to XY and 4XY human DNA, and to DNA from a human-mouse hybrid cell line that had retained only the human X-chromosome (A9/HRBC2). cDNA clones sharing homology with DNA from the X chromosome annealed to A9/HRBC2-DNA and hybridized more intensely to 4XY DNA than to XY DNA. Eleven such clones were identified. Of these, three hybridized only to X chromosomal DNA while the rest also annealed to DNA from one or more autosomes. Chromosomal assignment of the autosomal DNA fragments showed that, in addition to hybridization to X chromosomal DNA, four of the clones hybridized to DNA sequences from chromosome 2 and two clones to chromosome 7. Subregional mapping of the relevant X chromosomal DNA fragments indicated that one clone is homologous to DNA sequences located at Xp21-Xp22, whereas the others are located in the telomeric region of the long arm. The cDNA clones were used to search for restriction fragment length polymorphisms. Several restriction-site polymorphisms were detected. Some corresponded to variants of X chromosomal DNA sequences while others were from autosomes such as chromosomes 2 and 7.

Chromosome Mapping↗
PubMed1984

A human autosomal phosphoglycerate kinase locus maps near the HLA cluster.

The human cDNA probe pPGK824 , of Singer-Sam et al. [Singer-Sam, J., Simmer , R. L., Keith, D. H., Shively , L., Teplitz , M., Itakura , K., Gartler , S. M. & Riggs, A. D. (1983) Proc. Natl. Acad. Sci. USA 80, 802-806] was used to isolate a genomic clone lambda PGK-1 containing a portion of an autosomal locus for phosphoglycerate kinase (PGK). A unique sequence subclone (pGK-1) of lambda PGK-1 was then used to map this locus to the region p23-q12 of human autosome 6--i.e., to the interval that contains the major human histocompatibility locus (HLA). Since an autosomal gene coding for testis-specific PGK in the mouse has been shown to be closely linked to the H2 locus and to the T/t-complex locus [ Eicher , E. M., Cherry, M. & Flaherty , L. (1978) Mol. Gen. Genet. 158, 225-228], it is suggested that the lambda PGK-1 recombinant clone contains part of the human gene for the testis-specific isozyme of PGK. In addition, the subcloned pGK-1 detects an EcoRI restriction fragment length variant and may therefore prove useful for further genetical analysis of the HLA region and specifically for testing the hypothesis that spina bifida and anencephaly may be the human equivalent of the murine defects due to the T/t-complex locus. Our findings support the generally held hypothesis that a large number of structural loci clustered around the histocompatibility genes have been conserved in a close linkage association throughout a large evolutionary interval.

Animals↗