A new DNA marker tightly linked to the fragile X locus (FRAXA).

The fragile X syndrome is the most common cause of familial mental retardation. Genetic counseling and gene isolation are hampered by a lack of DNA markers close to the disease locus. Two somatic cell hybrids that each contain a human X chromosome with a breakpoint close to the fragile X locus have been characterized. A new DNA marker (DXS296) lies between the chromosome breakpoints and is the closest marker to the fragile X locus yet reported. The Hunter syndrome gene, which causes iduronate sulfatase deficiency, is located at the X chromosome breakpoint that is distal to this new marker, thus localizing the Hunter gene distal to the fragile X locus.

Deletions in the COL4A5 collagen gene in X-linked Alport syndrome. Characterization of the pathological transcripts in nonrenal cells and correlation with disease expression.

The type IV collagen alpha 5 chain (COL4A5) gene of 88 unrelated male patients with X-linked Alport syndrome was tested for major gene rearrangements by Southern blot analysis, using COL4A5 cDNA probes. 14 different deletions were detected, providing a 16% deletion rate in the COL4A5 gene in the patient population. The deletions are dispersed all over the gene with different sizes, ranging from 1 kb to the complete absence of the gene (> 250 kb) in one patient. In four patients with intragenic deletions, absence of the alpha 3 (IV) chain in the glomerular basement membrane was demonstrated by immunohistochemical studies. This finding supports the hypothesis that abnormalities in the alpha 5 (IV) chain may prevent normal incorporation of the alpha 3 (IV) chain into the glomerular basement membrane. Direct sequencing of cDNA amplified from lymphoblast mRNA of four patients with internal gene deletions, using appropriate combinations of primers amplifying across the predicted boundaries of the deletions, allowed us to determine the effect of the genomic rearrangements on the transcripts and, by inference, on the alpha 5 (IV) chain. Regardless of the extent of deletion and of the putative protein product, the 14 deletions occur in patients with juvenile-type Alport syndrome.

The gene for Bazex-Dupré-Christol syndrome maps to chromosome Xq.

Bazex-Dupré-Christol syndrome is an inherited condition with skin cancer predisposition characterized by follicular atrophoderma, hypotrichosis, and early onset of multiple basal cell carcinomas. Previous reports suggested an X-linked mode of inheritance. We therefore performed linkage analysis with microsatellite markers of the X chromosome in three families. We obtained evidence for X-linkage and regional assignment to Xq24-q27 of this syndrome (maximal lod score = 5.26 with a recombination fraction of 0% at the DXS1192 locus). This represents a first step towards the identification of a gene involved in hair follicle development and skin tumor formation.

New polymorphism and a new chromosome breakpoint establish the physical and genetic mapping of DXS369 in the DXS98-FRAXA interval.

Recently some of us cloned a new probe RN1 (DXS369), which appears a close marker for the fragile X locus (FRAXA) [Oostra et al.: Genomics 1990]. We present here new evidence for its physical and genetic mapping in the DXS98--FRAXA interval. We used 2 different somatic cell hybrid lines with breakpoints in the Xq27-q28 region: L10B Rea and PeCHN, and we established the order: (DXS105, DXS98)-L10B Rea-DXS369-PeCHN- (DXS304, DXS52). We detected an additional TaqI RFLP at the DXS369 locus which increases its informativeness up to 57%. Two point linkage analysis in a large set of families gave high lod scores for the FRAXA-DXS369 linkage (z(theta) = 10.1 at theta = 0.044) and for DXS369-DXS304, a marker distal to FRAXA (z = 19.2 at theta = 0.070). By multipoint analyses we established the localization of DXS369 in the DXS98-FRAXA interval. DXS369 is a much closer proximal marker for FRAXA than DXS105 or DXS98 and any new probe mapping between the breakpoints in L10B Rea and PeCHN will be of potential interest as a marker for FRAXA.

[Bovine chromosome mapping with the cell hybridization technic. Localization on the x chromosome of glucose-6-phosphate dehydrogenase, phosphoglycerate kinase, alpha-galactosidase A and hypoxanthine phosphoribosyltransferase]. / Carte génétique des bovins par la technique d'hybridation cellulaire. Localisation sur le chromosome X de la glucose-6-phosphate déhydrogénase, la phosphoglycérate kinase, l'alpha-galactosidase A et l'hypoxanthine guanine phosphoribosyl transférase.

Twelve hamster x cattle (Bos taurus) cell hybrids showed loss of cattle chromosomes, which allows their use for chromosome mapping of cattle. Genes coding for G6PD, alpha-GAL A, PGK, and (indirectly) HGPRT are synteneic and localized on the X chromosome.

Time dependence of X gene reactivation induced by 5-azacytidine: possible progressive restructuring of chromatin.

According to the theoretical mechanism of DNA demethylation by 5-azacytidine, the complete demethylation of one site will require two cell divisions. If reexpression is directly related to demethylation, a maximal reexpression is expected after two cell divisions. In a hamster X human hybrid cell line containing an inactive human X chromosome treated by 5-azacytidine, we show that HPRT reactivation frequency is increased more than 10-fold when cells are allowed to divide 14 times before the selection for the HPRT reactivants. We suggest that the delay corresponds to changes in chromatin conformation.

Coreactivation of four inactive X genes in a hamster x human hybrid and persistence of late replication of reactivated X chromosome.

Hamster-human hybrids which contained an inactive human X chromosome were treated by 5-azacytidine. Hypoxanthine guanine phosphoribosyltransferase derepressed hybrids were selected and derepression of three other loci, phosphoglycerate kinase, alpha-galactosidase, and glucose-6-phosphate dehydrogenase were studied. Among 32 hybrids selected for hypoxanthine guanine phosphoribosyltransferase, two were found to be reactivated at four X loci. The independence or nonindependence of the reactivation events will be discussed. No correlation was found between the time of replication and the expression or nonexpression of the X chromosome genes: X chromosomes reactivated at four loci remained late replicating; conversely early replication can exist without the expression of some X genes.

[Intracellular glycosaminoglycans of diploid and heteroploid human cells and of hybrid cell lines issued from their fusion]. / Glycosaminoglycannes intracellulaires de cellules humaines diploïdes et hétéroploïdes et des lignées hybrides issues de leur fusion

The human heteroploid cell line D98/AH-2 shows a low level of glycosaminoglycans (GAG) synthesis as compared with normal human diploid fibroblasts. Cellular hybrids were obtained by fusion of the D98/AH-2 cell line with normal fibroblasts, and fibroblasts derived from patients with mucopolysaccharidosis type II. In these hybrids the rate of GAG synthesis is intermediate between those of the parental cell lines.

Sheep gene mapping by somatic cell hybridization. III. Synteny between pyruvate kinase M2 (PKM2) and nucleoside phosphorylase (NP) in domestic sheep.

Twenty-five independent chinese hamster x sheep hybrids were analysed for ovine pyruvate kinase M2 (PKM2), nucleoside phosphorylase (NP) and mannosephosphate isomerase (MPI). An independent segregation is observed between PKM2 and MPI, whereas the results show a synteny between PKM2 and NP.

[Ovine histocompatibility complex OLA and enzymatic markers in hamster X sheep hybrids. I. Asynteny between PGM3-ME1 and OLA. (author's transl)]. / Complexe d'histocompatibilité ovin (ola) et marqueurs enzymatiques dans des hybrides hamster x mouton. I. Asynténie entre PGM3-ME1 et OLA.

Eighteen independent hamster X sheep fibroblast hybrids have been obtained. OLA typing of parental cells has shown that ovine fibroblasts carry the OLA A1, A8, B7, and B9 specificities. Analysis of the segregation of the OLA genes compared with the segregation of the 16 enzymatic markers studied previously indicated that, in contrast to the situation and man and certain other mammals, the genes of the histocompatibility complex of ovines (OLA) and those coding for the enzymes PGM3 and and ME1 are asyntenic. No synteny between OLA and any other enzymatic marker studied has been established.

Fine deletion mapping of the p22 region of the human X chromosome using a radiation-induced hybrid panel.

Radiation-induced somatic cell hybrids containing fragments of the human X chromosome were constructed. A panel of 17 hybrids was selected with the help of known markers in the Xp22 region. These hybrids identified 11 different breakpoints between Xp22.2 and Xp21.3. Eight markers were located in eight of the nine corresponding intervals, resulting in the following physical map: tel...DXS89-DXS278-DXS85-(DXS1224, DXS16)-(GLRA2, DXS987)-DXS207-(DXS-197, DXS1053)-(DXS43, DXS1195)-(DXS1229, DXS-999)-(DXS1052, DXS92, DXS274)-(DXS41, DXS1226)-DXS1198-DXS28...cen.

Inactive X chromosome gene reactivation induced by 5-azacytidine, is not correlated with the modification of the pattern of replication.

The replication pattern of a human inactive X chromosome reactivated for one to four genes by 5-azacytidine has been studied by the BrdU-33258-Hoechst-Giemsa technique in four subclones of a somatic hamster-human hybrid. In one of them the pattern was clearly modified. In the three others the changes were not significant. No correlation was found between expression and replication.

Isolation and characterisation of five new anonymous X-specific probes. One of them detecting a TaqI RFLP.

The authors isolated five single-copy X-specific probes from an X-enriched library. These probes were regionally localized on the X chromosome by using somatic hybrid cell lines obtained from patients carrying different X-autosome translocations. Three clones were located between Xq23 and Xq26, the two others were mapped between Xp21 and Xp11.2. Analyses with different restriction enzymes indicated that one of them detects a TaqI polymorphism with a polymorphism information content (PIC) of 0.28.

[Gene mapping in the pig (Sus scrofa 1.). I. Study of two syntenic groups G6PD, PGK, HPRT and PKM2, MPI (author's transl)]. / Carte génique du porc (Sus scrofa .1). I. Etude de deux groupes synténiques G6PD, PGK, HPRT et PKM2, MPI.

Thirteen pig-hamster and fifteen pig-mouse hybrid ceil lines were developed. These lines eliminate the pig chromosomes preferentially. The following syntenies were demonstrated: G6PD, PGK, HPRT, and PKM2, MPI.

Confirmation of the assignment of the gene for arylsulfatase A to chromosome 22 using somatic cell hybrids.

Human/hamster hybrid cell cultures were examined for the presence of ARSA and other marker enzymes. Many of these hybrids were also analyzed for human chromosomes. Our results confirm the assignment of ARSA to chromosome 22.

Normal lipid composition of fibroblasts from a case of type II achondrogenesis.

Fibroblasts from a case of achondrogenesis type II and fibroblasts from a normal control donor were subcultivated in vitro in parallel. The lipid study on these cells showed similar total lipid content, free cholesterol level, phospholipid distribution and fatty acid patterns, while neutral glycerides were slightly more elevated in the control fibroblasts. The histological finding of Laxova et al. (1973) could not be confirmed.

Actin-like sequences are present on human X and Y chromosomes.

The human genome contains greater than 20 actin-related sequences, six of which at least are expressed as protein. We have shown by blot hybridization the presence of actin-like sequences on both the X and the Y chromosomes. These sequences can be detected in HindIII digests of genomic DNA, using as probe cDNA clones corresponding to human alpha skeletal actin or to a hamster (beta or gamma) cytoskeletal actin; they show more homology to the latter probe. The actin probes also detect a polymorphic DNA fragment showing autosomal inheritance with a frequency for the major allele of 0.55 in the population studied. The X-linked actin sequence has been assigned to a centromeric region between Xp11 and Xq11 by hybridization to DNAs from a panel of human-mouse hybrid cell lines, and thus lies outside the postulated region of homology between the X and Y chromosomes. The Y-linked actin sequence can serve as a marker to analyse anomalies of sex determination or of gametogenesis in man. It was found in all XY males studied but was absent from the genomic DNA of four unrelated 'XX male' subjects and two XX hermaphrodites. This shows that the region of chromosome Y which contains the actin sequence is not translocated onto the X chromosome (or onto autosomes) in these patients.