Mapping of rare X-linked becker muscular dystrophy through double crossovers identified by DNA polymorphisms and by haplotype characterization in somatic cell hybrids

The analysis of 10 X-linked DNA polymorphisms (five mapping on the short arm and five on the long arm) in two Becker muscular dystrophy pedigrees has been used to localize this gene in the known sequence of DNA polymorphic markers on the X chromosome. In the first pedigree, the carrier mother, whose phase for Becker and for five informative polymorphisms is known, has transmitted a double recombinant X chromosome to one of her two affected sons. The discordance between these two affected brothers for four of the five informative polymorphisms indicates that the Becker gene is located between RC8 or D2 on one side and pDP34 on the other. In the second pedigree, where the maternal grandfather is dead and two maternal first cousins are affected, the phase of DNA polymorphic alleles has been identified in somatic cell hybrids resulting from the fusion of hamster fibroblasts with lymphocytes of the mothers and aunt of the patients. The discordance between the two first cousins for two of the four informative DNA polymorphisms is best explained by the occurrence of a single recombination in the X chromosome carried by one of them. This result further restricts the localization of the Becker gene to a region of the short arm delimited by B24 and L 1.28. Regional and fine gene mapping through the approach described in this paper should become useful in the future for X-linked as well as for autosomal genes.     

The pronatriodilatin gene is located on the distal short arm of human chromosome 1 and on mouse chromosome 4.

Atrial natriuretic factors (ANF) are polypeptides having natriuretic, diuretic, and smooth muscle-relaxing activities that are synthesized from a single larger precursor: pronatriodilatin. Chromosomal assignment of the gene coding for human pronatriodilatin was accomplished by in situ hybridization of a [3H]-labeled pronatriodilatin probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs with normal and rearranged chromosomes 1. The human pronatriodilatin gene was mapped to the distal short arm of chromosome 1, in band 1p36. Southern blot analysis of mouse X Chinese hamster somatic cell hybrids was used to assign the mouse pronatriodilatin gene to chromosome 4. This assignment adds another locus to the conserved syntenic group of homologous genes located on the distal half of the short arm of human chromosome 1 and on mouse chromosome 4.     

The human thyroglobulin gene: A polymorphic marker localized distal to C-MYC on chromosome 8 band q24

Summary We have used a cDNA clone for human phosphoglycerate kinase (PGK) to examine the chromosomal localization of three members of the human PGK gene family. Using somatic cell hybrids segregating portions of several X-autosome translocations as well as a clone panel of hybrids segregating radiation-induced fragments of the human X chromosome, we assign a PGK pseudogene to the region Xq11–Xq13, proximal to the functional X-linked PGK gene located in Xq13. In addition, using a panel of 24 somatic cell hybrids, we assign an autosomal PGK-related DNA sequence to human chromosome 19.     

Noninactivation of a selectable human X-linked gene that complements a murine temperature-sensitive cell cycle defect.

The human gene A1S9T, which complements the temperature-sensitive cell-cycle defect in the murine cell line tsA1S9 and which has previously been assigned to the X-chromosome short arm, is expressed from the inactive X chromosome in human/tsA1S9 somatic cell hybrids grown at the nonpermissive temperature. The Y chromosome cannot complement the defect; thus, unlike at least two other noninactivated X loci, A1S9T has no functional Y-linked homologue. As A1S9T is readily selectable in somatic cell hybrids with the tsA1S9 mouse line, this marker should be useful in isolating somatic cell hybrids containing inactive X chromosomes, or abnormal X's (active or inactive) retaining the short arm.     

A sensitive and dependable assay for distinguishing hamster and human X-linked steroid sulfatase activity in somatic cell hybrids

Summary A radioisotopic assay is described to distinguish between Chinese hamster and human steroid sulfatase activity in extracts prepared from hamsterxhuman somatic cell hybrids. This assay is based on different pH optima and provides a sensitive and unambiguous biochemical marker for the short arm of the human X chromosome and as well as for otherwise genetically inactivated X chromosomes in rodentxhuman hybrids.     

Assignment of Three Gene Loci (Pgk, Hgprt, G6pd) to the Long Arm of the Human X Chromosome by Somatic Cell Genetics

The intrachromosomal localization of three X-linked gene loci (PGK, HGPRT and G6PD) has been determined using a somatic cell genetic approach. A human cell line possessing an X/14 translocation was used as one parent in the formation of human/mouse hybrids. The translocation separates the human X into two parts: Xp and t(Xq14q). The data indicate that all three X-linked loci segregate with the t(Xq14q) rearrangement product thus permitting their assignment to the X chromosome's long arm. Secondary rearrangements and data from other laboratories suggest that the order of the the three markers from the centromere to the distal end of the X long arm is PGK, HGPRT, G6PD. It was also observed that NP, an autosomal locus, segregated with the t(Xq14q) chromosome. This provides strong support for the assignment of NP to 14.     

Toward a complete linkage map of the human X chromosome: regional assignment of 16 cloned single-copy DNA sequences employing a panel of somatic cell hybrids.

Closely linked restriction fragment length polymorphisms (RFLPs) are potentially useful as diagnostic markers of genetic defects, and, in principle, RFLPs can be employed to construct a complete linkage map of the human genome. On the X chromosome, linkage studies are particularly rewarding because in man more than 120 X-linked genes are known. Thus, it is probable that each X-specific RFLP will be of use as a genetic marker of one or several X-linked disorders. To facilitate the search for closely linked RFLPs, we have regionally assigned 16 cloned DNA sequences to various portions of the human X chromosome, employing a large panel of somatic cell hybrids. These probes have been used to correlate genetic and physical distances on Xp, and it can be extrapolated from these data that the number and distribution of available Xq sequences will also suffice to span the long arm of the X chromosome.     

Regional localization of DNA sequences on chromosome 21 using somatic cell hybrids.

We have used a panel of Chinese hamster X human somatic cell hybrids, each containing various portions of chromosome 21 as the only detectable human chromosome component, for regional mapping of cloned, chromosome 21-derived DNA sequences. Thirty unique and very low-repeat sequences were mapped to the short arm and three sections of the long arm. Three unique sequences map to the proximal part of the terminal band 21q22.3, and five to the distal part of this band. Some of these may represent parts of gene sequences that may be relevant to the pathogenesis of Down syndrome, as 21q22 is the area required to be present in triplicate for the full clinical picture.     

Assignment of cathepsin E (CTSE) to human chromosome region 1q31 by in situ hybridization and analysis of somatic cell hybrids

A full length cathepsin E (CTSE) cDNA clone was used to assign the corresponding gene to human chromosome region 1q31 by in situ hybridization. Southern blot analysis of DNA from three independent human x rodent somatic cell hybrids containing X;1 translocations confirmed the assignment of the CTSE gene to the distal region of the long arm of chromosome 1.     

Localization of HeLa cell tumor-suppressor gene to the long arm of chromosome II.

Cytogenetic and molecular genetic analyses of human intraspecific HeLa x fibroblast hybrids have provided evidence for the presence of a tumor-suppressor gene(s) on chromosome 11 of normal cells. In the present study, we have carried out extensive RFLP analysis of various nontumorigenic and tumorigenic hybrids with at least 50 different chromosome 11-specific probes to determine the precise location of this tumor-suppressor gene(s). Two different hybrid systems, (1) microcell hybrids derived by the transfer of a normal chromosome 11 into a tumorigenic HeLa-derived hybrid cell and (2) somatic cell hybrids derived by the fusion of the HeLa (D98OR) cells to a retinoblastoma (Y79) cell line, were particularly informative. The analysis showed that all but one of the nontumorigenic hybrid cell lines contained a complete copy of the normal chromosome 11. This variant hybrid contained a segment of the long arm but had lost the entire short arm of the chromosome. The tumorigenic microcell and somatic cell hybrids had retained the short arm of the chromosome but had lost at least the q13-23 region of the chromosome. Thus, these results showed a perfect correlation between the presence of the long arm of chromosome 11 and the suppression of the tumorigenic phenotype. We conclude therefore that the gene(s) involved in the suppression of the HeLa cell tumors is localized to the long arm (q arm) of chromosome 11.     

Human and mouse amelogenin gene loci are on the sex chromosomes

Enamel is the outermost covering of teeth and is the hardest tissue in the vertebrate body. The enamel matrix is composed of enamelin and amelogenin classes of protein. We have determined the chromosomal locations for the human and mouse amelogenin (AMEL) loci using Southern blot analyses of DNA from human, mouse, or somatic cell hybrids by hybridization to a characterized mouse amelogenin cDNA. We have determined that human AMEL sequences are located on the distal short arm of the X chromosome in the p22.1----p22.3 region and near the centromere on the Y chromosome, possibly at the proximal long arm (Yq11) region. These chromosomal assignments are consistent with the hypothesis that perturbation of the amelogenin gene is involved in X-linked types of amelogenesis imperfecta, as well as with the Y-chromosomal locations for genes that participate in regulating tooth size and shape. Unlike the locus in humans, the mouse AMEL locus appears to be assigned solely to the X chromosome. Finally, together with the data on other X and Y chromosome sequences, these data for AMEL mapping support the notion of a pericentric inversion occurring in the human Y chromosome during primate evolution.     

Localization of the ornithine aminotransferase gene and related sequences on two human chromosomes

Summary We have used a full length cDNA clone to determine the chromosomal location ofthegene encoding human ornithine aminotransferase (OAT), a mitochondrial matrix enzyme. Southern blot analysis of ScaI-digested DNA from 34 human-mouse somatic cell hybrids revealed 11 human fragments. Three fragments mapped to chromosome 10q23-10qter, confirming the previous provisional assignment of the functional gene to this autosome by analysis of OAT expression in somatic cell hybrids (O'Donnell et al. 1985). The remaining eight fragments were assigned to the X chromosome, and regionally assigned to Xp21-Xp11 by use of an X-chromosome mapping panel. These X chromosome sequences could represent pseudogenes, or related members of a multigene family. Two of the X chromosome fragments are alternate alleles of a restriction fragment length polymorphism (RFLP) making this OAT-related locus an excellent genetic marker. The RFLP may now be used to determine any possible relationship between this locus and several X-linked eye defects.     

Chromosome loss is responsible for segregation at the HPRT locus in Chinese hamster cell hybrids

The phenomenon of segregation of gene expression has been examined in intraspecific somatic cell hybrids. Specifically, segregation at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus has been studied in hybrids of Chinese hamster cell lines. The role of chromosome segregation, or other chromosomal events has been assessed by detailed comparison of karyotypes in the 6-thioguanine resistant segregants with those of the parental hybrid lines. The results clearly demonstrate that loss of an entire X chromosome is the primary event responsible for segregation at the HPRT locus, while deletion of a portion of the short arm of an X chromosome was also a frequent event. The results provide the first direct evidence for the assignment of the mapping of this locus to the distal region of the short arm. Analysis of chromosome number distributions in the hybrids and segregants suggests that in selecting chromosomal segregants one may also select for hybrid lines with reduced chromosome stability.     

Localization of a gene that escapes inactivation to the X chromosome proximal short arm: implications for X inactivation.

The process of mammalian X chromosome inactivation results in the inactivation of most, but not all, genes along one or the other of the two X chromosomes in females. On the human X chromosome, several genes have been described that "escape" inactivation and continue to be expressed from both homologues. All such previously mapped genes are located in the distal third of the short arm of the X chromosome, giving rise to the hypothesis of a region of the chromosome that remains noninactivated during development. The A1S9T gene, an X-linked locus that complements a mouse temperature-sensitive defect in DNA synthesis, escapes inactivation and has now been localized, in human-mouse somatic cell hybrids, to the proximal short arm, in Xp11.1 to Xp11.3. Thus, A1S9T lies in a region of the chromosome that is separate from the other genes known to escape inactivation and is located between other genes known to be subject to X inactivation. This finding both rules out models based on a single chromosomal region that escapes inactivation and suggests that X inactivation proceeds by a mechanism that allows considerable autonomy between different genes or regions on the chromosome.     

Integration of Ecogpt and SV40 early region sequences into human chromosome 17: a dominant selection system in whole cell and microcell human-mouse hybrids

The dominant selectable gene, Ecogpt, has been introduced, by the calcium phosphate precipitation technique, into normal human fibroblasts, along with the SV40 early region genes. In one transfectant clone, integration of these sequences into human chromosome 17 was demonstrated by the construction of human-mouse somatic cell hybrids, selected for by growth in medium containing mycophenolic acid and xanthine. A whole cell hybrid, made between the human transfectant and a mouse L cell, was used as donor of the Ecogpt-carrying human chromosome 17 to 'tribrids' growing in suspension, made by whole cell fusion between a mouse thymoma cell line, and to microcell hybrids made with a mouse teratocarcinoma cell line. Two tribrids contained karyotypically normal human chromosomes 17 and a small number of other human chromosomes, while a third tribrid had a portion of the long arm of chromosome 17 translocated to mouse as its only human genetic material. Two independent microcell hybrids contained a normal chromosome 17 and no other human chromosome on a mouse teratocarcinoma background. These experiments demonstrate the ability to construct human-mouse somatic cell hybrids using a dominant selection system. By applying this approach it should be possible to select for a wide range of different human chromosomes in whole cell and microcell hybrids. In particular, transfer of single human chromosomes to mouse teratocarcinoma cells will allow examination of developmentally regulated human gene sequences after differentiation of such hybrids.     

The gene for T11 (CD2) maps to chromosome 1 in humans and to chromosome 3 in mice

The chromosomal locations of the human and murine T11 (CD2) gene have been determined. Using recently cloned cDNA to probe Southern blots of mouse X human and Chinese hamster X mouse somatic cell hybrids, we have localized the human T11 gene to chromosome 1 and the murine T11 gene to chromosome 3. Based on previously determined blocks of homology between human chromosome 1 and mouse chromosome 3, it is suggested that the human T11 gene may lie on the short arm of chromosome 1 proximal to p221. Thus, the T11 gene is not linked to any other genes for T cell markers that have been mapped to date.