Cloning of the human and mouse type X collagen genes and mapping of the mouse type X collagen gene to chromosome 10.

Type X collagen, a homotrimer of alpha 1 (X) polypeptide chains, is specifically expressed by hypertrophic chondrocytes in regions of cartilage undergoing endochondral ossification. We have previously described the isolation of a small fragment of the human type X collagen gene (COL10A1) and its localization to the q21-q22 region of human chromosome 6 [Apte, S., Mattei, M.-G. & Olsen, B. R. (1991) FEBS Lett. 282, 393-396]. Using this fragment as a probe to screen genomic libraries, we report here the isolation of human and mouse genomic clones which contain the major part of the human and mouse type X collagen genes. In both species, the 14-kb genomic clones which were isolated contain a long open reading frame (greater than 2000 bp in length) which codes for the entire C-terminal non-collagenous (NC1) domain, the entire collagenous (COL) domain and part of the N-terminal non-collagenous (NC2) domain of the alpha 1(X) collagen chain. The human genomic clone contains the major part of the COL10A1 gene, in addition to the region we have previously cloned, and is highly similar to the corresponding portions of the mouse genomic clone (84.5% similarity at the nucleotide level, and 86.1% at the level of the conceptual translation product). The identification of the mouse genomic clone as the alpha 1(X) collagen gene (Col10a1) was confirmed by in situ hybridization of a fragment of the mouse genomic clone to sections from newborn mice. Hybridization was restricted to the hypertrophic chondrocytes of developing chondroepiphyses, being absent in small chondrocytes and in other tissues. Using interspecific backcross analysis, the locus for the mouse alpha 1 (X) collagen gene was assigned to chromosome 10. The cloning and chromosomal mapping of the human and mouse alpha 1 (X) collagen genes now permit the investigation of the possible role of type X collagen gene defects in the genesis of chondrodysplasias in both species and provide data essential for the generation of transgenic mice deficient in type X collagen.     

Gyrate atrophy of the choroid and retina: assignment of the ornithine aminotransferase structural gene to human chromosome 10 and mouse chromosome 7.

Gyrate atrophy of the choroid and retina is an autosomal recessive, blinding human disease caused by a deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT). Since human OAT cDNA hybridizes to DNA sequences on both human chromosomes 10 and X, a locus coding for OAT enzyme activity may be present on one or both of these human chromosomes. We have used a series of mouse-human somatic cell hybrids, in combination with starch gel electrophoresis and a histochemical stain for OAT enzyme activity, to assign the structural gene for OAT to human chromosome 10. Our results suggest that the human X chromosome does not contain a locus coding for OAT enzyme activity. In addition, we have used a panel of Chinese hamster-mouse hybrids to assign the murine Oat structural gene to mouse chromosome 7. Our findings, combined with recent molecular studies, indicate that human OAT probes specific for chromosome 10 will be useful for the diagnosis and genetic counseling of individuals at risk for gyrate atrophy.     

Gene linkage analysis in the mouse by somatic cell hybridization: assignment of adenine phosphoribosyltransferase to chromosome 8 and alpha-galactosidase to the X chromosome.

Somatic cell hybridization techniques were applied to gene linkage analysis in the laboratory mouse. Cells of an established line of Chinese hamster lung fibroblasts were fused with mouse embryo fibroblasts and with mouse peritoneal macrophages obtained from different inbred strains. From 3 hybridization experiments, 123 primary and secondary clones were isolated in HAT selective medium and 24 were back-selected in 8-azaguanine. Hybrid clones were characterized for the expression of 16 murine isozymes by starch, acrylamide, and Cellogel electrophoresis, and on the basis of segregation data, 3 syntenic associations could be made. Malate oxidoreductase decarboxylating (MOD) and mannose phosphate isomerase (MPI) segregated concordantly, confirming an established linkage relationship; adenine phosphoribosyltransferase (APRT) segregated concordantly with glutathione reductase (GR) which is known to be on chromosome 8; alpha-galactosidase was observed to be syntenic with hypoxanthine phosphoribosyltransferase (HPRT), and X-linked enzyme. All other isozymes examined segregated independently of one another.     

Bioautographic visualization of aminoacylase-1: assignment of the structural gene ACY-1 to chromosome 3 in man.

A bioautographic assay was developed for the visualization of aminoacylase-1 (N-acylamino acid aminohydrolase, ACY-1; EC 3.5.1.14) after zone electrophoresis. Bioautography and species differences in electrophoretic mobility of ACY-1 made it possible to investigate the chromosome assignment of the gene for human ACY-1 using human--mouse somatic cell hybrids. Human ACY-1 segregated concordantly with beta-galactosidase-A (beta GALA; EC 3.2.1.23) but showed discordant segregation with 32 other markers representing 23 linkage groups. The beta GALA gene has been previously assigned to chromosome 3. From this evidence and confirming chromosome analyses, ACY-1 has been assigned to chromosome 3. A genetic polymorphism in the electrophoretic mobility of ACY was observed in mouse strains, demonstrating that this enzyme can be mapped in genetic crosses of Mus musculus.     

Number and size of human X chromosome fragments transferred to mouse cells by chromosome-mediated gene transfer.

Labeled probes of unique-sequence human X chromosomal deoxyribonucleic acid, prepared by two different procedures, were used to measure the amount of human X chromosomal deoxyribonucleic acid in 12 mouse cell lines expressing human hypoxanthine phosphoribosyltransferase after chromosome-mediated gene transfer. The amount of X chromosomal deoxyribonucleic acid detected by this procedure ranged from undetectable levels in the three stable transformants and some unstable transformants examined to about 20% of the human X chromosome in two unstable transformants. Reassociation kinetics of the X chromosomal probe with deoxyribonucleic acid from the two unstable transformants containing 15 to 20% of the human X chromosome indicate that a single copy of these sequences is present. In one of these lines, the X chromosomal sequences exist as multiple fragments which were not concordantly segregated when the cells were selected for loss of hprt.     

Bipartite structure of the inactive mouse X chromosome

BackgroundIn mammals, one of the female X chromosomes and all imprinted genes are expressed exclusively from a single allele in somatic cells. To evaluate structural changes associated with allelic silencing, we have applied a recently developed Hi-C assay that uses DNase I for chromatin fragmentation to mouse F1 hybrid systems.ResultsWe find radically different conformations for the two female mouse X chromosomes. The inactive X has two superdomains of frequent intrachromosomal contacts separated by a boundary region. Comparison with the recently reported two-superdomain structure of the human inactive X shows that the genomic content of the superdomains differs between species, but part of the boundary region is conserved and located near the Dxz4/DXZ4 locus. In mouse, the boundary region also contains a minisatellite, Ds-TR, and both Dxz4 and Ds-TR appear to be anchored to the nucleolus. Genes that escape X inactivation do not cluster but are located near the periphery of the 3D structure, as are regions enriched in CTCF or RNA polymerase. Fewer short-range intrachromosomal contacts are detected for the inactive alleles of genes subject to X inactivation compared with the active alleles and with genes that escape X inactivation. This pattern is also evident for imprinted genes, in which more chromatin contacts are detected for the expressed allele.ConclusionsBy applying a novel Hi-C method to map allelic chromatin contacts, we discover a specific bipartite organization of the mouse inactive X chromosome that probably plays an important role in maintenance of gene silencing.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0728-8) contains supplementary material, which is available to authorized users.     

The human chromogranin A gene: chromosome assignment and RFLP analysis.

Chromogranin A/secretory protein I (CgA) is a glycoprotein that is stored and released along with peptide hormones and neurotransmitters from several tissues, although its exact function is not known. A cDNA (gene symbol CHGA) clone was used as a probe in Southern blot analyses of human-rodent somatic cell hybrid DNAs. Discordancy analysis allowed confirmation of the assignment of the gene to chromosome 14. These results were extended using in situ chromosome hybridization, and a signal was found at 14q32. BglII digestion of genomic DNA from 28 unrelated Caucasian individuals probed with CHGA detected a two-allele RFLP with allelic frequencies of .34 and .66.     

Human delta-aminolevulinate synthase: assignment of the housekeeping gene to 3p21 and the erythroid-specific gene to the X chromosome

delta-Aminolevulinate synthase (ALAS) catalyzes the first committed step of heme biosynthesis. Previous studies suggested that there were erythroid and nonerythroid ALAS isozymes. We have isolated cDNAs encoding the ubiquitously expressed housekeeping ALAS isozyme and a related, but distinct, erythroid-specific isozyme. Using these different cDNAs, the human ALAS housekeeping gene (ALAS1) and the human erythroid-specific (ALAS2) gene have been localized to chromosomes 3p21 and X, respectively, by somatic cell hybrid and in situ hybridization techniques. The ALAS1 gene was concordant with chromosome 3 in all 26 human fibroblast/murine(RAG) somatic cell hybrid clones analyzed and was discordant with all other chromosomes in at least 6 of 26 clones. The regional localization of ALAS1 to 3p21 was accomplished by in situ hybridization using the 125I-labeled human ALAS1 cDNA. Of the 43 grains observed over chromosome 3, 63% were localized to the region 3p21. The gene encoding ALAS2 was assigned by examination of a DNA panel of 30 somatic cell hybrid lines hybridized with the ALAS2 cDNA. The ALAS2 gene segregated with the human X chromosome in all 30 hybrid cell lines analyzed and was discordant with all other chromosomes in at least 8 of the 30 hybrids. These results confirm the existence of two independent, but related, genes encoding human ALAS. Furthermore, the mapping of the ALAS2 gene to the X chromosome and the observed reduction in ALAS activity in X-linked sideroblastic anemia suggest that this disorder may be due to a mutation in the erythroid-specific gene.     

Species-specific monoclonal antibodies in the assignment of the gene for human fibronectin to chromosome 2.

Using three different species-specific monoclonal antibodies we have studied, in human-mouse and human-hamster somatic cell hybrids, the correlation between the presence of different human chromosomes and the ability to release human fibronectin into the tissue culture medium. Presence of human fibronectin was determined by an affinity-radioimmunoassay. In addition, tissue culture media of the different hybrids were separated on SDS-polyacrylamide gels, the proteins were blotted onto a nitrocellulose sheet and human fibronectin visualized by an immunoenzymatic technique. Karyology and determination of isoenzyme markers of specific human chromosomes show that the ability to produce human fibronectin segregated with the presence of human chromosome 2.     

Localization of the human X-linked gene for chronic granulomatous disease to the mouse X chromosome: implications for X-chromosome evolution

The gene encoded at the human X-linked chronic granulomatous disease locus (cytochrome b245 beta subunit) has been mapped to the mouse X chromosome using an interspecific Mus domesticus x M. spretus cross. The localization of this gene provides detailed information on one of the proposed ancestral breakpoints that account for the divergent evolution of the mouse and human X chromosomes.