Direct assignment of citrate synthase (CS) gene to human chromosome 12 in man-mouse somatic cell hybrids

Summary A panel of twenty independently derived clones of man-mouse somatic cell hybrids isolated from fusions involving eight different parent cell combinations simultaneously analyzed for human chromosomes, citrate synthase, and a large number of other enzyme markers firmly or tentatively assigned to individual human chromosomes have provided direct evidence for a firm assignment of the structural gene coding for citrate synthase (CS) to human chromosome 12.     

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.     

Confirmation of the human cathepsin B gene (CTSB) assignment to chromosome 8

Summary Human cathepsin B gene (CTSB) has been mapped to two locations: 8p22 and 13q14. Here we confirm the chromosome 8 assignment by three independent methods: (1) analysis of human-hamster somatic cell hybrid DNA by polymerase chain reaction; (2) comparison of hybridization signals to cathepsin B in interphase nuclei of normal fibroblasts and fibroblasts with a chromosome 8 deletion; and (3) fluorescence in situ hybridization to metaphase spreads using cathepsin B cosmid clones. Our results indicate that human CTSB is located at 8p22-p23.1.     

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.     

A case report of a patient with retinoblastoma and chromosome 13q deletion: assignment of a new gene (gene for LCP1) on human chromosome 13

Summary Retinoblastoma (Rb) occurs in hereditary, non-hereditary, and chromosomal deletion forms and the locus for the Rb gene (Rb-1) is closely linked to the locus for esterase D (ESD) assigned to the chromosome 13q14.11. We describe a patient who was predicted to have Rb from the genetic analysis of the chromosome and ESD phenotype. Furthermore, the gene for lymphocyte cytosol polypeptide with molecular weight of 64,000 (LCP1: McKusick catalogue No. 15343, 1983) was assigned to chromosome 13 by deletion mapping. A 3-month-old female had many characteristics of chromosome 13q-syndrome, including dolichocephaly, epicanthus, ptosis, depressed nasal bridge, micrognathia, short webbed neck, and short fifth fingers with clinodactyly and single crease. The karyotype of the patient was 46,XX,del(13) (q14.1–q32), though both the parents had normal karyotypes. As expected, the phenotype of ESD derived from one of the parents, the father in this case, was not detected in peripheral blood lymphocytes by two-dimensional gel electrophoresis (two-DE), indicating that ESD from the father was deleted in the abnormal chromosome 13. The possibility of paternity was calculated to be 0.996 based on the data using 22 genetic markers. Bilateral retinoblastomas could be diagnosed by ophthalmologic examinations before the manifestation of any clinical signs of the tumor and immediately intensive care was taken. In addition, the phenotype of LCP1 derived from the father was not expressed in the lymphocyte proteins from the patient. These data indicate that the gene for LCP1 (LCP1) is located in the region q14.1–q32 of chromosome 13 and may be a useful genetic marker for preclinical diagnosis of Rb.     

Regional assignment of the human cell cycle control gene CDC2 to chromosome 10q21 by in situ hybridization

Summary The human homologue of the fission yeast cell control gene CDC2 has been assigned to human chromosome 10 at band q21 by in situ hybridization.     

Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation.

The tsBN2 cell line, a temperature-sensitive (ts) mutant of baby hamster kidney cell line BHK21/13, seems to possess a mutation in the gene that controls initiation of chromosome condensation. At the nonpermissive temperature (39.5 degrees C), the chromatin of tsBN2 cells is prematurely condensed, and the cells die. Using tsBN2 cells as a recipient of DNA-mediated gene transfer, we investigated a human gene that is responsible for regulation of chromosome condensation and cell proliferation. We found that the human gene complementing the tsBN2 mutation resides in the area of the 40- to 50-kilobase HindIII fragment, derived from HeLa cells. Based on this finding, we initiated cloning of a human gene complementing the tsBN2 mutation. From lambda and cosmid libraries carrying partial digests of DNA from the secondary transformants, the 41.8-kilobase HindIII fragment containing the human DNA was isolated. The cloned human DNA was conserved in ts+ transformants through primary and secondary transfections. Two cosmid clones convert the ts- phenotype of tsBN2 cells to ts+ with more than 100 times a higher efficiency, compared with cases of transfection with total human DNA. Thus, the cloned DNA fragments contain an active human gene that complements the tsBN2 mutation.     

Localization of the choline acetyltransferase (CHAT) gene to human chromosome 10

A cDNA clone encoding the complete sequence of porcine choline acetyltransferase (CHAT) isolated by S. Berrard et al. (1987, Proc. Natl. Acad. Sci. USA 84: 9280-9284) was hybridized to TaqI digests of a panel of 25 human-rodent somatic cell hybrids and to a complementary panel of 10 human-rodent hybrids in order to determine the chromosomal localization of human CHAT. To enhance the detection of the human signal, hybridization and washings were performed under low stringency conditions on membranes presaturated with sonicated DNA from parental rodent strains. All informative human fragments had the same distribution among the hybrids, mapping CHAT to a single human chromosome. CHAT was assigned to chromosome 10 because all other chromosomes were eliminated by exclusion based on the analysis of the signal segregation. This result indicates that mutation of the CHAT gene cannot be responsible for the primary defect in familial Alzheimer's disease.     

Regional assignment of the human gene for platelet-type phosphofructokinase (PFKP) to chromosome 10p: novel use of polyspecific rodent antisera to localize human enzyme genes

Human phosphofructokinase (PFK; EC 2.7.1.11) is under the control of three structural loci which encode muscle-type (M), liver-type (L), and platelet or fibroblast-type (P) subunits; human diploid fibroblasts and leukocytes express all three loci. In order to assign the human PFKP locus to a specific human chromosome, in this study, we have examined ten human X rodent somatic cell hybrids for the expression of human P subunits using a mouse anti-human P subunit-specific antiserum in an active-enzyme-immunoprecipitation technique. In nine of ten hybrids studied, the expression of the PFKP locus segregated concordantly with chromosome 10 and none other, indicating that PFKP is located on chromosome 10; the discordancy rates for all the other chromosomes were 0.2 or greater. In the one discordant hybrid, only the long arm of chromosome 10 was retained and PFKP was not expressed. Human fibroblasts from a patient with duplication of the short arm of chromosome 10 consistently exhibited PFK activity values 180% of normal. These data indicate that human PFKP is located on the short arm of chromosome 10, and that a gene dosage effect is demonstrable in fibroblasts with a duplication of 10p. The use of rodent antihuman antibody combined with immunoprecipitation aided by staphylococci-bearing protein A may find general application in mapping human enzyme genes, when human and rodent gene-products are not distinguishable by other means.     

Assignment of human pancreatic lipase gene (PNLIP) to chromosome 10q24-q26.

Human pancreatic lipase (EC 3.1.1.3) is a 56-kDa protein secreted by the acinar pancreas and is essential for the hydrolysis and absorption of long-chain triglyceride fatty acids in the intestine. In vivo, the 12-kDa protein cofactor, colipase, is required to anchor lipase to the surface of lipid micelles, counteracting the destabilizing influence of bile salts. Southern blot analysis, using a pancreatic lipase cDNA to probe DNA from mouse-human somatic cell hybrids, indicated that the pancreatic lipase gene (PNLIP) resides on human chromosome 10. In situ hybridization to human metaphase chromosomes confirmed the cell hybrid results and further localized the gene to the 10q24-qter region with the strongest peak at q26.1.     

REN: a novel,developmentally regulated gene that promotes neural cell differentiation

Expansion and fate choice of pluripotent stem cells along the neuroectodermal lineage is regulated by a number of signals, including EGF, retinoic acid, and NGF, which also control the proliferation and differentiation of central nervous system (CNS) and peripheral nervous system (PNS) neural progenitor cells. We report here the identification of a novel gene, REN, upregulated by neurogenic signals (retinoic acid, EGF, and NGF) in pluripotent embryonal stem (ES) cells and neural progenitor cell lines in association with neurotypic differentiation. Consistent with a role in neural promotion, REN overexpression induced neuronal differentiation as well as growth arrest and p27Kip1 expression in CNS and PNS neural progenitor cell lines, and its inhibition impaired retinoic acid induction of neurogenin-1 and NeuroD expression. REN expression is developmentally regulated, initially detected in the neural fold epithelium of the mouse embryo during gastrulation, and subsequently throughout the ventral neural tube, the outer layer of the ventricular encephalic neuroepithelium and in neural crest derivatives including dorsal root ganglia. We propose that REN represents a novel component of the neurogenic signaling cascade induced by retinoic acid, EGF, and NGF, and is both a marker and a regulator of neuronal differentiation.     

Possible assignment of the glyoxalase I (GLO) gene to chromosome 6 using man-mouse somatic cell hybrids

Summary A correlation between the expression or absence of human glyoxalase I and chromosome 6 (as well its markers ME₁, IPO-B, and PGM₃) was observed in man-mouse somatic cell hybrids. This segregation pattern indicates that the GLO gene is situated on chromosome 6.

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Zusammenfassung In Hybriden somatischer Zellen zwischen Maus und Mensch wurde eine Korrelation zwischen Vorhandensein bzw. Abwesenheit der menschlichen Glyoxalase I und von Chromosom 6 (sowie seinen Markern ME₁, IPO-B und PGM₃) ermittelt. Diese Korrelation spricht dafür, daß das GLO-Gen auf Chromosome 6 liegt.

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Supported by the Deutsche Forschungsgemeinschaft BE 352/8 and GR 373/6.     

Evolutionary conservation of the human homologue of the yeast cell cycle control gene cdc2 and assignment of Cd2 to chromosome 10

Summary The human homologue of the fission yeast Schizosaccharomyces pombe cell cycle control gene cdc2 has been assigned to chromosome 10. DNA hybridization reveals that this gene is highly conserved in vertebrates. The human CDC2 gene probe detects a simple two-allele polymorphism in Taq1-digested DNA.     

Somatic cell mapping of the human cyclophilin B gene (PPIB) to chromosome 15.

The polymerase chain reaction (PCR) technique was used to generate a unique probe complementary to the hydrophobic 5' end of the human cyclophilin B gene. This unique probe was hybridized to DNAs from human x hamster hybrid somatic cell lines retaining different combinations of human chromosomes. The gene was assigned to chromosome 15.     

Assignment of the gene for human sphingolipid activator protein-2 (SAP-2) to chromosome 10.

Sphingolipid activator protein-2 (SAP-2) has been found to stimulate the enzymatic hydrolysis of glucosylceramide, galactosylceramide, and sphingomyelin. When human skin fibroblast extracts were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by electroblotting and immunochemical staining using monospecific antibodies against SAP-2, two or three major bands with estimated mol. wts. of 9,000-10,000 were found. These antibodies did not crossreact with purified SAP-1, another activating protein, or with extracts of CHO-K1 cells. A series of 22 human/Chinese hamster ovary cell hybrids containing different human chromosomes were examined by this method. All eight hybrid clones containing human chromosome 10 were found to have crossreacting protein in this region. Other chromosomes could be excluded by this method. From these results, we conclude that the gene coding for human SAP-2 is located on chromosome 10.     

Confirmation of the assignment of the gene for galactose-1-phosphate uridylyltransferase (E.C. 2.7.7.12) to human chromosome 9.

The gene for galactose-1-phosphate uridylyltransferase (GALT) has previously been assigned to human chromosomes 2, 3, and 9. We have studied a further series of human-mouse hybrids and are able to confirm that the human gene for GALT is located on human chromosome 9.