Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neo ^r-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.     

Mapping of the thrombospondin gene to human chromosome 15 and mouse chromosome 2 by in situ hybridization

We have mapped the thrombospondin gene (THBS1) to a single locus on human chromosome 15 (band q15) and on mouse chromosome 2 (region F). Thrombospondin has been implicated in a variety of cell-matrix and cell-cell interactions. The finding of a single locus suggests that the different functions of thrombospondin are not due to a closely related family of genes. These results also confirm a region of homology between the proximal part of human chromosome 15 and region F of mouse chromosome 2.     

Assignment of the gene coding for the alpha 2-macroglobulin receptor to mouse chromosome 15 and to human chromosome 12q13-q14 by isotopic and nonisotopic in situ hybridization.

The assignment of the gene encoding the alpha 2-macroglobulin receptor (A2MR), which was first described as the low-density lipoprotein receptor-related protein, was confirmed by nonisotopic and isotopic in situ hybridizations on normal human metaphases to the region 12q13-q14. The same human cDNA, which has 95% sequence identity with the mouse A2mr, was hybridized to metaphases containing the Robertsonian translocation Rb(6;15)1Ald. The mouse A2mr gene was assigned to chromosome 15 in the region B2-D1. This locus and other loci on mouse chromosome 15 have been shown to be homologous with loci on human chromosome 12q.     

Chromosome mapping of the growth hormone receptor gene in man and mouse

Pituitary growth hormone (GH) is essential for normal growth and development in animals and GH deficiency leads to dwarfism. This hormone acts via specific high-affinity cell surface receptors found in liver and other tissues. The recent cloning and sequencing of cDNAs encoding human and rabbit GH receptors (GHR) has demonstrated that this receptor is unrelated to any previously described cell membrane receptor or growth factor receptor. We have used the cloned human GHR cDNA to map the GHR locus to the proximal short arm of human chromosome 5, region p13.1----p12, and to mouse chromosome 15 by Southern blot analysis and in situ hybridization. While human chromosome 5 carries several genes for hormone and growth factor receptors, GHR is the only growth-related gene so far mapped to the short arm. Inasmuch as GHR is the first gene with apparently homologous loci on human chromosome 5 and mouse chromosome 15, it identifies a new homologous conserved region. In humans, deficiency of GH receptor activity probably causes Laron-type dwarfism, an autosomal recessive disorder prevalent in Oriental Jews. In mice, the autosomal recessive mutation miniature (mn) is characterized by severe growth failure and early death and has been mapped to chromosome 15. Our assignment of Ghr to mouse chromosome 15 suggests this as a candidate gene for the mn mutation.     

A regulatory locus,Hdc-e,determines the response of mouse kidney histidine decarboxylase to estrogen

Levels of histidine decarboxylase (HDC; EC 4.1.1.22) activity in female mouse kidney are modulated by estrogen (administered as implanted pellets). In some inbred strains HDC activity is induced by estrogen, while in others the enzyme is repressed. Immunoprecipitation with an anti-fetal rat HDC antiserum has shown that induction and repression of HDC levels are due to changes in enzyme concentration. Segregation analysis has identified a single additively inherited regulatory locus, Hdc-e, which determines the response to estrogen. The allele Hdc-eb (C57BL/10) determines induction, and the allele Hdc-ed (DBA/2) determines repression. Preliminary evidence indicates cosegregation of Hdc-e alleles with alleles of another regulatory locus, Hdc-c (determining kidney HDC concentration), and therefore putative linkage of Hdc-e with the HDC gene complex on chromosome 2. This is the first report of a mammalian regulatory gene controlling two opposite mechanisms, induction and repression in response to a single effector.     

Assignment of the gene for neuroendocrine protein 7B2 (SGNE1 locus) to mouse chromosome region 2[E3-F3] and to human chromosome region 15q11-q15

The gene for 7B2, a protein found in the secretory granules of neural and endocrine cells (gene symbol SGNE1) was localized to the E3-F3 region of mouse chromosome 2 and to the q11-q15 region of human chromosome 15. This was determined by in situ hybridization, using a mouse 7B2 cDNA and an intronic fragment of the corresponding human gene as probes. The respective locations of SGNE1 in the two species correlate with the conservation of loci between these subregions of mouse chromosome 2 and human chromosome 15. Clinically, the human SGNE1 DNA fragment may serve as a molecular probe of this locus in both the Prader-Willi and the Angelman syndromes, which are often accompanied by submicroscopic chromosomal deletions in the 15q11-15q13 region.     

The mouse L-histidine decarboxylase gene: structure and transcriptional regulation by CpG methylation in the promoter region

To investigate the regulation of mouse L-histidine decarboxylase (HDC) gene expression, we isolated genomic DNA clones encoding HDC. Structural analysis revealed that the mouse HDC gene was composed of 12 exons, spanning approximately 24 kb. Northern blotting analysis indicated that, among the cell lines examined, a high level of HDC gene expression was restricted to mature mast cell lines and an erythroblastic cell line. The gene was induced strongly in the mouse immature mast cell line P815 after incubation in the peritoneal cavity of BDF1 mice. We observed that the promoter region was demethylated in the HDC-expressing cell lines and in induced P815 cells. Interestingly, forced demethylation by 5-azacytidine (5-azaC) treatment induced high expression of HDC mRNA in P815 cells. The activity of a mouse HDC promoter-reporter construct stably transfected in P815 cells was repressed by in vitro patch-methylation. This low promoter activity of the patch-methylated reporter construct was restored after 5-azaC treatment, which demethylated the patch-methylated promoter. These results indicate that DNA methylation state of the promoter region controls HDC gene expression.     

Autistic disorder and chromosome 15q11-q13: construction and analysis of a BAC/PAC contig

Autistic disorder (AD) is a neurodevelopmental disorder that affects approximately 2-10/10,000 individuals. Chromosome 15q11-q13 has been implicated in the genetic etiology of AD based on (1) cytogenetic abnormalities; (2) increased recombination frequency in this region in AD versus non-AD families; (3) suggested linkage with markers D15S156, D15S219, and D15S217; and (4) evidence for significant association with polymorphisms in the gamma-aminobutyric acid receptor subunit B3 gene (GABRB3). To isolate the putative 15q11-q13 candidate AD gene, a genomic contig and physical map of the approximately 1.2-Mb region from the GABA receptor gene cluster to the OCA2 locus was generated. Twenty-one bacterial artificial chromosome (BAC) clones, 32 P1-derived artificial chromosome (PAC) clones, and 2 P1 clones have been isolated using the markers D15S540, GABRB3, GABRA5, GABRG3, D15S822, and D15S217, as well as 34 novel markers developed from the end sequences of BAC/PAC clones. In contrast to previous findings, the markers D15S822 and D15S975 have been localized within the GABRG3 gene, which we have shown to be approximately 250 kb in size. NotI and numerous EagI restriction enzyme cut sites were identified in this region. The BAC/PAC genomic contig can be utilized for the study of genomic structure and the identification and characterization of genes and their methylation status in this autism candidate gene region on human chromosome 15q11-q13.     

The genes for the highly homologous Ca(2+)-binding proteins oncomodulin and parvalbumin are not linked in the human genome.

The chromosomal loci of the human parvalbumin and oncomodulin single-copy genes that encode structurally and evolutionarily closely related Ca(2+)-binding proteins were determined by somatic cell hybrid analysis. Southern blot analysis of genomic DNA from 25 human-hamster somatic cell hybrids showed that the human gene for oncomodulin resides on chromosome 7. Analysis of human-mouse hybrids selectively retaining human chromosome 7 or a portion of it allowed specific assignment of the gene locus to the p11-p13 region of chromosome 7 known to be mutated or deleted in patients with the Greig cephalopolysyndactyly syndrome. By gene dosage analysis on Southern blots, we showed that the gene for human parvalbumin maps distally to the cat eye syndrome marker D22S9 on chromosome 22q. Using somatic cell hybrids containing parts of human chromosome 22, the parvalbumin gene was sublocalized to the region 22q12-q13.1. This region contains a linkage group that maps to mouse chromosome 15, region E, and includes the SIS, ARSA, and DIA 1 genes. Our findings are consistent with the recent localization of the mouse parvalbumin gene to this region by two independent methods (C. H. Zühlke et al., 1989, Genet. Res. 54:37-43; S. Adolph et al., 1989, Cytogenet. Cell Genet. 52:177-179).     

Physical mapping of the genes for three components of the mouse DNA replication complex: polymerase alpha to the X chromosome, primase p49 subunit to chromosome 10, and primase p58 subunit to chromosome 1

DNA polymerase alpha and primase are two key enzymatic components of the eukaryotic DNA replication complex. In situ hybridization of cloned cDNAs for mouse DNA polymerase alpha and for the two subunits of mouse primase has been utilized to physically map these genes in the mouse genome. The DNA polymerase alpha gene (Pola) was mapped to the mouse X chromosome in region C-D. The gene encoding the p58 subunit of primase (Prim2) was located to mouse chromosome 1 in region A5-B and the p49 subunit gene (Prim1) was found to be on mouse chromosome 10 in the distal part of band D that is close to the telomere. Current knowledge of mouse and human conserved chromosomal regions along with the findings presented here lead to predictions of where the genes for the DNA primase subunits may be found in the human genome: the p58 subunit gene may be on human chromosome 2 and the p49 subunit gene on human chromosome 12. The mapping of Pola to region C-D of the mouse X chromosome adds a new marker in a conserved region between the mouse X chromosome and region Xp21-22.1 of the human X chromosome.     

Localization of the human gene for 230-kDal bullous pemphigoid autoantigen (BPAG1) to chromosome 6pter----q15.

Chromosome mapping of the human gene encoding the 230-kDal autoantigen of an autoimmune skin disease, bullous pemphigoid, was performed using flow-sorted human chromosomes of cells of normal karyotype and cells carrying a reciprocal translocation t(6;16)(q15;q24). The cDNA of the autoantigen hybridized with intact chromosome 6 and translocation chromosome 6p- (6pter----q15::16q24----qter). The gene (BPA230) was located to the chromosome region 6pter----q15.     

Chromosomal assignments of the genes for neuroendocrine convertase PC1 (NEC1) to human 5q15-21, neuroendocrine convertase PC2 (NEC2) to human 20p11.1-11.2, and furin (mouse 7[D1-E2] region).

The chromosomal localization of the genes coding for the pro-protein and pro-hormone convertases PC1, PC2, and Furin has been achieved by in situ hybridization. The genes for PC1 and PC2 were located on human chromosomes 5q15-21 and 20p11.1-11.2, respectively. The gene for Furin was assigned to the mouse chromosome 7D1-7E2 region. These data complete the chromosomal localization of these three convertases in both human and mouse. The results confirm the regional correspondence of the human chromosomes 15 and mouse chromosomes 7, as well as between human chromosome 20 and mouse chromosome 2. Furthermore, the identification of the NEC1 locus on human chromosome 5 and mouse chromosome 13 suggests a conservation of synthenic regions between these regions of the human and mouse genomes.     

Chromosomal assignment and genomic structure of Il15

Interleukin-15 (IL-15) is a novel cytokine whose effects on T-cell activation and proliferation are similar to those of interleukin-2 (IL-2), presumably because IL-15 utilizes the β and γ chains of the IL-2 receptor. Murine IL-15 cDNA and genomic clones were isolated and characterized. The murine Il15 gene was found to consist of eight exons spanning at least 34 kb and was localized to the central region of mouse chromosome 8 by interspecific backcross analysis. Intron positions in a partial human IL15 genomic clone were identical with positions of corresponding introns in the murine gene. The human IL15 gene was mapped to human chromosome 4q31 by fluorescence in situ hybridization.     

Chromosomal localization of seven neuronal nicotinic acetylcholine receptor subunit genes in humans.

We have determined the chromosomal location of seven human neuronal nicotinic acetylcholine receptor subunit genes by genomic Southern analysis of hamster/human somatic cell hybrid DNAs. The beta 2 subunit gene was localized to human chromosome 1, the alpha 2 and beta 3 subunit genes were localized to human chromosome 8, the alpha 3, alpha 5, and beta 4 subunit genes were localized to human chromosome 15, and the alpha 4 subunit gene was localized to human chromosome 20. Mapping of the beta 2 subunit gene to chromosome 1 establishes a syntenic group with the amylase gene locus on human chromosome 1 and mouse chromosome 3, while mapping of the alpha 3 subunit gene to chromosome 15 confirms the existence of a syntenic group with the mannose phosphate isomerase gene locus on human chromosome 15 and mouse chromosome 9.     

Monoclonal antibodies specific for human chromosome 5 obtained with a monochromosomal hybrid can be used to sort out cells containing the chromosome with a FACS

Using a human-mouse monochromosomal hybrid, BG15-6, that contains an intact human chromosome 5, we isolated four monoclonal antibodies, 2A10, 3H9, 5G9, and 6G12, as chromosome marker antibodies recognizing cell surface antigens specific for human chromosome 5. The binding patterns of these antibodies to BG15 subclones containing fragments of human chromosome 5 indicated that 2A10, 3H9, and 6G12 recognized the antigens produced by genes located on 5pterq22, and that 5G9 recognized the antigen produced by a gene located on 5q23. Cells containing human chromosome 5 were very effectively sorted in a fluorescence-activated cell sorter (FACS) using monoclonal antibody 6G12. This method for sorting cells containing human chromosome 5 or an appropriate fragment of this chromosome from among human-rodent hybrid cells should be very useful in studies on gene expression, gene cloning and gene mapping.by M. Trendelenburg     

Mapping of the c-myc, pvt-1 and immunoglobulin kappa genes in relation to the mouse plasmacytoma-associated variant (6;15) translocation breakpoint.

A variant mouse plasmacytoma (MPC)-associated translocation chromosome has arisen by pericentric inversion and exchange of the distal segments of a Robertsonian 6;15 fusion chromosome in the CAK TEPC 1198 mouse plasmacytoma, as described earlier. In situ hybridization was performed on the normal and the inverted Rb chromosomes, using myc and kappa probes. On the normal Rb chromosome, myc was in the 15 D2/3 region, whereas kappa hybridized in the 6 C2 area, as expected. On the inverted Rb chromosome, myc remains on the centrometric side of the translocation breakpoint on the chromosome 15-derived portion, whereas kappa has moved to the chromosome 6-derived segment that joined the same breakpoint on the telomeric side. Taken together with our recent demonstration that the murine c-myc locus is oriented 'head up' on chromosome 15, and with the results of Cory and co-workers concerning the relationship between the kappa gene and the associated pvt-1 region in the CAK TEPC 1198 tumor, the following conclusions can be drawn: (i) in the variant translocation of the CAK TEPC 1198 MPC, the breakage occurs 3' of the c-myc gene, as in the human Burkitt lymphoma-associated variant translocations; (ii) the pvt-1 gene on chromosome 15 is distal to the myc gene; (iii) the kappa light chain locus is oriented 'head up' on mouse chromosome 6 and faces pvt-1 and, beyond it, c-myc, in a head-to-tail configuration.