Mapping of the silver fox genome. III. Determination of the chromosomal localization of the GOT2, AK1, ALDOC, ACP1, ITPA, PGP and BLVR genes

Evidence is presented for the chromosome localization of seven silver fox genes obtained with the help of panel of fox x Chinese hamster somatic cell hybrids. Thus, the AK1, GOT2 and ALDOC are assigned to chromosome VFU2, PGP to chromosome VFU3, BLVR to chromosome VFU5, ACP1 to chromosome VFU8 and ITPA to chromosome VFU14. The genetic map of 29 fox genes is compared with those reported for man and other animals. The results obtained support and extend our previous suggestion that formation of the Canidae branch of the Carnivora phylogenetic tree was associated with great increase in the rate of reorganization of the ancestral karyotype.     

Mapping of the silver fox genes: assignments of the genes for ME1, ADK, PP, PEPA, GSR, MPI, and GOT1

Evidence is presented for the assignment of seven fox genes on the basis of the segregation data for chromosomes and enzymes of fox x Chinese hamster somatic cell hybrids. The chromosomal loci of the following enzyme genes were determined: ME1, VFU1; ADK and PP, VFU4; PEPA, VFU5; GSR, VFU7; and MPI and GOT1, VFU15. The localization of these genes now extends the fox genetic map to 22 mapped genes. Based on comparative analysis of mammalian genetic maps, karyotype evolution in Carnivora is discussed.     

Genome mapping of the silver fox. I. Determination of the chromosomal location of 8 fox genes and the search for homologous regions on fox and human chromosomes

Twenty-three silver fox-Chinese hamster somatic cell hybrids were analysed for the expression of fox enzyme loci and the segregation of fox chromosomes. This analysis made it possible to assign the gene PGD to chromosome 2, MDH2 to chromosome 3. NP to chromosome 10. APRT, ENO1, PGM1 to chromosome 12, MDH1 and IDH1 to chromosome 16. Possible use of the above-mentioned clone panel for fox gene mapping is analysed. An attempt to reveal homologous regions on fox and human chromosomes was made by comparative analysis of prometaphase fox and human chromosomes containing the homologous genes. The means and perspectives of verification of the hypothesis proposed are discussed.     

Genetic effects of chromosomal rearrangements in Chinese hamster ovary cells: expression and chromosomal assignment of TK, GALK, ACP1, ADA, and ITPA loci.

Polyethylene glycol-mediated fusion of Chinese hamster ovary (CHO) cells with mouse Cl1D cells produced interspecific somatic cell hybrids which slowly segregated CHO chromosomes. Cytogenetic and isozyme analysis of HAT- and bromodeoxyuridine-selected hybrid subclones and of members of a hybrid clone panel retaining different combinations of CHO chromosomes enabled provisional assignments of the following enzyme loci to CHO chromosomes: TK, GALK, and ACP1 to chromosome 7; TK and GALK to chromosome Z13; ACP1, ADA, and ITPA to chromosome Z8; and ADA and ITPA to chromosome Z9. These genetic markers reflect the origin of each of these Z group chromosomes and indicate the functional activity of alleles located on rearranged chromosomes. Identification of diploid electrophoretic shift mutations for ADA and ITPA was consistent with those observations. Assignment of the functional TK locus in TK+/- CHO-AT3-2 cells indicated that gene deletion may be responsible for TK hemizygosity in this subline.     

Bone morphogenetic protein: chromosomal localization of human genes for BMP1, BMP2A, and BMP3

Bone morphogenetic protein (BMP) induces endochondral bone formation in vivo. The human genes have been cloned for a group of proteins containing BMP activity (BMP1, BMP2A, and BMP3). Two of the proteins are members of the transforming growth factor-beta supergene family (BMP2A and BMP3), while BMP1 is a novel regulatory protein. Using somatic cell hybrid lines, cDNA probes were used to map BMP1 to chromosome 8, BMP2A to chromosome 20, and BMP3 to the p14-q21 region of chromosome 4. This analysis reveals that the BMP2A and BMP3 genes map to conserved regions between mouse and human, while the BMP1 gene does not. The locations of the BMP genes were found to overlap with the loci for several disorders of cartilage and bone formation.     

Genomic structure and chromosomal localization of the human hepatocyte growth factor activator inhibitor type 1 and 2 genes.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are recently discovered Kunitz-type serine protease inhibitors which can be purified and cloned from human stomach cancer cell line MKN45 as specific inhibitors against hepatocyte growth factor activator (HGFA). HAI-2 was identical with the protein originally reported as placental bikunin. Both proteins contain two Kunitz inhibitor domains (KDs), of which the first domain (KD1) is mainly responsible for the inhibitory activity against HGFA, and are expressed ubiquitously in various tissues. In this study, we cloned the genes coding for these two structurally similar proteins by screening of human genomic bacterial artificial chromosome (BAC) library and their genomic structures were compared. HAI-1 and -2 genes consist of 11 and 8 exons spanning 12 kbp and 12.5 kbp, respectively. Three exons were inserted between KD1 and KD2 of each gene, of which the middle one was the low-density lipoprotein (LDL) receptor-like domain (HAI-1) and the testis specific exon (HAI-2). Apparently homologous regions between HAI-1 and -2 were not found in 5'-flanking region and neither TATA nor CAAT box was present. The genes were mapped to chromosome 15q15 (HAI-1) and 19q13.11 (HAI-2). These results suggested that although HAI-1 and -2 genes might be derived from same ancestor gene, they acquired distinctive in vivo roles during their evolution.     

Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization.

Human Tumor Necrosis Factor and Lymphotoxin are cytotoxic proteins which have similar biological activities and share 30 percent amino acid homology. The single copy genes which encode these proteins share several structural features: each gene is approximately three kilobase pairs in length and is interrupted by three introns. In addition, these genes are closely linked and have been mapped to human chromosome 6. However, only the last exons of both genes, which code for more than 80 percent of each secreted protein, are significantly homologous (56 percent).     

Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and functional expression

N-Acetylation by hepatic arylamine N-acetyltransferase (NAT, EC 2.3.1.5) is a major route in the metabolism and detoxification of numerous drugs and foreign chemicals. NAT is the target of a common genetic polymorphism of clinical relevance in human populations. We have used our recently isolated rabbit cDNA rnat to clone three human NAT genes from human leukocyte DNA. None of the three genomic coding sequences was interrupted by introns. Two genes, designated NAT1 and NAT2, each possessed open reading frames of 870 bp. Both genes have been assigned to human chromosome 8, pter-q11. Following transfection they were transiently expressed in monkey kidney COS-1 cells. NAT1 and NAT2 gave rise to functional NAT proteins, as judged by their NAT enzyme activity with the arylamine substrate sulfamethazine. Western blots with NAT-specific antisera detected proteins of apparent molecular weight of 33 and 31 kD in NAT1- and NAT2-transfected cultures, respectively. The product of NAT2 had an identical apparent molecular weight as that of NAT detected in human liver cytosol. The deduced amino acid sequence of NAT2 also contained 6 peptide sequences which had previously been determined from tryptic peptides of the polymorphic NAT purified from human liver. These data suggest that NAT2 encodes the polymorphic NAT protein. The third gene, NATP, had multiple deleterious mutations and did not encode a functional NAT protein; it most likely represents a pseudogene.     

Chromosomal and regional localization of the genes for UMPH2, APRT, PEPD, PEPS, PSP, and PGP in mink: comparison with man and mouse

Segregation of mink biochemical markers uridine 5'-monophosphate phosphohydrolase-2 (UMPH2), adenine phosphoribosyltransferase (APRT), phosphoserine phosphatase (PSP), phosphoglycolate phosphatase (PGP), peptidases D (PEPD) and S (PEPS), as well as mink chromosomes, was investigated in a set of mink x mouse hybrid clones. The results obtained allowed us to make the following mink gene assignments: UMPH2, chromosome 8; PEPD and APRT, chromosome 7; PEPS, chromosome 6; and PSP and PGP, chromosome 14. The latter two genes are the first known markers for mink chromosome 14. For regional mapping, UMPH2 was analyzed in mouse cell clones transformed by means of mink metaphase chromosomes (Gradov et al., 1985) and also in mink x mouse hybrid clones carrying fragments of mink chromosome 8 of different sizes. Based on the data obtained, the gene for UMPH2 was assigned to the region 8pter----p26 of mink chromosome 8. The present data is compared with that previously established for man and mouse with reference to the conservation of syntenic gene groups and G-band homoeologies of chromosomes in mammals.     

Genomic organization, chromosomal localization, and independent expression of human cyclin D genes.

Murine cDNA clones for three cyclin D genes that are normally expressed during the G1 phase of the cell cycle were used to clone the cognate human genes. Bacteriophage and cosmid clones encompassing five independent genomic loci were partially sequenced and chromosomally assigned by an analysis of somatic cell hybrids containing different human chromosomes and by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes. The human cyclin D1 gene (approved gene symbol, CCND1) was assigned to chromosome band 11q13, cyclin D2 (CCND2) to chromosome band 12p13, and cyclin D3 (CCND3) to chromosome band 6p21. Pseudogenes containing sequences related to cyclin D2 and cyclin D3 mapped to chromosome bands 11q13 and 6p21, respectively. Partial nucleotide sequence analysis of exons within each gene revealed that the authentic human cyclin D genes are more related to their mouse counterparts than to each other. These genes are ubiquitously transcribed in human tumor cell lines derived from different cell lineages, but are independently and, in many cases, redundantly expressed. The complex patterns of expression of individual cyclin D genes and their evolutionary conservation across species suggest that each family member may play a distinct role in cell cycle progression.     

Methacrylate polymerization by AzoRNA: potential usefulness for chromosomal localization of genes

An azo pyrimidine nucleotide has been prepared and enzymatically attached to oligo(A) primers. The nucleotide's azo pyrimidine group has previously been shown to initiate polymerization of methacrylate esters designed to bind marker groups for visualization by microscopy. When attached to RNA molecules complementary to a chromosomal DNA segment, these nucleotides may allow localization of the DNA segment following in situ hybridization of the probe, methacrylate polymerization, and marker attachment. Since mRNA molecules of potential interest as probes bear a 3′-poly(A) tail, the modified nucleotides were added to oligo(A) primers as models. First, N⁴-ureidocytosine nucleotides were enzymatically added to ApApA, (Ap)₉A, or [5′-³²P]-(pA)₁₀, using the modified cytidine 5′-diphosphate and “primer-dependent” polynucleotide phosphorylase (M. luteus). In the case of the ApApA-primed reaction, the N⁴-ureidocytosine nucleotides in the product polynucleotide were converted into azo nucleotides by oxidation with N-bromosuccinimide. The other two primers were employed to study the time course of polynucleotide formation and to verify that primer was indeed being utilized by the enzyme. The suitability of the modified nucleotide for in situ hybridization studies was examined. Poly(N⁴-ureidocytidylic acid) was prepared from poly(C) and semicarbazide by the bisulfite-catalyzed transamination reaction. It was found that 95% of the N⁴-ureidocytosine nucleotides in this polynucleotide survive the elevated temperatures typically required for DNA:DNA denaturation and RNA:DNA annealing. When poly(N⁴-ureidocytidylic acid) was mixed with poly(I) in buffered aqueous salt solutions, no evidence for hybridization was found, so binding of the probe RNA to the denatured chromosomal DNA molecule via the modified nucleotides is not expected. Upon oxidation of poly(N⁴-ureidocytidylic acid) with N-bromosuccinimide, the azo nucleotides were formed, as judged by the appearance of a characteristic peak at approximately 350 nm in the uv-absorption spectrum of the yellow-orange product, azoRNA. The azo nucleotides in azoRNA exhibited the expected acid lability, which is known to be accompanied by 1-glyceryl methacrylate polymerization in the case of the simple azo pyrimidine. Because 1-glyceryl metharcylate bears substituent glycol groups for attaching heavy atoms or fluorescent markers, it is possible that probe RNA molecules bearing azo nucleotides may be useful for localizing low-multiplicity genes along eukaryotic chromosomes.     

Genomic organization and chromosomal localization of the TAPA-1 gene.

TAPA-1 is a 26-kDa integral membrane protein expressed on many human cell types. Antibodies against TAPA-1 induce homotypic aggregation of cells and can inhibit their growth. The murine homologue of TAPA-1 was cloned from both cDNA and genomic DNA libraries. A very high level of homology was found between human and mouse TAPA-1. The 5' untranslated region of the TAPA-1 gene resembles housekeeping gene promoters with respect to G + C content and the presence of potential Sp1 binding sites. The chromosomal localization of human and murine TAPA-1 genes was determined by Southern blot experiments using DNA from somatic cell hybrids. The genes were found to be part of a conserved syntenic group in mouse chromosome 7 and the short arm of human chromosome 11. The organization of the TAPA-1 gene and the projection of the exon boundaries on the proposed protein structure are presented.     

Mapping AK1, ACONs, and AK3 to chromosome 9 in man employing and X/9 translocation and somatic cell hybrids.

The adenylate kinase 1 (AK1), adenylate kinase3 (AK3), and aconitaseS (ACONS) genes have been assigned to chromosome 9 in man by employing an X/9 translocation segregating in man-mouse somatic cell hybrids. Segregation was controlled by taking advantage of the HAT/8-azaguanine selection-counterselection strategy directed at the X-linked HPRT locus. Assignment of AK1 to chromosome 9 has suggested the assignment of the ABO blood-group locus and the nail-patella (Np) locus to 9, since both loci are linked to AK1 by family studies.