Human-rodent Somatic Cell Hybrids Research Articles

Molecular Characterization of the 5′-Flanking Region of Human Genomic ETA Gene

A clone containing the promoter, the exon 1 and exon 2 of human genomic ETA gene was isolated and sequenced. The transcription initiation site and TATA box were identified at 528 bp upstream of the initiation codon and 24 bp upstream of the transcription initiation site, respectively. The 534 bp fragment containing 5′-flanking sequence and most of the exon 1 of the human ETA gene showed a promoter activity corresponding to 55% of SV40 early promoter activity when placed upstream of the luciferase gene and transfected into CHO cells. Thus, the TATA box which consists of the TAAAAA sequence is functional for transcription of the human ETA gene. The coding region of the human ETA receptor, therefore, starts from exon 2 which contained the first and the second transmembrane regions. The human ETA gene was demonstrated to be a single pair of copy by Southern blot analysis, and it was localized in chromosome 4 by analysis with a panel of human-rodent somatic cell hybrid lines.

Localization of the hydroxyindole-O-methyltransferase gene to the pseudoautosomal region: implications for mapping of psychiatric disorders.

The human hydroxyindole-O-methyltransferase (HIOMT) gene has been mapped to the X chromosome using cDNA probes from the 3' and 5' regions of a human cDNA clone. Southern blot analysis of a panel of human-rodent somatic cell hybrid DNAs revealed that the gene was localized to the short arm of chromosome X, and most probably the pseudoautosomal region of the human X (Xp22.3) and Y (Yp11.3) chromosomes. Several multiallelic restriction fragment length polymorphisms were detected at this locus allowing further localization of the gene by two-point and multipoint linkage analysis in the 40 CEPH families. These results confirmed the pseudoautosomal localization of the HIOMT gene and allowed ordering of the gene in close proximity to DXYS17 at a position about 600-800 kb from the pseudoautosomal boundary and about 1800 to 2000 kb from the telomere. It will be possible to readily identify or exclude the involvement of this gene in genetic diseases by linkage analysis of the disease locus with the high frequency multiallelic polymorphisms at this locus.

A human histone H2B.1 variant gene, located on chromosome 1, utilizes alternative 3' end processing.

A variant human H2B histone gene (GL105), previously shown to encode a 2300 nt replication independent mRNA, has been cloned. We demonstrate this gene expresses alternative mRNAs regulated differentially during the HeLa S3 cell cycle. The H2B-Gl105 gene encodes both a 500 nt cell cycle dependent mRNA and a 2300 nt constitutively expressed mRNA. The 3' end of the cell cycle regulated mRNA terminates immediately following the region of hyphenated dyad symmetry typical of most histone mRNAs, whereas the constitutively expressed mRNA has a 1798 nt non-translated trailer that contains the same region of hyphenated dyad symmetry but is polyadenylated. The cap site for the H2B-GL105 mRNAs is located 42 nt upstream of the protein coding region. The H2B-GL105 histone gene was localized to chromosome region 1q21-1q23 by chromosomal in situ hybridization and by analysis of rodent-human somatic cell hybrids using an H2B-GL105 specific probe. The H2B-GL105 gene is paired with a functional H2A histone gene and this H2A/H2B gene pair is separated by a bidirectionally transcribed intergenic promoter region containing consensus TATA and CCAAT boxes and an OTF-1 element. These results demonstrate that cell cycle regulated and constitutively expressed histone mRNAs can be encoded by the same gene, and indicate that alternative 3' end processing may be an important mechanism for regulation of histone mRNA. Such control further increases the versatility by which cells can modulate the synthesis of replication-dependent as well as variant histone proteins during the cell cycle and at the onset of differentiation.

Localization of the human gene for μ-crystallin to chromosome 16p

The mapping of the human [mu]-crystallin gene was accomplished using polymerase chain reaction (PCR) amplification of a sequence tagged site (STS) in somatic cell hybrids. Two oligonucleotides from the 3[prime]-untranslated region were synthesized and used as primers for the amplification of a 260-bp DNA sequence from genomic DNA. For localization to a specific chromosome, a commercially available panel of human-rodent somatic cell hybrids, panel 2 from NIGMS (Camden, NJ), was used. Amplification of the appropriate size STS was observed only in somatic cell hybrid GM/NA10567, which contained human chromosome 16 as the only human chromosomal material. To further define the mapping position of the [mu]-crystallin gene on human chromosome 16, DNA from another panel of mouse-human somatic cell hybrids containing portions of human chromosome 16 was used. After PCR amplification the results were consistent with localization of the [mu]-crystallin gene on the short arm of chromosome 16 between breakpoints from hybride CY175 and CY145(D)-CY13-CY123 on chromosomal bands p13.11-p12.3. 5 refs., 1 fig.

Rapid generation of region-specific genomic clones by chromosome microdissection: Isolation of DNA from a region frequently deleted in malignant melanoma

Malignant melanoma is frequently characterized by the deletion of the long arm of chromosome 6 (usually encompassing 6q16-q21). In an effort to saturate this region with DNA markers, microdissection and molecular cloning of DNA from banded human metaphases recent development of a novel chromosome microdissection scheme that omits microchemical manipulation of DNA. Microdissection was targeted on band 6q21. Direct PCR amplification of dissected DNA was first used as a probe in chromosomal in situ hybridization of normal metaphases to confirm the specificity of material excised for cloning. A genomic library of 20,000 clones, which is highly enriched for sequences encompassing 6q21, was then constructed. Clones from this library have been mapped against a human-rodent somatic cell hybrid mapping panel that divides chromosome 6 into seven regions, confirming the localization of probes within the target region. Direct PCR amplification of DNA excised by microdissection greatly simplifies and facilitates this chromosome band-specific cloning strategy. The isolation of microclones from this region of chromosome 6 should assist in establishing a physical map of the melanoma deletion region.

A bone morphogenetic protein subfamily: Chromosomal localization of human genes for BMP5, BMP6, and BMP7

Bone morphogenetic proteins (BMPs) were originally identified by the ability of a demineralized bone extract to induce endochondral osteogenesis in vivo. Seven BMP cDNAs (BMP1 through BMP7) have been recovered through molecular cloning. Recombinant protein products from six of these clones (BMP2 through BMP7) are members of the transforming growth factor beta (TGF-beta) superfamily of regulatory molecules. Based upon a high degree of amino acid sequence homology, BMP5, BMP6, and BMP7 constitute a subfamily within the BMPs. Using human-rodent somatic cell hybrid lines and cDNA probes, we mapped the three members of this subfamily of genes to the human chromosomes. BMP5 and BMP6 are syntenic on human chromosome 6, while BMP7 is syntenic with previously localized BMP2 on human chromosome 20. This analysis reveals that BMP6 maps to a conserved region between the mouse and human genomes. Sequence analysis suggests that the Drosophila 60A gene is the dipteran homolog of this BMP subfamily and may provide clues to the physiologic functions of the products of these genes in human biology.

Isolation, mapping, and characterization of two cDNA clones expressed in the cerebellum

In an effort to characterize genes expressed in the cerebellum, we have isolated two cDNA clones, H11B (D16S286) and 507 (D5S344), that hybridized to a cerebellar cDNA probe. Using a panel of human-rodent somatic cell hybrids, cDNA clone H11B was mapped to human chromosome 16, and clone 507 was mapped to human chromosome 5. TaqI RFLPs were identified with both clones and were used for linkage analysis in the CEPH families. D16S286 was tightly linked to several markers near chromosome 16p13, and D5S344 was tightly linked to several markers on chromosome 5q. Sequence tagged sites or expressed sequence tags were generated from the 3' untranslated regions of both cDNA clones.

Neural retina-specific leucine zipper gene NRL (D14S46E) maps to human chromosome 14q11.1-q11.2

The product of a neural retina-specific gene, NRL, belongs to the "leucine zipper" family of DNA-binding proteins and has a strong similarity to the v-maf oncogene product. The NRL gene maps to human chromosome 14 by Southern blot analysis of genomic DNA from a human-rodent somatic cell hybrid panel. In situ hybridization to metaphase chromosomes has further sublocalized the gene to the region 14q11.1-q11.2. D14S46E has now been assigned to the NRL gene. Because of its specific pattern of expression, NRL is a candidate gene for retinal diseases.

Determination of the nucleotide sequence and chromosomal localization of the ATP2B2 Gene Encoding Human Ca 2+-Pumping ATPase Isoform PMCA2

The plasma membrane Ca(2+)-pumping ATPase (Ca(2+)-ATPase) is responsible for maintaining calcium homeostasis in eukaryotic cells. The Ca(2+)-ATPase is a family of pumps that are encoded by at least four genes. A cDNA for the human version of Ca(2+)-ATPase isoform PMCA2 was isolated and characterized. Comparison of the human and rat cDNA sequences showed that they were 95% homologous in the coding domain, and this homology was reflected in the deduced protein sequence where greater than 98% homology between the human and rat sequences was found. The amino acid differences that were found were almost all conservative. The PMCA2 cDNA was used to probe Southern blots of human-rodent somatic cell hybrid DNAs; the results indicated that the human PMCA2 gene was located on chromosome 3.

The human loricrin gene.

Loricrin is the major protein component of the cornified cell envelope of terminally differentiated mammalian epidermal (stratum corneum) cells. Using a specific human cDNA clone, we have isolated and characterized the human loricrin gene. We show that it has a very simple structure of a single intron of 1188 base pairs (bp) in the 5'-untranslated region; there are no introns in coding sequences. By use of rodent-human somatic cell hybrids, followed by in situ hybridization with a biotin-labeled genomic DNA clone, the single-copy gene maps to chromosome location 1q21. Polymerase chain reaction analyses of genomic DNAs from different individuals show that human loricrin consists of two allelic size variants, due to sequence variations in its second glycine loop domain, and these variants segregate in the human population by normal Mendelian mechanisms. Furthermore, there are multiple sequence variants within these two size class alleles due to various deletions of 12 bp (4 amino acids) in the major loop of this glycine loop domain. By use of a specific loricrin antibody, we show by immunogold electron microscopy that loricrin initially appears in the granular layer of human epidermis and forms composite keratohyalin granules with profilaggrin, but localizes to the cell periphery (cell envelope) of fully differentiated stratum corneum cells.

In situ mapping of the gene coding for a leucine zipper DNA binding protein (CDR62) to 16p12–16p13.1

A cDNA clone encoding the major antigen (CDR62) associated with the antibody-induced paraneoplastic cerebellar degeneration has been used to identify the chromosomal location of the corresponding structural gene(s) by screening for its retention in a panel of rodent-human somatic cell hybrids. Having established the synteny of the gene with the autosome 16, we proceeded to its precise subregional mapping by in situ fluorescence hybridization with a recombinant lambda phage containing the genomic region homologous to the cDNA gene. The latter studies pin-pointed the chromosomal localization of the structural CDR62 gene within the interval 16p12–16p13.1.

Assignment of the gene for human spasmolytic protein (hSP/SML1) to chromosome 21.

A human cDNA corresponding to the porcine pancreatic spasmolytic protein (PSP) was isolated, and the recombinant clone was originally termed hSP for human spasmolytic protein. Later, the term SML1 for spasmolysin was suggested for the human gene. This protein shows a remarkable sequence homology to pS2, a protein coded by an estrogen-induced gene isolated from the breast carcinoma cell line MCF-7. Although, at the DNA level, the gene sequences pS2 and hSP/SML1 display insufficient homology for cross-hybridization, their expression in tumor cells occurs with remarkable coordination. The human pS2 gene sequence has been assigned to chromosome 21, and we have therefore attempted to map the hSP/SML1 gene by using cDNA and Southern blotting of genomic DNAs from a panel of human-rodent somatic cell hybrids carrying different complements of human chromosomes. Interestingly, the hSP/SML1 gene is also localized on chromosome 21.

An unusual expression of a squamous cell marker, small proline-rich protein gene, in tracheobronchial epithelium: differential regulation and gene mapping.

An unusual expression of a putative squamous cell marker, small proline-rich protein (spr1), in mucociliary epithelial cells of conducting airways was demonstrated in a serum-free culture system. A cDNA clone was isolated from the cDNA library of monkey tracheobronchial epithelial (TBE) cells by differential hybridization. This cDNA clone, MT5, exhibited 98% homology to a DNA sequence obtained from human keratinocytes treated with either UV light or phorbol esters (T. Kartasova et al., 1988, Mol. Cell. Biol. 8:2195-2230). The predicted peptide of MT5 is unusual for its high content of proline (29%), glutamine (18%), and cysteine (9%) and its repeated PKVPEPC units. The level of spr1 mRNA in cultured cells was inhibited more than 90% by vitamin A. In contrast, phorbol 12-myristate 13-acetate (PMA) stimulated the level of spr1 mRNA by 3- to 8-fold. This differential regulation coincided with the effects of these chemicals on the cornification of cultured TBE cells. Using MT5 as a probe, we have localized the tracheal spr1 gene on the human chromosome 1 by a Southern blot analysis using a panel of human-rodent somatic cell hybrid DNAs. The gene was further sublocalized to bands q22-23 by in situ hybridization.

Banding profiles of LTR of human endogenous retrovirus HERV-A in 24 chromosomes in somatic cell hybrids

The human genome carries multiple copies of sequences related to endogenous retroviral genomes. We investigated the distribution of one of these sequences, HERV-A, in 24 human chromosomes by Southern analyses using DNAs from flowsorted chromosomes or rodent cells carrying a single human chromosome. The results showed that HERV-A is distributed among all human chromosomes and that each chromosome has a specific Southern blot profile. The chromosome-specific pattern did not show significant polymorphism, except in a few cases, when the same chromosome obtained from different individuals was compared. These chromosome-specific Southern hybridization profiles may be useful for chromosome karyotyping. This would allow the integrity of human chromosomes in human-rodent somatic cell hybrids to be monitored without using conventional cytogenetic methods.

Molecular cloning and chromosomal assignment of human calbindin-D9K

Human calbindin-D9k, the vitamin D-dependent calcium binding protein, has been cloned and sequenced following initial amplification of intestinal cDNA sequences by the polymerase chain reaction using mixed oligonucleotide primers. The derived amino acid sequence of 79 residues has a calculated molecular weight of 9015 and is 89% homologous with the bovine and porcine sequences. Probing DNA from human-rodent somatic cell hybrids mapped human calbindin-D9k to chromosome Xp. A single abundant mRNA transcript was detectable in proximal small intestine but not in kidney, uterus or placenta.

Chromosomal assignment of human phenol and bilirubin UDP-glucuronosyltransferase genes (UGT1A-subfamily).

DNA probes were prepared from the 5'-terminal portion of four cDNA clones encoding human phenol and bilirubin UDP-glucuronosyltransferases (UGTs). An additional sequence common to all four clones was isolated from the 3'-terminal portion of one of the clones (UGT1A1). The four specific and the one common DNA sequences were used as probes on a panel of 16 human--rodent somatic cell hybrid DNAs by Southern-blot analysis. The results obtained indicate that all four cDNA clones are encoded by gene(s) located on human chromosome 2.

Chromosomal assignment of 46 brain cDNAs

Expressed sequence tags (ESTs) have been obtained from several hundred brain cDNAs as an initial effort to characterize expressed brain genes. These ESTs will become tools for human genome mapping and they will also provide candidate causative genes for inherited disorders affecting the central nervous system. We have developed a procedure for the rapid chromosomal assignment of these ESTs: cDNA sequences are first analyzed by a computer program to determine regions likely not to be interrupted by introns in the genomic DNA. A pair of oligonucleotide primers is then designed to amplify this region by the polymerase chain reaction using DNA template from human-rodent somatic cell hybrid chromosomal panels. The chromosomal assignment of the cDNA is determined by studying the segregation of the amplified products in these panels. In this paper we describe the mappig of 46 brain ESTs, as well as observations on the amplification of rodent sequences.

Assignment of the gene encoding DNA ligase I to human chromosome 19q13.2–13.3

The gene encoding DNA ligase I has been mapped on human chromosome 19 by analysis of rodent-human somatic cell hybrids informative for this chromosome and by two-color fluorescence in situ hybridization. The DNA ligase I gene ( LIG1) is localized to 19q13.2–13.3 and is distal to ERCC1, the most telomeric of three DNA repair genes on this chromosome.

The NM23 gene maps to human chromosome band 17q22 and shows a restriction fragment length polymorphism with BglII.

The NM23-Hl gene is a putative tumor suppressor gene that may be important in the metastasic process. Recent genetic and immunological data indicate that the NM23-Hl gene encodes a protein with nucleoside diphosphate (NDP) kinase activity. The mapping of NM23-Hl by panels of rodent-human somatic cell hybrids and in situ hybridization showed that the gene is located in human chromosome band 17q22. A two-allele polymorphism with BglII was demonstrated.

Mapping of the human gene for the human immunodeficiency virus type 1 enhancer binding protein HIV-EP2 to chromosome 6q23–q24

The human gene encoding the human immunodeficiency virus type 1 enhancer binding protein HIV-EP2 has been isolated. Using Southern analysis of human-rodent somatic cell hybrid DNA with a human HIV-EP2-specific cDNA probe, the HIV-EP2 gene was assigned to chromosome 6. The gene was further localized to the region 6q23–24 by fluorescence in situ hybridization.