Human-rodent Somatic Cell Hybrids Research Articles

Cytochrome P450 2D6

The cytochrome P450 2D6 (CYP2D6) is an enzyme of great historical importance for pharmacogenetics and is now thought to be involved in the metabolism of up to 25% of the drugs that are in common use in the clinic [1]. Several years before the gene was cloned, researchers observed that Caucasian population responded in a bimodal pattern to certain drugs such as debrisoquine and sparteine [2,3], with most patients exhibiting ‘normal’ pharmacokinetics, whereas others seemed to have great difficulty in metabolizing debrisoquine or sparteine. Debrisoquine and sparteine became examples of so-called probe drugs, and were used to phenotype patients [4]. This finding led researchers to conclude that there were common polymorphisms in an as yet unidentified metabolic gene that contributed to the variable pharmacokinetics of these drugs. The protein responsible for the altered metabolism was later purified from human liver microsomes by Distlerath et al. [5]. The gene encoding this protein was initially localized to chromosome 22 by Eichelbaum et al. [6]. The cDNA was cloned by Gonzalez et al. [7,8] from human liver cDNA libraries using an antibody against the rat ortholog. The deduced human protein revealed 73% sequence similarity with the rat protein and by the use of human–rodent somatic cell hybrids the gene was localized to human chromosome 22 [8], confirming the earlier study [6]. This gene came to be called CYP2D6, and is part of the cytochrome P450 gene family – a group of enzymes that is responsible for phase I metabolism and elimination of numerous endogenous substrates and a diverse array of drugs. Among the drug-metabolizing cytochrome P450s, CYP2D6 is the only noninducible enzyme, which results in a large contribution of genetic variation to the interindividual variation in enzyme activity [1]. CYP2D6 is highly polymorphic, with over 90 known allelic variants [9]. A selection of these variants and haplotypes is described in this summary, and the full list of all named alleles can be found at: http://www.cypalleles.ki.se/cyp2d6.htm.

C16orf5, a novel proline-rich gene at 16p13.3, is highly expressed in the brain.

A novel gene has been characterized, designated C16orf5, with an unusually high content of proline residues (40% over 104 residues) at the N-terminus of the protein. The C-terminus of the protein is also cysteine rich with 14 cysteine residues present. Analysis using Northern and dot blots showed that the highest expression of this gene is in the brain. The gene was located on chromosome 16 at band p13.3 by FISH to metaphase chromosomes. Southern blot analysis with a human-rodent somatic cell hybrid panel showed a location between the somatic hybrid breakpoints 23HA and CY196. This gene comprises at least four exons and an open reading frame of 786 bp encoding a predicted protein of 261 amino acids. Analysis of this protein using PSORTII predicted a nuclear localization.

Chromosomal localization of three human poly(A)-binding protein genes and four related pseudogenes.

In humans, the poly(A)-binding proteins (PABPs) comprise a small nuclear isoform and a conserved gene family that displays at least three functional proteins: PABP1, inducible PABP (iPABP), and PABP3, plus four pseudogenes (1, 2, 3, and PABP4). In situ hybridization of PABP3 cDNA as the probe on metaphasic chromosomes have revealed five possible loci for this gene family at 2q21-q22, 13q11-q12, 12q13.3-q15, 8q22, and 3q24-q25. Amplifications of specific DNA fragments from a human-rodent somatic cell hybrid panel have allowed us to associate PABP1 and PABP3 with 8q22 and 13q11-q12, respectively. The iPABP gene has been assigned to chromosome 1. This result, compared with radiation hybrid database information, strengthens the location of this gene to 1p32-p36. The pseudogenes PABP4, 1, and 2 have been assigned to chromosomes 15, 4, and 14, respectively. Three loci detected on chromosome spreads are not associated with any amplified fragment. They might represent other related PABP genes not yet identified.

Generation and chromosome mapping of expressed sequence tags (ESTs) from a human infant thymus.

In an effort to identify novel genes that are expressed differentially in an infant thymus, we constructed an oligo-d(T) primed cDNA library from a human infant thymus followed by single-run partial sequencing to generate expressed sequence tags (ESTs). Characterization of more than 1400 sequences enabled us to convert human thymus transcripts into 1223 useful ESTs. These ESTs consisted of 613 (50.1%) showing homology to known human genes, 51 (4.2%) matching to genes from other species, 289 (23.6%) matching ESTs of unknown functions, and 182 (14.9%) being novel transcripts. The expression profile of an infant thymus features a high number of genes related to cell division-DNA synthesis and gene-protein expression, indicating the active growth stage of an infant thymus. To identify the chromosomal localization of 43 thymus ESTs, PCR-based mapping was performed using a human-rodent somatic cell hybrid or radiation hybrid mapping panel. The results indicated that several novel genes were determined to be located in the vicinity of previously mapped disease loci; histidinemia loci, plasminogen Tochigi disease loci, Ehlers-Danlos syndrome, hypertriglyceridemia, thyroid resistance locus, ocular albinism, galactosemia, porphyria variegata, Charcot-Marie-tooth disease, FEOM (fibrosis of extraocular muscles), Prader-Willi syndrome.

The human NADH: ubiquinone oxidoreductase NDUFS5 (15 kDa) subunit: cDNA cloning, chromosomal localization, tissue distribution and the absence of mutations in isolated complex I-deficient patients.

We have cloned the cDNA of the NDUFS5 subunit (15 kDa) of the human mitochondrial respiratory chain complex NADH: ubiquinone oxidoreductase (complex I). The open reading frame consists of 321 base-pairs, coding for 106 amino acids, with a calculated molecular mass of 12.5 kDa. There is an 81.0% identity with the bovine equivalent on cDNA level and 74.5% identity on amino acid basis. PCR analysis of rodent-human somatic cell hybrids revealed that the human NDUFS5 gene maps to chromosome 1. The NDUFS5 mRNA is expressed ubiquitously in human tissues, with a relative higher expression in human heart, skeletal muscle, liver, kidney and fetal heart. A mutation detection study of twenty isolated enzymatic complex I-deficient patients revealed no mutations, nor polymorphisms.

Regional mapping panels for human chromosomes 1, 2, and 7.

The NIGMS Human Genetic Cell Repository has assembled regional mapping panels for human chromosomes 1, 2, and 7 from human rodent somatic cell hybrids submitted to the collection by researchers from 14 different laboratories. All hybrids were characterized initially by the submitters and verified by the Repository. Each hybrid carries a stable defined human segment as a derivative or deletion chromosome. These panels define 8-10 intervals for each chromosome. The panel for chromosome 2 is a new resource. The panels for chromosomes 1 and 7 complement previously published panels. The Repository distributes these regional mapping panels as cell cultures or as DNA. Information about these panels as well as for panels for chromosomes 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 21, 22, 22, and X may be viewed in the NIGMS Repository electronic catalog (http://locus.umdnj.edu/nigms).

Gene conversion is a likely cause of mutation in PKD1.

Approximately 70% of the gene responsible for the most common form of autosomal dominant polycystic kidney disease ( PKD1 ) is replicated in several highly homologous copies located more proximally on chromosome 16. We recently have described a novel technique for mutation detection in the duplicated region of PKD1 that circumvents the difficulties posed by these homologs. We have used this method to identify two patients with a nearly identical cluster of base pair substitutions in exon 23. Since pseudogenes are known to be reservoirs for mutation via gene conversion events for a number of other diseases, we decided to test whether these sequence differences in PKD1 could have arisen as a result of this mechanism. Using changes in restriction digest patterns, we were able to show that these sequence substitutions are also present in N23HA, a rodent-human somatic cell hybrid that contains only the PKD1 homologs. Moreover, these changes were also detected in total DNA from several affected and unaffected individuals that did not harbor this mutation in their PKD1 gene copy. This is the first example of gene conversion in PKD1 , and our findings highlight the importance of using gene-specific reagents in defining PKD1 mutations.

Mapping interleukin enhancer binding factor 3 gene (ILF3) to human chromosome 19 (19q11-qter and 19p11-p13.1) by polymerase chain reaction amplification of human-rodent somatic cell hybrid DNA templates.

Interleukin-2 (IL-2) is the first lymphokine secreted following T cell activation. Several transcription factors regulate IL-2 gene expression, including the nuclear factor of activated T cells (NFAT). NFAT acts at the antigen receptor response element-2 (ARRE-2) sequence in the IL-2 enhancer and is the nuclear target of T cell stimulation signals and the immunosuppressant drugs cyclosporine (Sandimmune) and FK-506 (tacrolimus), which are potent inhibitors of IL-2 gene transcription. NFAT has been cloned and found to consist of two subunits, NF45 (ILF2) and NF90 (ILF3). We have recently assigned the gene encoding the small NFAT subunit, NF45 (ILF3) to human chromosome 1 (1q11-qter and 1p11-p12). This communication reports the assignment of the gene encoding the large NFAT subunit, NF90 or interleukin enhancer binding factor 3 gene (ILF3), to human chromosome 19 (19q11-qter and 19p11-p13.1) by polymerase chain reaction (PCR) amplification of ILF3-specific DNA sequences from well-characterized human-rodent somatic cell hybrid DNAs.

Reactivation of XIST in normal fibroblasts and a somatic cell hybrid: abnormal localization of XIST RNA in hybrid cells.

The XIST gene, expressed only from the inactive X chromosome, is a critical component of X inactivation. Although apparently unnecessary for maintenance of inactivation, XIST expression is thought to be sufficient for inactivation of genes in cis even when XIST is located abnormally on another chromosome. This repression appears to involve the association of XIST RNA with the chromosome from which it is expressed. Reactivated genes on the inactive X chromosome, however, maintain expression in several somatic cell hybrid lines with stable expression of XIST. We describe here another example of an XIST-expressing human-hamster hybrid that lacks X-linked gene repression in which the human XIST gene present on an active X chromosome was reactivated by treatment with 5-aza-2'-deoxycytidine. These data raise the possibility that human XIST RNA does not function properly in human-rodent somatic cell hybrids. As part of our approach to address this question, we reactivated the XIST gene in normal male fibroblasts and then compared their patterns of XIST RNA localization by subcellular fractionation and in situ hybridization with those of hybrid cells. Although XIST RNA is nuclear in all cell types, we found that the in situ signals are much more diffuse in hybrids than in human cells. These data suggest that hybrids lack components needed for XIST localization and, presumably, XIST-mediated gene repression.

A human forkhead/winged-helix transcription factor expressed in developing pulmonary and renal epithelium.

Members of the forkhead/winged-helix transcription factor family play crucial roles during vertebrate development. A human hepatocyte nuclear factor/forkhead homolog (HFH)-4 cDNA encoding a 421-amino acid protein was isolated from a human fetal lung cDNA library. By Southern blot analysis of human-rodent somatic cell hybrid genomic DNA, the human HFH-4 gene localizes to chromosome 17q23-qter. This is the locus of another forkhead/winged-helix gene, the interleukin enhancer binding factor gene. RNA blot analysis revealed a 2.5-kilobase human HFH-4 transcript in fetal lung, kidney, and brain as well as in adult reproductive tissues, lung, and brain. By in situ hybridization, HFH-4 expression is associated with differentiation of the proximal pulmonary epithelium, starting during the pseudoglandular stage of human lung development. During human renal morphogenesis, HFH-4 is expressed in the developing epithelial cells of the ureteric duct, glomerulus, and epithelial vesicles. The unique pattern of HFH-4 expression during human fetal development suggests a role for this forkhead/winged-helix factor during pulmonary and renal epithelial development.

Neuropeptide Y Receptor Genes Mapped in Human and Mouse: Receptors with High Affinity for Pancreatic Polypeptide Are Not Clustered with Receptors Specific for Neuropeptide Y and Peptide YY

Ppyr1, Npy5r,andNpy6r,the genes encoding mouse type 4, type 5, and type 6 members of the neuropeptide Y receptor family, have been mapped by interspecific backcross analysis to conserved linkage groups on mouse Chr 14, Chr 8, and Chr 18, respectively. The human genes,PPYR1andNPY5R,have been localized to chromosomes 10q and 4q, respectively, by analysis of a panel of rodent–human somatic cell hybrids and yeast artificial chromosomes. These studies complete the mapping of the cloned NPY receptor subtypes in human and mouse and, together with previous studies, establish that the genes encoding receptors with high affinity for pancreatic polypeptide are not clustered with the genes encoding receptors specific for neuropeptide Y and peptide YY. The physical association of these receptor genes correlates with ligand-binding properties, rather than sequence identity, and suggests a complex evolutionary relationship.

Expression, genomic structure and high resolution mapping to 19p13.2 of the human smooth muscle cell calponin gene

Smooth muscle cells (SMC) express a battery of cell-restricted differentiation genes, many of which are down-regulated during the course of vascular disease. Here, we present the mRNA expression, genomic structure and chromosomal mapping of the gene encoding human smooth muscle cell calponin (SMCC). Human SMCC transcripts are restricted to tissues and cells of SMC origin and, in the latter case, appear to be uniquely controlled in two distinct human SMC lines of uterine and aortic origin. Restriction mapping, Southern blot and PCR analysis of a 70-kb human bacterial artificial chromosome (BAC) revealed a genomic structure (seven exons spanning >11 kb) very similar to that reported for the mouse SMCC gene. Using a variety of human–rodent somatic cell hybrid and radiation hybrid mapping panels, the human SMCC gene was mapped to a genomic interval of less than 1.32 Mb in 19p13.2. These results provide new information concerning the regulation of SMCC gene expression and demonstrate the utility of two human SMC lines for the further characterization of this gene's expression control. The identification of a BAC harboring the entire human SMCC locus represents an important reagent for future analysis of SMCC regulatory sequences. Finally, the localization of SMCC to a defined genomic interval will facilitate an analysis of its potential as a candidate gene for disease phenotypes mapping to 19p13.2.

CDNA Cloning, Chromosomal Localization, and Expression Pattern ofEPLG8,a New Member of theEPLGGene Family Encoding Ligands of EPH-Related Protein-Tyrosine Kinase Receptors

By screening a human fetal brain cDNA library under low stringency using cDNA encoding the mouse ligand of Cek5 as a probe, we have isolated a novel cDNA belonging to theEPLGgene family. This family encodes ligands of EPH-related tyrosine kinase receptors. Since the novel gene is the eighth member of theEPLGgene family, it is designatedEPLG8.The deduced amino acid sequence ofEPLG8suggests that it encodes a transmembrane protein that is most related to those encoded byEPLG2andEPLG5.We mapped theEPLG8gene to human chromosome 17p11.2–p13.1 by PCR screening of human–rodent somatic cell hybrid panels. In the midterm fetus,EPLG8mRNA is expressed at the highest level in brain, followed by heart, kidney, and lung. In the adult,EPLG8mRNA expression is restricted to brain. These data suggest that LERK-8, the protein encoded byEPLG8,is important in brain development as well as in its maintenance. Moreover, since levels ofEPLG8expression were particularly high in several forebrain subregions compared to other brain subregions, LERK-8 may play a pivotal role in forebrain function.

Cloning of the rat and human mitochondrial branched chain aminotransferases (BCAT m)

The rat and human mitochondrial branched chain aminotransferase (BCAT m) cDNAs have been isolated and shown to encode mature proteins of 41.2 and 41.3 kDa with presequences of 27 amino acids. When rat BCAT m is overexpressed in COS-1 cells, the protein exhibits BCAT activity and correct processing of the mitochondrial targeting sequence. Southern blot analysis of genomic DNA from a panel of rodent-human somatic cell hybrids revealed that the human BCAT m gene resides on chromosome 19 and the human cytosolic enzyme (BCAT c) gene on chromosome 12. Finally, the nomenclature BCAT1 for the cytosolic gene and BCAT2 for the mitochondrial BCAT gene is proposed.

ECK,a HumanEPH-Related Gene, Maps to 1p36.1, a Common Region of Alteration in Human Cancers

Mouseeck,a member of theEPHgene family, has been mapped to mouse chromosome 4. The syntenic relationship between this chromosome and human chromosome 1 suggests that the humanECKgene maps to the distal short arm of human chromosome 1 (1p). Since this region is frequently deleted or altered in certain tumors of neuroectodermal origin, it is important to define the specific chromosomal localization of the humanECKgene. PCR screening of a rodent–human somatic cell hybrid panel byECK-specific primers showed thatECKis indeed localized to human chromosome 1. Additional PCR screening of a regional screening panel for chromosome 1p indicated thatECKis localized to 1p36, distal toFUCA1.Furthermore, fluorescencein situhybridization analysis with anECK-specific P1 clone showed thatECKmaps proximal to genetic markerD1S228.Taken together, the data suggest thatECKmaps to 1p36.1, a region that is frequently deleted in neuroblastoma, melanoma, and other neuroectodermal tumors.

Mapping interleukin enhancer binding factor 2 gene (ILF2) to human chromosome 1 (1q11-qter and 1p11-p12) by polymerase chain reaction amplification of human-rodent somatic cell hybrid DNA templates.

Interleukin-2 (IL-2) is the first lymphokine secreted following T cell activation. Several transcription factors regulate IL-2 gene expression, including the nuclear factor of activated T cells (NFAT). NFAT acts at the antigen receptor response element-2 (ARRE-2) sequence in the IL-2 enhancer and is the nuclear target of T cell stimulation signals and the immunosuppressant drugs cyclosporine and FK506, which are potent inhibitors of IL-2 gene transcription. NFAT has been cloned and found to consist of two subunits, NF45 (ILF2) and NF90 (ILF3). This communication reports the assignment of NF45, interleukin enhancer binding factor 2 gene (ILF2), to human chromosome 1 (1q11-qter and 1p11-p12) by polymerase chain reaction (PCR) amplification of ILF2-specific DNA sequences from well-characterized human-rodent somatic cell hybrid DNA.

Chromosomal Assignment of the Human Gene Encoding the Fos-Related Antigen-2 (FRA2) to Chromosome 2p22–p23

The four members of the Fos gene family give rise to proteins that are part of the AP-1 transcription factor complex. When studying cAMP-induced apoptosis in a leukemia cell line from rat, we found that theFra-2gene (coding for the Fos-related antigen-2) became strongly upregulated as the leukemia cells started to die. It was therefore of interest to determine the cytogenetic localization of the humanFra-2gene (FRA2), including a comparison to chromosomal aberrations observed in leukemia patients. Based on sequence information from the rat and chickenFra-2homologs, we were able to PCR-amplify a 4.5-kb genomic fragment covering exon 4 ofFRA2.This fragment was employed as probe for both radioactive and fluorescencein situhybridization to human metaphase chromosomes, allowing us to assignFRA2to 2p22–p23. The localization of the gene to chromosome 2 was independently verified by PCR amplification of aFRA2-specific fragment from a panel of rodent–human somatic cell hybrids.

Cloning, Chromosomal Localization, Physical Mapping, and Genomic Characterization ofHKR3

TheKrüppel-type zinc finger proteins are members of a conserved family of transcription factors that are important in developmental regulation. Altered expression of several of these proteins has been implicated in human diseases, including cancer. We report the cloning, mapping, and characterization of the zinc finger geneHuman Krüppel-Related 3(HKR3). Genomic clones ofHKR3were isolated from a P1 library and localized to human chromosome subband 1p36.3 by human–rodent somatic cell hybrid mapping and fluorescencein situhybridization. The gene was physically mapped to within 40 kb ofD1S214by YAC content and long-range restriction mapping.HKR3spans 9.5 kb of genomic DNA and is contained in 11 exons. Sequencing defined each of the exon/intron splice site junctions and identified a CpG island in the 5′ region of the gene.HKR3is ubiquitously expressed in human tissues as at least two major transcripts, the shorter of which excludes a conserved finger-associated box and a putative acidic activation domain contained in the full-length transcript.HKR3is a novel zinc finger gene that maps to a region of the genome commonly rearranged or deleted in human cancers.

Localization of Human Flavin-Containing Monooxygenase GenesFMO2andFMO5to Chromosome 1q

The human flavin-containing monooxygenase (FMO) gene family comprises at least five distinct members (FMO1toFMO5) that code for enzymes responsible for the oxidation of a wide variety of soft nucleophilic substrates, including drugs and environmental pollutants. Three of these genes (FMO1, FMO3,andFMO4) have previously been localized to human chromosome 1q, raising the possibility that the entire gene family is clustered in this chromosomal region. Analysis by polymerase chain reaction of DNA isolated from a panel of human–rodent somatic cell hybrids demonstrates that the two remaining identified members of theFMOgene family,FMO2andFMO5,also are located on chromosome 1q.

Plasmacytoma-Associated Neuronal Glycoprotein,Pang,Maps to Mouse Chromosome 6 and Human Chromosome 3

A new member of the immunoglobulin/fibronectin superfamily of adhesion molecules,Pang(plasmacytoma-associated neuronal glycoprotein), was recently isolated from a plasmacytoma. In previous studies,Pangwas found to be normally expressed in the brain and ectopically activated by intracisternal A-type particle long terminal repeats in plasmacytomas. In this study,Pangwas initially mapped to mouse Chr 6 by somatic cell hybrid analysis and further positioned on the chromosome betweenWnt7aandPcp1.Southern blot analysis of human–rodent somatic cell hybrids together with predictions from the mouse map location indicate that human PANG is located at 3p26.