Somatic Hybrid Panel Research Articles

Assignment of the porcine MAM domain containing glycosylphosphatidylinositol anchor 1 (MDGA1) gene on chromosome 7q11→13 by in situ hybridisation and somatic hybrid panel mapping

In course of a genome analysis project with the aim to map and characterize type I markers on porcine Chromosome 7 we mapped the porcine MDGA1 (MAM domain containing glycosylphosphatidylinositol anchor 1) gene. The human orthologue was only recently described and the original name GPIM (GPI and MAM protein) has received the above official gene name and symbol (De Juan et al., 2002). The MDGA1 gene appears to be highly conserved among mammals. The protein contains several immune-globulin (Ig) domains, one fibronectin (FnIII) domain, and one MAM (meprin, A5 protein, receptor proteintyrosine phosphatase mu) domain. According to these structural features, showing characteristics of a cell adhesion molecule, the protein is possibly involved in cell-cell communication and tumor progression.

Comparative porcine gene mapping relative to human chromosomes 9, 10, 20 and 22.

Comparative anchor tagged sequence (CATS) consensus primers from loci mapped to human chromosomes 9, 10, 20, and 22 have been used to amplify homologous loci in pigs. Of 53, CATS primers tested in pigs, only 23 yielded products homologous to the human locus (42% success). Ten loci were physically mapped (43% success rate for verified products, but only 19% for primers tested). Due to lack of polymorphism, linkage mapping was possible only for AMBP. Map locations were consistent with human/pig ZOO-FISH, except for ADRA1A, whose position is still equivocal in humans. These CATS primers have made very limited contributions to pig/human comparative gene mapping because of low efficiency of amplification of orthologous porcine product, frequent amplification from rodent template in a somatic hybrid panel and low level of polymorphism.

The MGEA6 Multigene Family Has an Active Locus on 14q and at Least Nine Pseudogenes on Different Chromosomes

The meningioma expressed antigen-6 (MGEA6) was originally identified as an immunogenic antigen in meningioma patients. Somatic hybrid panel mapping and fluorescence in situ hybridization revealed MGEA6-related sequences on different human chromosomes. Here we carry out database analysis to investigate the complexity of the MGEA6-related sequences and demonstrate the existence of a multigene family. We localized the active gene (spanning over 83 kb) to chromosome 14q and elucidated its exon/intron structure. We identified and characterized 9 processed pseudogenes on 9 different chromosomes including chromosomes 2, 3, 6, 7, 9, 10, 12, 13, and 18. We performed phylogenetic analysis and concluded that the MGEA6 pseudogenes may result from more than one retrotransposition event; we calculated divergence times of the pseudogenes to be between 21.5 and 28.9 million years ago.

Mapping dispersed repetitive loci using semi-specific PCR cloning and somatic cell hybrid mapping.

A simple and effective method based upon semi-specific PCR followed by cloning has been developed. Chromosomal mapping of the generated fragment on a somatic cell hybrid panel identifies the chromosomal position, and yields a unique sequence tag for the site. Using this method, the chromosomal location of one porcine endogenous retrovirus (PERV) was determined. The porcine genomic sequences were first amplified by PCR using a PERV-specific primer and a porcine short interspersed nuclear element (SINE)-specific primer. PCR products were cloned, and those sequences that contained PERV plus flanking regions were selected using a second round of PCR and cloning. Sequences flanking the PERV were determined and a PERV-B was physically mapped on porcine chromosome 17 using a somatic hybrid panel. The general utility of the method was subsequently demonstrated by locating PERVs in the genome of PERV infected human 293 cells. This method obviates the need for individual library construction or linker/adaptor ligation, and can be used to quickly locate individual sites of moderately repeated, dispersed DNA sequences in any genome.

Cloning and Chromosomal Localization of a Human Class III Myosin

Two class III myosins have been identified to date: NINAC from Drosophila melanogaster and MyoIIILim from Limulus polyphemus. Both have N-terminal kinase domains and are expressed exclusively in photoreceptors. Mutations in NINAC have been shown to alter the photoresponse and compromise photoreceptor survival. We report the cloning and chromosomal localization of a human class III myosin, MYO3A, from retina and a retinal pigment epithelial cell line. Human MYO3A (which we will refer to simply as MYO3A) possesses an N-terminal kinase domain and three consensus calmodulin-binding (IQ) motifs, two in the neck and one in the tail domain. We detected two MYO3A splice variants differing by 52 amino acids near the kinase/myosin junction. On Northern blots, MYO3A probes detected a 6.5-kb transcript in human and monkey retina, in a cultured human RPE cell line (RPE-19), and at much lower levels in human pancreas. A somatic hybrid panel PCR screen localized MYO3A to human chromosome 10, and a radiation hybrid screen further localized it proximal to marker D10S197, which is located at 10p11.1 on the human cytogenetic map. Since mutations in NINAC have been shown to alter the photoresponse and compromise photoreceptor survival, the human homologue MYO3A may also play a role in photoreceptor function and/or maintenance.

Isolation of a Pancreas-Specific Gene Located on Human Chromosome 14q31: Expression Analysis in Human Pancreatic Ductal Carcinomas

We have isolated a novel cDNA (SEL1L) that shows sequence similarities to SEL-1, a gene identified as an extragenic suppressor of thelin-12hypomorphic mutant fromCaenorhabditis elegans(7, 8). SEL1L exhibits a tissue-specific pattern of expression: a single poly(A)+RNA species of 7.5 kb is abundantly expressed only in the pancreas of healthy individuals, whereas low to undetectable levels are observed in other adult and in some fetal tissues. Somatic hybrid panel and fluorescencein situhybridization positioned this gene in the q31 band of human chromosome 14. The tissue-specific expression of this gene induced us to study its role in human pancreatic carcinomas. Our analysis revealed that 17% of adenocarcinomas of the pancreas did not express SEL1L to a detectable level; however, no gross genomic alterations were apparent in the few hundred kilobases of the relevant region.

Chromosomal location of the human immunoglobulin lambda variable 8 (IGLV8) gene family outside the major λ locus on chromosome 22q11.2

Physical mapping of the human immunoglobulin λ locus (IGL) on chromosome 22q11 has shown the existence of at least 52 variable region gene segments. These Vλ genes are associated with EcoRI fragments detectable in Southern blots of genomic DNA samples. The current physical map of the IGL locus includes a unique Vλ8 gene (IGL8a, accession no. Z73650) in a 3.7 kb EcoRI fragment. However, our Southern blot- EcoRI-restriction fragment length polymorphism studies on the Brazilian population using a specific probe for the Vλ8 gene (pVL8 probe) have revealed the presence of two additional fragments bearing Vλ8 sequences (8.0 kilobase (kb) at 100% frequency and 6.0 kb at 10% frequency). We have used human/rodent somatic cell hybrid DNAs to locate these new Vλ8 genes outside the major locus on chromosome 22q11.2. Polymerase chain reactions using specific primers for the IGLV8a gene on the somatic hybrid panel showed that chromosome 8 (besides 22q11) also comprises Vλ8 sequences. This finding represents evidence for the dispersion of the human IGLV8 gene family outside the major locus (orphan genes).

CDNA cloning and chromosomal mapping of a predicted coiled-coil proline-rich protein immunogenic in meningioma patients.

There is increasing evidence that tumor expressed genes induce immune responses in cancer patients. To identify meningioma expressed antigens, we established a meningioma expression library which was screened with autologous serum. Out of 20 positive cDNA clones eight share high sequence homologies as determined by sequence analysis. These eight clones can be grouped into three classes which differ in length and which are characterized by specific sequence variations. The longest open reading frame was found to be 2412 bp encoding an immunoreactive antigen termed meningioma expressed antigen 6 (MEA6). Using five sequence specific primer pairs, somatic hybrid panel mapping revealed locations of the three classes on several human chromosomes including chromosomes 2, 3, 6, 7, 9, 13 and 14. The mapping results were confirmed by fluorescence in situ hybridization. RT-PCR showed consistent expression of all classes in several meningiomas and additional tissues using the same set of primer pairs as for chromosomal mapping. The expression data were confirmed by northern blot analysis. For the predicted amino acid sequence BLASTX revealed a homology to a human C219-reactive peptide which was previously isolated by an antibody directed against p-glycoprotein. Sequence properties of the MEA protein include an acidic activation domain, a proline-rich region and two coiled-coil domains indicating protein binding and activation functions.

CDNA Cloning and Gene Structure of a Novel Water Channel Expressed Exclusively in Human Kidney: Evidence for a Gene Cluster of Aquaporins at Chromosome Locus 12q13

A 1.8-kb cDNA clone (designed hKID, gene symbol AQP2L) with homology to the aquaporins was isolated from a human kidney cDNA library. The longest open reading frame of 846 bp encoded a 282-amino-acid hydrophobic protein that contained the conserved NPA motifs of MIP family members. Cell-free translation produced a nonglycosylated protein migrating at 29 kDa. Amino acid alignment showed the greatest homology of hKID to human MIP (48% identity) and AQP-2 (52%), with lesser homology to human MIWC (AQP-4, 34%), CHIP28 (AQP-1, 38%), and GLIP (AQP-3, 22%). Northern blot analysis revealed a 2.2-kb transcript expressed only in human kidney. PCR/Southern blot analysis of human kidney cDNA using primers flanking the hKID coding sequence revealed expression of a full-length mRNA and short transcripts with partial exon 1 and partial exon 4 deletions. Expression of hKID cRNA inXenopusoocytes did not increase glycerol or urea permeability, but increased osmotic water permeability from (2.8 ± 0.5) × 10−4to (7.4 ± 0.7) × 10−4cm/s (10°C) in a mercurial-sensitive manner. Sequence comparison of hKID cDNA with a cloned 21-kb genomic DNA indicated three introns (lengths 0.7, 0.25, and 0.4 kb) separating four exons with boundaries at amino acids 121, 174, and 201. The hKID promoter was identified and contained TATA, SP1, E-box, and AP1 and AP2 elements; primer extension revealed hKID transcription initiation 654 bp upstream from the translational initiation site. Genomic Southern blot indicted a single-copy hKID gene. PCR analysis of a human/rodent somatic hybrid panel localized the hKID gene to chromosome 12. Chromosomal fluorescencein situhybridization mapped the hKID (AQP2L) gene to chromosome locus 12q13, the same location as the AQP-2 and MIP genes. The high sequence homology, similar genomic structure, and identical chromosomal loci of hKID, MIP, and AQP-2 suggest a MIP family gene cluster at chromosome locus 12q13. Further work is needed to establish the physiological significance of hKID.

Assignment of markers by using polymerase chain reaction on pools of swine flow-sorted chromosomes.

Gene chromosomal assignment can be realized not only by somatic hybrid panels but also by spot-blot hybridization or polymerase chain reaction (PCR) of flow-sorted chromosomes. We propose a swine chromosome assignment strategy by PCR amplification on pooled chromosomal DNA, which allows assignment despite possible chromosomal contamination during sorting. Each pool contains three different chromosomes, each chromosome being present in one or two pools. We present concordant results obtained for eight markers already mapped to different swine chromosomes and we assign the somatostatin gene to chromosome 13, a new marker in the pig genome.

Localization of the Human Achaete-Scute Homolog Gene (ASCL1) Distal to Phenylalanine Hydroxylase (PAH) and Proximal to Tumor Rejection Antigen (TRA1) on Chromosome 12q22–q23

ASCL1, the human achaete-scute homolog, is a helix–loop–helix transcription factor that was previously assigned to chromosome 12 using a rodent–human somatic hybrid panel. We now placed this gene on a yeast artificial chromosome contig encompassing position 119 cM of the Généthon genetic map between the two genes phenylalanine hydroxylase (PAH) and tumor rejection antigen 1 (TRA1). We also localized ASCL1 in the 12q22–q23 cytogenetic interval by using fluorescencein situhybridization.

Assignment of the 49-kDa (PRIM1) and 58-kDa (PRIM2A and PRIM2B) Subunit Genes of the Human DNA Primase to Chromosome Bands 1q44 and 6p11.1-p12

DNA primase is an essential replication protein that catalyzes the synthesis of oligoribonucleotide primers. DNA primase, consisting of two subunits (p49 and p58), plays a key role in both the initiation of DNA replication and the synthesis of Okazaki fragments for lagging strand synthesis. We mapped the locations of human chromosomes of the genes coding for both subunits [p49 (PRIM1) and p58 (PRIM2)] by PCR amplification using DNAs of a panel of somatic hybrids, to chromosomes 1 and 6, respectively. The PRIM1 gene was mapped to 1q44, and two PRIM2 loci (PRIM2A and PRIM2B) were detected at 6p11.1-p12 by fluorescence in situ hybridization using several genomic DNA probes.

Thioredoxin, a mediator of growth inhibition, maps to 9q31

The human thioredoxin gene has been provisionally mapped to 3p11–p12. Recently thioredoxin cDNA has been isolated in a procedure that detects transcripts coding for growth-suppressing proteins, and thus the chromosomal location of the gene is of particular interest. Chromosome 3 is believed to harbor several tumor suppressor genes important in the development of lung and other common epithelial tumors. To establish more firmly the chromosomal location of the human thioredoxin gene, a somatic hybrid panel was used; it identified chromosome 9 as the location of the transcribed thioredoxin gene. Fluorescence in situ hybridization of a YAC encoding the transcribed thioredoxin gene refined the localization to 9q31.

Localization of the gene for a novel human adenylyl cyclase (ADCY7) to chromosome 16.

A novel form of human adenylyl cyclase (ADCY7) has been discovered in the human erythroleukemia cell line (HEL). This cell line has been widely used as a model for studies of the characteristics of human platelets. Data from HEL cells suggests that ADCY7 may be the major AC form in human platelets. In the current study polymerase chain reaction (PCR) techniques coupled with use of human/rodent somatic hybrid panels and a yeast artificial chromosome (YAC) library were used to determine the chromosomal localization of the gene (adcy7) for ADCY7 enzyme. A 251-bp product from the 3' untranslated region of human adcy7 was amplified for PCR mapping and the results localize the adcy7 gene to region 16q12-16q13 of the human genome. The AC enzyme family is characterized by the presence of 12 membrane-spanning domains in its sequences, and this chromosomal region is known to contain other genes coding for proteins characterized by 12 membrane-spanning domains.

Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2–q24.3, 3q27, 13q12.3–q13, and 7q11.23

Replication factor C is a multimeric primer-recognition protein consisting of five subunits (p145, p40, p38, p37, and p36.5) and is essential for the processive elongation of DNA chains catalyzed by DNA polymerase δ or ϵ in human cells. We have mapped the locations on human chromosomes of the genes coding for the four smaller subunits [p36.5 (RFC5), p37 (RFC4), p38 (RFC3), and p40 (RFC2)] using both PCR amplification from DNAs of a panel of somatic hybrids and fluorescence in situ hybridization to bands 12q24.2–q24.3, 3q27, 13q12.3–q13, and 7q11.23, respectively.

Molecular analysis of a novel subtelomeric repeat with polymorphic chromosomal distribution

DNA from a 50-kb yeast artificial chromosome (YAC) containing one human telomere was characterized. Cloned sequences from the centromeric end of this YAC (designated yRM2001) localized to several human chromosomes by somatic hybrid panel mapping. The telomeric end of the YAC contained both (TTAGGG)n sequences and the previously characterized TelBam3.4 subterminal repeat element. A novel low-copy repeat element (designated HC1103) mapped 19 kb from the telomeric end of the YAC. This repeat was shown by fluorescence in situ hybridization to be present in several subtelomeric regions (3q, 12p, 15q, 19p, and 20p) and at an interstitial site (2q13-->q14) in all individuals studied, but to be polymorphically distributed at several other telomeres. The YAC vector-insert EcoRI cloning site was positioned 50 kb to 70 kb from chromosome termini in human genomic DNA using RecA-assisted restriction endonuclease (RARE) cleavage analysis. Our results suggest that the DNA segment cloned in yRM2001 contains a novel block of low-copy DNA consistently present at some human telomeres, but polymorphically distributed at others.

Mapping of 28 (CA)n microsatellite repeats and 13 Alu markers on human chromosome 11 using a panel of somatic cell hybrids.

We report the mapping of 28 (CA)n microsatellite repeats and 13 Alu repeats, most of which are highly polymorphic, in 24 intervals on chromosome 11 by PCR amplification from DNAs of a panel of somatic hybrids.

Organization and sequence of the gene encoding the human acrosin-trypsin inhibitor (HUSI-II)

A complete cDNA encoding the acrosin-trypsin inhibitor, HUSI-II, was used as a probe to isolate genomic clones from a human placenta library. Three clones which cover the entire HUSI-II gene were isolated and characterized. The exon-intron organization of the gene was determined and found to be identical to other known Kazal-type inhibitor-encoding genes. The striking similarity in the amino acid sequences which was found previously in HUSI-II and glycoprotein hormone β-subunits, is neither reflected in codon usage nor in the exon-intron arrangement of the genes. A 1.8-kb segment 5′ of the gene was sequenced. The analysis of this sequence showed that HUSI-II contains a G + C-rich region upstream from the transcription start point ( tsp) which fulfills the criteria for a CpG island. Furthermore, in the first intron, a potential glucocorticoid-responsive element was found as a half-palindrome flanked by two CACCC elements. Determination of the tsp by S1 mapping revealed that HUSI-II has multiple tsp. Genomic Southern hybridization was used to show that HUSI-II is a single-copy gene. The localization of the gene to chromosome 4 was determined by hybridization of a 5′ genomic fragment to the DNA of a panel of somatic hybrids between human and rodent cells.

Complementary physical and genetic techniques map the vinculin (VCL) gene on chromosome 10q

Vinculin is a cytoskeletal protein component of adherenstype cell junctions. The gene had been mapped to 10q11.2-qter. We have used a combination of physical and genetic mapping techniques to refine this localization. Hybridization of the vinculin cDNA probe, HV1, to a human-rodent somatic hybrid panel initially suggested a position of either 10q11.2 or 10q22.1–10q23. Genetic recombination mapping in three-generation families with multiple endocrine neoplasia type 2 (MEN2) indicated a position distal to D10S22 (10q21.1) in 10q22.1–10q23. This was confirmed by hybridization of the vinculin cDNA to flow-sorted translocation derivative chromosomes containing the q21-qter portion of chromosome 10. We conclude that the vinculin locus maps in 10q22.1–q23, distal to D10S22.

The human transmembrane secretory component (poly-Ig receptor): molecular cloning, restriction fragment length polymorphism and chromosomal sublocalization

The human transmembrane secretory component (SC) mediates glandular translocation of polymeric IgA and IgM into exocrine secretions. A 2898-bp cDNA clone, encoding the entire sequence of the human transmembrane SC, was isolated from a colonic adenocarcinoma cell line cDNA library. The deduced amino-acid sequence had a length of 764 residues and showed an overall similarity of 56% and 64% with the rabbit and rat counterpart, respectively. A restriction fragment length polymorphism (RFLP) was found with PvuII, revealing a two-alle RFLP with an autosomal codominant inheritance pattern and allele frequencies of 0.65 and 0.35. Southern blot analysis of human-rodent somatic hybrid panels, including hybrids with translocation chromosomes carrying different parts of chromosome 1, assigned the SC gene to 1q31-q42, thus confirming a previously reported provisional assignment.