Chromosomal Assignment Research Articles

Characterization and chromosome assignment of the canine gamma-sarcoglycan gene (SGCG) to CFA 25q21→q23

Mutations in the gene for gamma-sarcoglycan (SGCG) located on HSA 13q12 are responsible for limb girdle muscular dystrophy (LGMD2C) in human. Here we report the cloning of the canine SGCG gene together with its genomic structure and several intragenic polymorphisms. The coding part of the canine SGCG contains seven exons spanning at least 70 kb of genomic DNA. The chromosome assignment of the canine SGCG gene to CFA 25q21→q23 confirms that the canine syntenic group 10 corresponds to CFA 25 and also supports the findings of human-canine reciprocal chromosome painting.

The 90- and 110-kDa Human NFAR Proteins Are Translated from Two Differentially Spliced mRNAs Encoded on Chromosome 19p13

The NFAR gene (nuclear factor associated with dsRNA) encodes a putative transcription-associated factor that we have shown is a substrate for the interferon-inducible, dsRNA-dependent protein kinase, PKR. However, our protein expression analysis has revealed that NFAR exists as two major protein species of 90 kDa (NFAR-1) and 110 kDa (NFAR-2) in the cell. To resolve the genetic identity of NFAR-1 and -2, we carried out sequence analysis of genomic and cDNA NFAR clones and determined that the coding region of this gene spans 16.2 kb and comprises 21 exons. Our data indicate that NFAR-1 and -2 arise from a single gene on chromosome 19p13 and are generated through alternative splicing events. NFAR-1 (HGMW-approved symbol ILF3) was found to comprise 1 extra exon, 18, that contains several stop codons to ensure termination of the protein at amino acid 702. In contrast, NFAR-2 lacks this exon, though it comprises an additional 3 coding exons (exons 19, 20, and 21) located at the carboxyl region to generate an extended product of 894 amino acids. Our studies, the first to elucidate the gene structure and chromosomal assignment of NFAR, establish the genetic basis for future NFAR research in humans.

Molecular characterization and chromosome assignment of the porcine gene for leukemia inhibitory factor LIF

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine involved in early conceptus development in pig. We isolated a PAC clone containing the porcine LIF gene and determined the complete DNA sequence of the gene, which spans about 6.3 kb and consists of five exons including three alternative first exons (1D, 1M, 1T) spliced onto common second and third exons. The LIF-D transcript encodes a protein of 202 amino acids sharing 87, 84, and 78% identity with respectively human, ovine, and murine leukemia inhibitory factors. The LIF-M and LIF-T transcripts both encode a truncated protein of 158 amino acids. Two SNP markers within untranslated regions of the LIF cDNA were identified. One SNP is located in the 5′-UTR of the alternative exon 1T while the other SNP is located in the 3′-UTR of exon 3. Based on fluorescence in situ hybridization and radiation hybrid mapping, the porcine LIF gene was assigned to chromosome 14q2.1→q2.2.

Sequence and chromosomal assignment of a human novel cDNA: similarity to gamma-aminobutyric acid transporter

Gamma-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the mammalian brain. Although initially thought to be confined to the central nervous system, GABAergic activity has also been described in other tissues throughout the body. In the present study, we report the cloning and localization of human GABA transporter cDNA and document its expression in various human tissues. A human liver cDNA library was initially screened by a 32P-labeled murine brain GABA transporter 3 (GAT-3) cDNA probe, and full-length cDNA was cloned by employing Marathon-Ready human kidney cDNA. The human GABA transporter cDNA encoded a 569 amino acid hydrophobic protein with 12 transmembrane domains (TMs). Search of published sequences revealed high homology with rat GAT-2, murine GAT-3 cDNA, human solute carrier family 6 member 13 (SLC6A13), and a human peripheral betaine/GABA transporter. Northern blot analyses demonstrated that the human GABA transporter is expressed strongly in the kidney and to a lesser extent in the liver and brain. The sequence was well matched with human chromosome 12p13.3, suggesting the human GABA transporter contains 14 exons. The above findings confirm the existence of and further characterize a specific GABA transporter in human tissues.

Comparative genomic in situ hybridization (cGISH) analysis on plant chromosomes revealed by labelled Arabidopsis DNA.

A new approach for comparative cytogenetic banding analysis of plant chromosomes has been established. The comparative GISH (cGISH) technique is universally applicable to various complex genomes of Monocotyledonae (Triticum aestivum, Agropyron elongatum, Secale cereale, Hordeum vulgare, Allium cepa, Muscari armenaticum and Lilium longiflorum) and Dicotyledonae (Vicia faba, Beta vulgaris, Arabidopsis thaliana). Labelled total genomic DNA of A. thaliana generates signals at conserved chromosome regions. The nucleolus organizing regions (NORs) containing the majority of tandemly repeated rDNA sequences, N-band regions containing satellite DNA, conserved homologous sequences at telomeres and additional chromosome-characteristic markers were detected in heterologous FISH experiments. Multicolour FISH analysis with repetitive DNA probes simultaneously revealed the chromosome assignment of 56 cGISH signals in rye and 61 cGISH signals in barley. Further advantages of this technique are: (1) the fast and straightforward preparation of the probe; (2) the generation of signals with high intensity and reproducibility even without signal amplification; and (3) no requirement of species-specific sequences suitable for molecular karyotype analysis. Hybridization can be performed without competitive DNA. Signal detection without significant background is possible under low stringency conditions. The universal application of this fast and simple one-step fluorescence banding technique for plant cytogenetic and plant genome evolution is discussed.

Sequencing and chromosomal assignment of the rat endothelial-derived lipase gene (Lipg).

Part of the nucleotide sequence of the Lipg gene in the rat was established using primers based on the mRNA sequence described in the mouse. The rat intron sequence served as a template for designing primers for the specific amplification of rat Lipg. A rat-hamster radiation hybrid (RH) panel was used for chromosomal assignment of the rat Lipg gene. The Lipg gene was found to be located on rat chromosome 18 in the vicinity of the marker D18Mit11; a region reported to be homologous with both human and mouse chromosome 18.

Chromosome assignment of four plexin A genes (Plxna1, Plxna2, Plxna3, Plxna4) in mouse, rat, Syrian hamster and Chinese hamster

We determined chromosome locations of four plexin A subfamily genes, Plxna1, Plxna2, Plxna3 and Plxna4, in four rodent species, mouse, rat, Syrian hamster and Chinese hamster, by fluorescence in situ hybridization. Plxna1, Plxna2, Plxna3 and Plxna4 were localized to Chr 6E2, 1H6, XB-C1 and 6B1 in mouse, Chr 4q34.1, 13q26→q27, Xq37.1→q37.2 and 4q21.3→q22 in rat, Chr 8qb1.1→qb1.3, 11qb8, Xpb8 and 5qb3.3 in Syrian hamster, and Chr 8q1.2, 5q3.7, Xp2.7 and 1q2.2→q2.3 in Chinese hamster, respectively. All the mouse and rat plexin A genes were localized to chromosome regions where conserved homology has been identified among human, mouse and rat.

Isolation and characterization of Suv39h2, a second histone H3 methyltransferase gene that displays testis-specific expression.

Higher-order chromatin has been implicated in epigenetic gene control and in the functional organization of chromosomes. We have recently discovered mouse (Suv39h1) and human (SUV39H1) histone H3 lysine 9-selective methyltransferases (Suv39h HMTases) and shown that they modulate chromatin dynamics in somatic cells. We describe here the isolation, chromosomal assignment, and characterization of a second murine gene, Suv39h2. Like Suv39h1, Suv39h2 encodes an H3 HMTase that shares 59% identity with Suv39h1 but which differs by the presence of a highly basic N terminus. Using fluorescent in situ hybridization and haplotype analysis, the Suv39h2 locus was mapped to the subcentromeric region of mouse chromosome 2, whereas the Suv39h1 locus resides at the tip of the mouse X chromosome. Notably, although both Suv39h loci display overlapping expression profiles during mouse embryogenesis, Suv39h2 transcripts remain specifically expressed in adult testes. Immunolocalization of Suv39h2 protein during spermatogenesis indicates enriched distribution at the heterochromatin from the leptotene to the round spermatid stage. Moreover, Suv39h2 specifically accumulates with chromatin of the sex chromosomes (XY body) which undergo transcriptional silencing during the first meiotic prophase. These data are consistent with redundant enzymatic roles for Suv39h1 and Suv39h2 during mouse development and suggest an additional function of the Suv39h2 HMTase in organizing meiotic heterochromatin that may even impart an epigenetic imprint to the male germ line.

Identification, tissue specific expression, and chromosomal localisation of several human dynein heavy chain genes.

Sliding between adjacent microtubules within the axonema gives rise to the motility of cilia and flagella. The driving force is produced by dynein complexes which are mainly composed of the axonemal dynein heavy chains. We used cells of human respiratory epithelium after in vitro ciliogenesis to clone cDNA fragments of nine dynein heavy chain genes, one of which had never been identified before. Dynein heavy chains are highly conserved from protozoa to human and the evolutionary ancestry of these dynein heavy chain cDNA fragments was deduced by phylogenetic analysis. These dynein heavy chain cDNAs are highly transcribed in human tissues containing axonema such as trachea, testis and brain, but not in adult heart or placenta. PAC clones containing dynein heavy chains were obtained and used to determine by FISH their chromosomal position in the human genome. They were mapped to 2p12-p11, 2q33, 3p21.2-p21.1, 13q14, 16p12 and 17p12. The chromosomal assignment of these dynein heavy chain genes which was confirmed by GeneBridge 4 radiation hybrid screening, will be extremely useful for linkage analysis efforts in patients with primary ciliary dyskinesia (PCD).

Mapping of 14 expressed sequence tags (ESTs) from porcine skeletal muscle by somatic cell hybrid analysis.

Chromosomal assignments are reported for fourteen porcine expressed sequence tags (ESTs)--CALM1, CRYAB, MYH7, MYL1, PDK4, PGAM2, PYGM, REV3L, RFC1, SLN, SPTBN1, SRM160, TPM1 and YWHAG. The ESTs were derived from our porcine skeletal muscle cDNA library. The ESTs sequences selected for mapping included the presence of the 3'-untranslated region. The assignments were performed using two independent somatic cell hybrid panels providing the possibility of confirmation of the results obtained. The observed localizations are compared with the locations predicted from heterologous (human-pig, pig-human) chromosome painting data and knowledge of the map locations of the human homologues. These results add new information to the porcine genome transcript map.

Sequence analysis, gene expression, and chromosomal assignment of mouse Borg4 gene and its human orthologue.

The Borg (binder of Rho GTPases) family proteins interact with CDC42 and TC10 in a guanosine triphosphate (GTP)-dependent manner. We have isolated a full-length cDNA of the mouse Borg4 gene, which is a member of this family. Sequence analysis revealed that this gene encoded a putative 349-amino acid protein. By reverse transcription--coupled polymerase chain reaction (RT-PCR) analysis, we observed that Borg4 was expressed ubiquitously in adult tissues. Additionally, we determined the entire cDNA sequence of the putative human Borg4 orthologue. By fluorescence in situ hybridization, mouse Borg4 and the putative human orthologue have been assigned to mouse chromosome 11E and human chromosome 17q24-25, which has been described as syntenic to the mouse region.

Human Fas associated factor 1, hFAF1, gene maps to chromosome band 1p32.

Human Fas associated factor 1 protein (hFAF1) is involved in the positive regulation of Fas signaling even though it can not initiate the signal for itself. By chromosomal assignment using somatic cell hybrids (CASH), the hFAF1 gene was located on human chromosome 1 between markers D1S443 and D1S197. The hFAF1 gene was mapped to human chromosome band 1p32 by FISH utilizing a genomic PAC clone containing the gene. In genomic Southern analysis using hFAF1 cDNA as a probe, several bands appeared in three different restriction enzyme digestions. The single band appearance in FISH analysis compared to several bands in Southern blots implies that the hFAF1 gene would be rather big or that an additional hFAF1 gene isotype(s) might be present in close vicinity.

Analysis of the murine phosphoinositide 3-kinase γ gene

Phosphoinositide 3-kinase γ is preferentially expressed in leukocytes. PI3Kγ is activated by βγ subunits of heterotrimeric G-proteins, which thus link seven transmembrane helix receptor activation to phosphatidylinositol (3,4,5)-trisphosphate production. Here we describe the molecular cloning of the murine PI3Kγ cDNA, the PI3Kγ gene structure, its chromosomal assignment and the analysis of promoter activity. The mouse cDNA shares 86% identity to its pig and human orthologues at the nucleotide level. The MmPI3Kγ gene spans approximately 30 kb and comprises 11 exons. RACE-PCR indicated the presence of multiple start sites generating 5′ UTRs with different lengths, the longest being 874 bp. The putative promoter region contains no TATA box but several putative binding sites for hematopoietic specific transcription factors. A 1200 bp long sequence upstream the first transcription start site was found to possess tissue specific promoter activity. Deletion constructs revealed two contiguous regions, with activator function, ranging from positions −139 to −557, and with inhibitory function, ranging from positions −557 to −892. FISH analysis revealed that the MmPI3Kγ is located on chromosome 12 band B and that the human orthologue is positioned on chromosome 7q22.2–22.3. In spite of some differences in the ATP-binding site, recombinant murine PI3Kγ protein is equally sensitive to wortmannin as its human counterpart. This suggests that mouse models will provide reliable results in the assessments of novel PI3Kγ inhibitors.

A novel gene "Niban" upregulated in renal carcinogenesis: cloning by the cDNA-amplified fragment length polymorphism approach.

A modified AFLP (amplified fragment length polymorphism) method was employed to isolate genes differentially expressed in renal carcinogenesis of Tsc2 gene mutant (Eker) rats. One gene, selected for further investigation, was named “Niban” (“second” in Japanese), because it is the second new gene to be found after Erc (expressed in renal carcinoma) in our laboratory. Importantly, “Niban” is well expressed even in small primary rat Eker renal tumors, more than in progressed cell lines, and is also expressed in human renal carcinoma cells, but not in normal human or rat kidneys. Chromosome assignment was to RNO 13 in the rat, and HSA 1. This “Niban” gene is a candidate as a marker for renal tumor, especially early‐stage renal carcinogenesis.

Molecular analysis and chromosomal assignment of the canine CALC-I/alpha-CGRP gene.

We have isolated a recombinant phage harboring the canine CALC-I/alpha-CGRP gene. The gene spans a region of approx. 5.3 kb and consists of six exons with sizes ranging from 95 bp (exon 2) and 494 bp (exon 4). By alternative splicing, two transcripts with ORFs of 390 and 384 nt are generated. These encode either the 32-amino acid-long hormone calcitonin (CALC) or the neurotransmitter calcitonin gene-related peptide (alpha-CGRP) with a length of 37 amino acids after proteolytic processing of precursor molecules. The canine calcitonin precursor consists of 130 amino acids with a molecular mass of 14.05 kDa and a statistical pI of 8.0, whereas the deduced alpha-CGRP precursor harbors 128 amino acids with a molecular mass of 13.87 kDa and a statistical pI of 8.6. Both polypeptides have a common N-terminal region of 76 amino acids that is encoded by exons 2 and 3 and separated by different eight (CALC) or six (alpha-CGRP) amino acid spacers from the biologically active polypeptide. The CALC-I/alpha-CGRP gene is a member of the calcitonin gene family and was assigned to chromosome CFA 16q25.1. A comparative analysis of different dog breeds revealed a breed-specific allelic d(CAGGAG)-hexanucleotide expansion in exon 3. This expansion results in an elongation of the common N-terminal region by two amino acids (glutamine-glutamic acid) and alters the molecular mass to 14.31 kDa (pI 7.9) and 14.13 kDa (pI 8.5) of the calcitonin and alpha-CGRP precursor, respectively.

A Novel High Mobility Group Protein Gene Is a Candidate for Xp22 Abnormalities in Uterine Leiomyomas and Other Benign Tumors

Because genes of the high mobility group protein family HMGI(Y) are known to take part in the development of a variety of benign solid tumors, the aim of the present study was to search for further members of that family in the human genome. Analysis for HMGI(Y)-related sequences by the polymerase chain reaction (PCR) with the use of cDNA-specific primers offered evidence for HMGIY-like sequences, whereas HMGIC-related sequences were apparently absent. By chromosomal assignment of somatic cell hybrids PCR, HMGIY cDNA-related sequences were detected on seven chromosomes. Positive clones were obtained by screening of a P1-derived artificial chromosome library and mapped by fluorescence in situ hybridization. One of these clones assigned to Xp22.1 was chosen for further analysis because Xp22 is a target region for clonal aberrations in benign solid tumors. Sequence analysis of a DNA fragment of this clone, designated as HMGIYL1, revealed a 94.4% homology to the coding region of HMGIY. Within the HMGIYL1 sequence, no nucleotide sequence divergences leading to a frame shift or a new termination codon compared to HMGIY were found, and a TATA-box-like motif 5′ of it was detected. By reverse transcriptase PCR experiments with the use of HeLa cells and human fetal tissue, HMGIYL1 expression was not detectable. Nevertheless, if not active by itself, it is possible that HMGIYL1 may become activated by chromosomal rearrangements of Xp22 observed in benign solid tumors.

Fetuin-B, a second member of the fetuin family in mammals

A set of orthologous plasma proteins found in human, sheep, pig, cow and rodents, now collectively designated fetuin-A, constitutes the fetuin family. Fetuin-A has been identified as a major protein during fetal life and is also involved in important functions such as inhibition of the insulin receptor tyrosine kinase activity, protease inhibitory activities and development-associated regulation of calcium metabolism and osteogenesis. Furthermore, fetuin-A is a key partner in the recovery phase of an acute inflammatory response. We now describe a second protein of the fetuin family, called fetuin-B, which is found at least in human and rodents. On grounds of domain homology, overall conservation of cysteine residues and chromosomal assignments of the corresponding genes in these species, fetuin-B is unambiguously a paralogue of fetuin-A. Yet, fetuin-A and fetuin-B exhibit significant differences at the amino acid sequence level, notably including variations with respect to the archetypal fetuin-specific signature. Differences and similarities in terms of gene regulation were also observed. Indeed, studies performed during development in rat and mouse showed for the first time high expression of a member of the fetuin family in adulthood, as shown with the fetuin-B mRNA in rat. However, like its fetuin-A counterpart, the fetuin-B mRNA level is down-regulated during the acute phase of experimentally induced inflammation in rat.

Cloning, characterization and chromosomal assignment of NBC4, a new member of the sodium bicarbonate cotransporter family

We report the cloning, characterization and chromosomal assignment of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC4, from human heart. NBC4 maps to chromosome 2p13 and is a new candidate gene for Alstrom syndrome. NBC4 encodes a 1074-residue polypeptide with 12 putative membrane-spanning domains. Unlike other members of the NBC family, NBC4 has a unique glycine-rich region (amino acids 438–485). In addition, NBC4 lacks the lysine-rich C-terminus of NBC1 with which it is most homologous. The first of two putative stilbene binding motifs (K(M/L)(X)K) is lacking in NBC4 (amino acids 655–658). The ∼6 kb NBC4 transcript is moderately expressed in heart, with the highest expression in liver, testes and spleen.

Fetuin-B, a second member of the fetuin family in mammals

A set of orthologous plasma proteins found in human, sheep, pig, cow and rodents, now collectively designated fetuin-A, constitutes the fetuin family. Fetuin-A has been identified as a major protein during fetal life and is also involved in important functions such as inhibition of the insulin receptor tyrosine kinase activity, protease inhibitory activities and development-associated regulation of calcium metabolism and osteogenesis. Furthermore, fetuin-A is a key partner in the recovery phase of an acute inflammatory response. We now describe a second protein of the fetuin family, called fetuin-B, which is found at least in human and rodents. On grounds of domain homology, overall conservation of cysteine residues and chromosomal assignments of the corresponding genes in these species, fetuin-B is unambiguously a paralogue of fetuin-A. Yet, fetuin-A and fetuin-B exhibit significant differences at the amino acid sequence level, notably including variations with respect to the archetypal fetuin-specific signature. Differences and similarities in terms of gene regulation were also observed. Indeed, studies performed during development in rat and mouse showed for the first time high expression of a member of the fetuin family in adulthood, as shown with the fetuin-B mRNA in rat. However, like its fetuin-A counterpart, the fetuin-B mRNA level is down-regulated during the acute phase of experimentally induced inflammation in rat.

Genomic organization, chromosomal assignment, and expression analysis of the mouse suppressor of fused gene (Sufu) coding a Gli protein partner.

Suppressor of fused (Sufu) is a negative regulator of the Hedgehog pathway both in Drosophila and vertebrates. Here, we report the genomic organization of the mouse Sufu gene (mSufu). This gene comprises 11 exons spanning more than 30 kb and encodes a protein with a putative PEST sequence. DNA-consensus sequences recognized by basic helix-loop-helix (bHLH) proteins, referred to as E-box motifs, are found in the 5' flanking region. Analysis by single-strand conformation polymorphism and radiation hybrid positioned the Sufu locus to the distal end of mouse Chr 19 between D19Mit102 and D19Mit9, near the Fgf8 and dactylin genes. Mouse Sufu is expressed in various tissues, particularly in the nervous system, ectoderm, and limbs, throughout the developing embryo. Sufu binds with all three Gli proteins, with different affinities. This report, in conjunction with recent studies, points out the importance of Sufu in mouse embryonic development.