Cloning and functional expression of human retinal kir2.4, a pH-sensitive inwardly rectifying K(+) channel.

To identify novel potassium channel genes expressed in the retina, we screened a human retina cDNA library with an EST sequence showing partial homology to inwardly rectifying potassium (Kir) channel genes. The isolated cDNA yielded a 2,961-base pair sequence with the predicted open reading frame showing strong homology to the rat Kir2. 4 (rKir2.4). Northern analysis of mRNA from human and bovine tissues showed preferential expression of Kir2.4 in the neural retina. In situ hybridization to sections of monkey retina detected Kir2.4 transcript in most retinal neurons. Somatic hybridization analysis and dual-color in situ hybridization to metaphase chromosomes mapped Kir2.4 to human chromosome 19 q13.1-q13.3. Expression of human Kir2. 4 cRNA in Xenopus oocytes generated strong, inwardly rectifying K(+) currents that were enhanced by extracellular alkalinization. We conclude that human Kir2.4 encodes an inwardly rectifying K(+) channel that is preferentially expressed in the neural retina and that is sensitive to physiological changes in extracellular pH.

Genomic structure, expression, and chromosomal localization of the human glycine N-methyltransferase gene.

The glycine N-methyltransferase (GNMT) gene encodes a protein that not only acts as an enzyme to regulate the ratio of S-adenosylmethionine to S-adenosylhomocysteine, but also participates in the detoxification pathway in liver cells. Previously, we reported that the expression level of GNMT was diminished in human hepatocellular carcinoma. In this study, the human GNMT gene was cloned and characterized. It contains six exons and spans about 10 kb. Instead of a TATA box, it has a transcriptional initiator located 801 bp upstream from the translation start codon. The gene was localized to chromosome 6p12 using fluorescence in situ hybridization. Northern blot analysis of 16 tissues from different human organs showed that GNMT was expressed only in liver, pancreas, and prostate.

Structure, function, and genomic organization of human Na(+)-dependent high-affinity dicarboxylate transporter.

We have cloned and functionally characterized the human Na(+)-dependent high-affinity dicarboxylate transporter (hNaDC3) from placenta. The hNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the cloned transporter mediates the transport of succinate in the presence of Na(+) [concentration of substrate necessary for half-maximal transport (K(t)) for succinate = 20+/-1 microM]. Dimethylsuccinate also interacts with hNaDC3. The Na(+)-to-succinate stoichiometry is 3:1 and concentration of Na(+) necessary for half-maximal transport (K(Na(+))(0.5)) is 49+/-1 mM as determined by uptake studies with radiolabeled succinate. When expressed in Xenopus laevis oocytes, hNaDC3 induces Na(+)-dependent inward currents in the presence of succinate and dimethylsuccinate. At a membrane potential of -50 mV, K(Suc)(0.5) is 102+/-20 microM and K(Na(+))(0.5) is 22+/-4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer and radiolabeled succinate uptake in hNaDC3-expressing oocytes indicate a charge-to-succinate ratio of 1:1 for the transport process, suggesting a Na(+)-to-succinate stoichiometry of 3:1. pH titration of citrate-induced currents shows that hNaDC3 accepts preferentially the divalent anionic form of citrate as a substrate. Li(+) inhibits succinate-induced currents in the presence of Na(+). Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The human NaDC3 gene is located on chromosome 20q12-13.1, as evidenced by fluorescent in situ hybridization. The gene is >80 kbp long and consists of 13 exons and 12 introns.

Expression profile and chromosomal location of cDNA clones, identified from an enriched adult retina library.

PURPOSE: To delineate the profile of genes expressed in the adult human retina and assign chromosomal location of cDNA clones. METHODS: The end-sequence of random clones from an enriched human retinal cDNA library was analyzed by NCBI database search. Expression profile was established by northern blot analysis, database search, or both. Selected cDNA clones were localized to human chromosomes by somatic cell hybrid analysis, in situ hybridization to metaphase chromosomes, or both. Chromosomal location was also obtained by searching the databases. RESULT: One hundred and thirty-seven clones were isolated from the subtracted retinal library. Fifty-one clones were identical with 35 known human genes in GenBank, and 24 clones corresponded to 23 uncharacterized human expressed sequenced tags (ESTs), novel genes, or both. The remaining 59 clones were not pursued further because they contained bacterial sequences or repetitive elements. Several clones indicated a restricted pattern of expression with high levels of transcripts in the retina. Chromosomal location of novel retinal ESTs is also reported. CONCLUSIONS: This study provides a profile of genes expressed in the adult human retina. One round of subtraction eliminated most constitutively expressed genes and permitted partial normalization of the retinal library. Twenty-three novel genes were identified. The combined information obtained from expression analysis and chromosomal localization of retinal cDNAs should be valuable in identifying candidate genes for diseases involving retinal dysfunction.

Human placental Na+-dependent multivitamin transporter. Cloning, functional expression, gene structure, and chromosomal localization.

We have cloned the human Na+-dependent multivitamin transporter (SMVT), which transports the water-soluble vitamins pantothenate, biotin, and lipoate, from a placental choriocarcinoma cell line (JAR). The cDNA codes for a protein of 635 amino acids with 12 transmembrane domains and 4 putative sites for N-linked glycosylation. The human SMVT exhibits a high degree of homology (84% identity and 89% similarity) to the rat counterpart. When expressed in HRPE cells, the cDNA-induced transport process is obligatorily dependent on Na+ and accepts pantothenate, biotin, and lipoate as substrates. The relationship between the cDNA-specific uptake rate of pantothenate or biotin and Na+ concentration is sigmoidal with a Na+:vitamin stoichiometry of 2:1. The human SMVT, when expressed in Xenopus laevis oocytes, induces inward currents in the presence of pantothenate, biotin, and lipoate in a Na+-, concentration-, and potential-dependent manner. We also report here on the structural organization and chromosomal localization of the human SMVT gene. The SMVT gene is approximately 14 kilobase pairs in length and consists of 17 exons. The SMVT gene is located on chromosome 2p23 as evidenced by somatic cell hybrid analysis and fluorescence in situ hybridization.

Exon-intron structure, analysis of promoter region, and chromosomal localization of the human type 1 sigma receptor gene.

Sigma receptor is a protein that interacts with a variety of psychotomimetic drugs including cocaine and amphetamines and is believed to play an important role in the cellular functions of various tissues associated with the endocrine, immune, and nervous systems. Here we report on the structure and organization of the human gene coding for this receptor. The gene is approximately 7 kbp long and contains four exons, interrupted by three introns. Exon 3 is the shortest (93 bp), and exon 4 is the longest (1,132 bp). Among the introns, intron 3 is the longest (approximately 1,250 bp). Exon 2 codes for the single transmembrane domain present in the receptor. 5' rapid amplification of cDNA end reactions with mRNA from the JAR human trophoblast cell line have identified 56 bp upstream of the translation start codon as the initiation site for transcription. This transcription start site has been confirmed by RNase protection analysis. Structural analysis of the 5' flanking region has revealed that the gene is TATA-less. This region, however, contains a CCAATC box in the reverse complement and several GC boxes that are recognition sites for SP1. There are also consensus sequences for the liver-specific transcription factor nuclear factor-1/L, for a variety of cytokine responsive factors, and for the xenobiotic responsive factor called the arylhydrocarbon receptor. Southern blot analysis of the genomic DNA from Chinese hamster-human and mouse-human hybrid cell lines and fluorescent in situ hybridization with human metaphase chromosome spreads have shown that the gene is located on human chromosome 9, band p13, a region known to be associated with different psychiatric disorders.

Familial dup(5)(q15q21) associated with normal and abnormal phenotypes.

We studied a familial dup(5q) present in a phenotypically normal father and his monozygotic twin daughters with different abnormal phenotypes. High-resolution chromosome analysis suggested that the duplicated segment was of region q15-21, which seems to be the smallest dup(5q) reported thus far. This dup(5q) was confirmed by fluorescence in situ hybridization with a chromosome 5 painting library and 5q cosmid clones. The presence of the dup(5q) in a normal father suggested that the duplication itself may be harmless. The anomalies in the twins may be due to processes other than this chromosome change.

A thyroid hormone receptor coactivator negatively regulated by the retinoblastoma protein.

The retinoblastoma protein (Rb) plays a critical role in cell proliferation, differentiation, and development. To decipher the mechanism of Rb function at the molecular level, we have systematically characterized a number of Rb-interacting proteins, among which is the clone C5 described here, which encodes a protein of 1,978 amino acids with an estimated molecular mass of 230 kDa. The corresponding gene was assigned to chromosome 14q31, the same region where genetic alterations have been associated with several abnormalities of thyroid hormone response. The protein uses two distinct regions to bind Rb and thyroid hormone receptor (TR), respectively, and thus was named Trip230. Trip230 binds to Rb independently of thyroid hormone while it forms a complex with TR in a thyroid hormone-dependent manner. Ectopic expression of the protein Trip230 in cells, but not a mutant form that does not bind to TR, enhances specifically TR-dependent transcriptional activity. Coexpression of wild-type Rb, but not mutant Rb that fails to bind to Trip230, inhibits such activity. These results not only identify a coactivator molecule that modulates TR activity, but also uncover a role for Rb in a pathway that responds to thyroid hormone.

Cytogenetic and molecular studies of a familial paracentric inversion of Y chromosome present in a patient with ambiguous genitalia.

Here we describe the first reported case of a patient with a familial paracentric inversion in the long arm of the Y chromosome and ambiguous genitalia. FISH analyses with Y chromosome YACs demonstrated that the inversion breakpoints of the patients and the father's Ys appear to be the same and lie within interval 5B of the Y chromosome. PCR and sequence analysis indicated that our patient carries a normal SRY gene. For an additional comparison of the patient's inv(Y) with the father, two other Y chromosome sequences were examined. Molecular studies of this familial inverted Y chromosome showed no differences in the ZFY and TSPY genes between the father and the patient suggesting that the short arm of our patient's inv(Y) is identical to that of the patient's father. Southern analysis using a probe of the DAX-1 gene indicated that a single copy of DSS (dosage sensitive sex reversal) locus was present in the patient. Our results suggest that the abnormal sexual development in our patient is likely attributable to (an)other mechanism(s) than mutation in the SRY gene and dosage alteration of the DAX-1 gene.

A complex chromosome rearrangement with at least five breakpoints studied by fluorescence in situ hybridization.

A newborn infant with multiple congenital anomalies was diagnosed with an unbalanced translocation of chromosomes 1 and 5. Studies of parental chromosomes revealed a complex rearrangement in the patient's mother involving the exchange of terminal long arms between chromosomes 1 and 5 and the insertion of an interstitial segment from the same chromosome 5q into chromosome 2q by high-resolution G-banding. Further study of the mother's chromosomes by fluorescent in situ hybridization (FISH) detected an additional insertion between the rearranged chromosomes 2 and 5, which was not revealed by G-banding. This led to the identification of a complex translocation-insertion between 3 chromosomes with at least 5 breaks [t(1;5;2)(1pter--> 1q42.3::5q23.2-->5qter;5pter-->5q21.2:: 2q33--> 2q35::1q42.3-->1qter;2pter-->2q33::5q21 .2--> 5q23.2::2q35-->2qter)] and illustrates the value of FISH as an adjunct to standard cytogenetics, particularly in cases of complex rearrangements.

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, and Npy6r, 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, PPYR1 and NPY5R, 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.

Characterization of the human type 2 neuropeptide Y receptor gene (NPY2R) and localization to the chromosome 4q region containing the type 1 neuropeptide Y receptor gene.

Neuropeptide Y (NPY) signals through a family of G-protein-coupled receptors present in the brain and sympathetic neurons. To further our understanding of the genetic elements involved in the regulation of NPY receptor expression, we have cloned and characterized the human gene encoding the type 2 NPY receptor (Y2 receptor, HGMW-approved symbol NPY2R).2 The transcript spans 9 kb of genomic sequence and is encoded on two exons. As in the type 1 NPY receptor (Y1 receptor) gene, the 5'-untranslated region of the Y2 receptor is interrupted by an intervening sequence ( approximately 4.5 kb). However, the Y2 receptor gene does not contain an intron analogous to that present in the coding region of the Y1 receptor. The predicted transcript size ( approximately 4.5 kb) is consistent with the size observed by Northern analysis. The 381-amino-acid sequence deduced from the open reading frame is identical to that encoded by the cDNA. The Y2 receptor gene maps to human chromosome 4q31, the same region containing the Y1 receptor locus, suggesting that these subtypes may have arisen by gene duplication despite their structural differences.

Cloning of the sodium-dependent, broad-scope, neutral amino acid transporter Bo from a human placental choriocarcinoma cell line.

We have isolated a cDNA from a human placental choriocarcinoma cell cDNA library which, when expressed in HeLa cells, induces a Na+-dependent amino acid transport system with preference for zwitterionic amino acids. Anionic amino acids, cationic amino acids, imino acids, and N-methylated amino acids are excluded by this system. These characteristics are identical to those described for the amino acid transporter Bo. When expressed in Xenopus laevis oocytes that do not have detectable endogenous activity of the amino acid transporter Bo, the cloned transporter increases alanine transport in the oocytes severalfold and induces alanine-evoked inward currents in the presence of Na+. The cDNA codes for a polypeptide containing 541 amino acids with 10 putative transmembrane domains. Amino acid sequence homology predicts this transporter (hATBo) to be a member of a superfamily consisting of the glutamate transporters, the neutral amino acid transport system ASCT, and the insulin-activable neutral/anionic amino acid transporter. Chromosomal assignment studies with somatic cell hybrid analysis and fluorescent in situ hybridization have located the ATBo gene to human chromosome 19q13.3.

Prenatal detection of two different monosomic cell lines by chorionic villus sampling.

We present a prenatal case of mosaicism with at least two monosomy cell lines: one with monosomy 21 (45,XY,-21) and one missing the Y (45,X) and a possible third 46,XY in chorionic villus cell culture. Cytogenetic studies were initiated following the ultrasound detection at 11 weeks of a large cystic hygroma and in utero growth retardation. Spontaneous fetal demise occurred at 12 weeks and the pregnancy was terminated. To our knowledge, this is the first report of two different monosomic cell lines found in chorionic villus cells.

Frequent spontaneous deletions at a shuttle vector locus in transgenic mice.

Transgenic mice carrying multiple copies of a recoverable lambda phage shuttle vector (lambda supF) were constructed for the purpose of studying mutagenesis in a whole animal. Spontaneous mutations in rescued supF target genes from several different lines of transgenic mice were analyzed. One mouse line, 1139, was identified in which the frequency of spontaneous mutations was unusually high (3.15 x 10(-4)), 20-fold higher than in other transgenic mice carrying a similar number of copies of the lambda transgene (approximately 100). Over 75% of the spontaneous mutations from 1139 mice were found to be deletions, whereas mostly point mutations were recovered from the other mice. In 1139 no significant variation among adult tissues has been detected. However, embryonic tissue yielded a 3- to 4-fold lower frequency of mutations, most of which were point mutations rather than deletions. The frequency of mutations at another locus, the hypoxanthine phosphoribosyl transferase gene, was not elevated in fibroblast lines established in culture from the 1139 mice. Overall, these results suggest that the deletion mutagenesis affecting the transgene sequences in 1139 mice is a locus-specific effect occurring during growth and development. The increased mutagenesis could not be explained by the degree of methylation of the transgene sequences, since hypermethylation was seen in both 1139 mice and other mice with a low frequency of shuttle vector mutations. The integrated lambda vector DNA in 1139 mice was mapped to a single site on chromosome 7, but no mechanism for the mutagenesis was suggested by this localization. It is proposed that the lambda DNA may have either integrated into an unstable genomic site or created a newly unstable locus in the process of integration.

Genetic analysis of benign, low-grade, and high-grade ovarian tumors.

Genetic abnormalities were assessed in 56 benign, low-, and high-grade ovarian tumors using comparative genomic hybridization (CGH) and analysis of loss of heterozygosity (LOH). In addition, 95 epithelial tumors were analyzed for microsatellite repeat instability. DNA sequence copy number abnormalities (CNAs) were not detected in the benign tumors, and more were detected in high-grade than in low-grade cancers. Almost no microsatellite repeat instability was detected in these cancers. CNAs occurring in more than 30% of the cancers included increased copy number on 3q25-26 and 8q24 and reduced copy number on 16q and 17pter-q21. Another 14 CNAs occurred in more than 20% of the cancers. Increased copy number at 3q25-26 and 20q13 was the most frequent CNA in low-grade tumors, and increased copy number at 8q24 occurred preferentially in high-grade tumors. The presence of a large number of CNAs per tumor was significantly correlated with reduced patient survival duration. Reduced copy number on 17pter-q21 was most strongly associated with accumulation of a large number of CNAs. The overall concordance between LOH and reduced copy number detected by CGH was 84%, but only 31% of the LOH was associated with reduced copy number detected using CGH.

Allelic deletion mapping of putative tumor suppressor genes on 17q in sporadic ovarian cancer.

OBJECTIVES: 1) To study whether the same or different chromosome 17q genes may be involved in the oncogenesis of familial and sporadic ovarian malignancies. 2) To localize the candidate gene in the sporadic ovarian cancers. METHODS (STUDY DESIGN): Using DNA extracted from ovarian tumors and corresponding peripheral leukocyte, we examined the status of loss of heterozygosity (LOH) at 12 loci spanning chromosome 17q12-q25 by Southern hybridization and polymerase chain reaction. RESULTS: Comparison of the extent of LOH among 25 epithelial ovarian tumors showing allele loss at one or more loci on 17q, the smallest overlapping region of allelic deletion is between D17S579 and GIP, with a genetic distance of approximate 2 cM. CONCLUSIONS: From our allelic deletion analysis of chromosome 17q loci, it indicates that there are more than one ovarian cancer candidate genes on chromosome 17q.

A de novo satellited short arm of the Y chromosome possibly resulting from an unstable translocation.

A satellited long arm of the Y chromosome (Yqs) is considered a normal variation, whereas the presence of a satellite on the short arm of the Y (Yps) has never been described in the literature. A Yps chromosome could be clinically significant if the translocation resulting in Yps has relocated the testis-determining gene, SRY, to another chromosome. A carrier of such a translocation would therefore be at increased risk for having XX male and XY female offspring. Here we describe the first reported case of de novo Yps present in a phenotypically normal male. This Yps chromosome was positive for C-banding and nucleolus organizer region (NOR) staining and showed a hybridization signal for the beta-satellite sequence. Fluorescence in situ hybridization (FISH) analysis indicated that SRY was retained on the Yps and the translocation breakpoint on Yps was distal to the pseudoautosomal region. At prenatal diagnosis, a normal appearing Y chromosome was found in his son, and thus the satellite on Yps was lost during meiotic Xp-Yp pairing. This Yps chromosome was likely the product of an "unstable" translocation.

Proton/peptide cotransporter (PEPT 2) from human kidney: functional characterization and chromosomal localization.

We report here on the functional characterization of the H+/peptide cotransporter PEPT 2 cloned from human kidney and on the chromosomal localization of the PEPT 2 gene. PEPT 2, when functionally expressed in HeLa cells, induces the transport of the neutral dipeptide glycylsarcosine. The induced transport activity is markedly influenced by extracellular pH. The optimum pH for the transport process is 6.0-7.0. Kinetic analysis has revealed that PEPT 2 is a high-affinity transporter, the Michaelis-Menten constant for glycylsarcosine being 74 +/- 14 microM. The human intestinal H+/peptide cotransporter PEPT 1 has 4-fold less affinity for the dipeptide under identical experimental conditions. Studies with other chemically diverse dipeptides have established that PEPT 2 possesses higher affinity than PEPT 1 not only for neutral peptides but also for peptides consisting of anionic and/or cationic amino acids. Somatic cell hybrid analysis and in situ hybridization have shown that the gene encoding PEPT 2 maps to human chromosome 3q13.3-q21.