Teresa L. Yang-Feng

Molecular analysis and chromosomal mapping of amplified genes isolated from a transformed mouse 3T3 cell line.

We are exploring the origin and function of amplified DNA sequences associated with double minutes (DMs) in a spontaneously transformed derivative of mouse 3T3 cells. Toward that goal, we have constructed a cDNA library using RNA from these cells and have isolated cDNA clones representing sequences that are amplified and overexpressed in these 3T3-DM cells. From results of Northern- and Southern-blot analyses, we conclude that these cDNAs represent two distinct genes, which we have designated mdm-1and mdm-2.Using DNAs from a panel of Chinese hamster-mouse somatic cell hybrids together with in situ hybridization protocols for gene mapping studies, we have found that these DM-associated, amplified DNA sequences originate from mouse chromosome 10, region C1–C3. Sequences homologous to mdm-1and mdm-2are present in the genomes of several species examined, including that of man.

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.

Exon‐Intron Structure, Analysis of Promoter Region, and Chromosomal Localization of the Human Type 1 σ Receptor Gene

Abstract: σ 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 ∼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 (∼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.

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. TheSMVT gene is ∼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.

Human Skeletal Muscle Insulin Receptor Substrate-1: Characterization of the cDNA, Gene, and Chromosomal Localization

Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has >50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5ʹ-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36–37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000–180,000 Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues. Compared with rat protein, human IRS-1 contains more potential Ser/Thr phosphorylation sites and only one nucleotide binding site. The entire protein coding sequence is contained within a single exon.

Colocalization of the genes for the α3(IV) and α4(IV) chains of type IV collagen to chromosome 2 bands q35–q37

Abstract Each type of basement membrane in man contains between two and five genetically distinct type IV collagens: α1(IV)–α5(IV). Genes for α1(IV), α2(IV), α3(IV), and α5(IV) have been isolated. We have recently isolated partial cDNAs for the fifth member of the family, designated α4(IV). On the basis of comparison of the deduced peptide sequences of all five chains, the type IV collagens can be divided into two families: α1-like, comprising α1(IV), α3(IV), and α5(IV); and α2-like, comprising α2(IV) and α4(IV). Genes encoding the α1(IV) and α2(IV) chains (COL4A1 and COL4A2) both map to human chromosome 13q34 and have been shown to be transcribed from opposite DNA strands using a common bidirectional promoter that allows coordinate regulation of the two chains. Indeed, these two chains are commonly found together in basement membrane and form [ α 1] 2 ·[ α 2] heterotrimers. Whereas α1(IV) and α2(IV) have been found in all basement membranes studied hitherto, it has been shown that α3(IV) and α4(IV) are found in only a subset of basement membranes. In basement membranes where either of these molecules is present, however, they are found together. In view of this relationship and the structural similarities between α1(IV) and α3(IV) and between α2(IV) and α4(IV), we hypothesized that COL4A3 and COL4A4, the genes encoding α3(IV) and α4(IV), respectively, have a genomic organization similar to that of COL4A1 and COL4A2. As a first test of this hypothesis we decided to determine whether COL4A3 and COL4A4 map to the same region of the human genome. We have previously shown that COL4A3 maps to chromosome 2 band q35–q37. Here we show, by somatic cell hybrid analysis and in situ hybridization, that COL4A4 colocalizes with COL4A3. Further experiments will be required to determine the genomic organization of these genes.

Cytogenetic results of chorionic villus sampling: high success rate and diagnostic accuracy in the United States collaborative study.

Cytogenetic results of first-trimester chorionic villus sampling are reported from seven U.S. medical centers. For 6033 patients who had a successful chorionic villus sampling procedure, the rate for obtaining a cytogenetic diagnosis was 99.6% with the direct method, long-term culture, or both. There were no incorrect sex predictions and no diagnostic errors involving trisomies 21, 18, or 13, sex chromosome aneuploidies, or structural abnormalities. There were no cases of normal cytogenetic diagnosis followed by birth of a cytogenetically abnormal infant. Three cases of unusual aneuploidies (tetraploidy, trisomy 16, and trisomy 22) detected by the direct method only were not confirmed by cytogenetic follow-up. Mosaic cytogenetic abnormalities were observed in 0.83% of all cases in which chorionic villus sampling was done but were confirmed by amniocentesis or in fetal tissues in only 7 of 30 cases (23.3%). Maternal cell contamination occurred in 1.9% of long-term cultures, although this did not present any cytogenetic diagnostic difficulties. Overall, a very high degree of laboratory success and diagnostic accuracy was observed with either cytogenetic method, although fewer predictive errors were observed with the long-term culture method and none were observed when both methods were used.

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.

Mapping of genes for inhibin subunits α, βA, and βB on human and mouse chromosomes and studies of jsd mice

Abstract Inhibin (INH) is a gonadal glycoprotein hormone that regulates pituitary FSH secretion and may also play a role in the regulation of androgen biosynthesis. There are two forms of inhibin that strongly inhibit pituitary FSH secretion. These share the same α subunit that is covalently linked to one of two distinct β subunits ( β A or β B . However, dimers of two β subunits are potent stimulators of FSH synthesis and release in vitro . The β subunits share extensive sequence similarity with transforming growth factor β. Recently isolated cDNAs for all three inhibin subunits have been used to map their cognate loci on human and mouse chromosomes by Southern blot analysis of somatic cell hybrid DNAs and by in situ hybridization. INHα and INH β B genes were assigned to human chromosome 2, regions q33 → qter and cen → q13, respectively, and to mouse chromosome 1. The INH β A locus was mapped to human chromosome 7p15 → p14 and mouse chromosome 13. The region of mouse chromosome 1 that carries other genes known to have homologs on human chromosome 2q includes the jsd locus (for juvenile spermatogonial depletion). Adult jsd jsd mice have elevated levels of serum FSH and their testes are devoid of spermatogonial cells. The possibility that the mutation in jsd involves the INHα or INH β B gene was investigated by Southern blotting of DNA from jsd jsd mice, and no major deletions or rearrangements were detected.

Chromosomal localization of human Na+,K+-ATPase α- and β-subunit genes

Abstract Na+,K+-ATPase is a heterodimeric enzyme responsible for the active maintenance of sodium and potassium gradients across the plasma membrane. Recently, cDNAs for several tissue-specific isoforms of the larger catalytic α-subunit and the smaller β-subunit have been cloned. We have hybridized rat brain and human kidney cDNA probes, as well as human genomic isoform-specific DNA fragments, to Southern filters containing panels of rodent × human somatic cell hybrid lines. The results obtained have allowed us to assign the loci for the ubiquitously expressed α-chain (ATP1A1) to human chromosome 1, region 1p21→cen, and for the α2 isoform that predominates in neural and muscle tissues (ATP1A2) to chromosome 1, region cen→q32. A common PstI RFLP was detected with the ATP1A2 probe. The α3 gene, which is expressed primarily in neural tissues (ATP1A3), was assigned to human chromosome 19. A fourth α gene of unknown function (αD) that was isolated by molecular cloning (ATP1AL1) was mapped to chromosome 13. Although evidence to date had suggested a single gene for the β-subunit, we found hybridizing restriction fragments derived from two different human chromosomes. On the basis of knowledge of conserved linkage goups on human and murine chromosomes, we propose that the coding gene ATP1B is located on the long arm of human chromosome 1 and that the sequence on human chromosome 4 (ATP1BL1) is either a related gene or a pseudogene.