Biomedical subjects

C L Hsieh

Publications and source records attributed to C L Hsieh.

At least 109 records · Page 6Linked to original sources

PubMed1991

V(D)J recombination: evidence that a replicative mechanism is not required.

We examined a series of extrachromosomal DNA substrates for V(D)J recombination under replicating and nonreplicating conditions. Complete and partial replications were examined by monitoring the loss of prokaryote-specific adenine methylation at 14 to 22 MboI-DpnI restriction sites (GATC) on the substrates. Some of these sites are within 2 bases of the signal sequence ends. We found that neither coding joint nor signal joint formation requires substrate replication. After ruling out replication as a substrate requirement, we determined whether replication had any effect on the efficiency of V(D)J recombination. Quantitation of V(D)J recombination efficiency on nonreplicating substrates requires some method of monitoring the entry of substrate molecules into the cells. We devised such a method by monitoring DNA repair of substrates into which we had substituted deoxyuridine for 10 to 20% of the thymidine nucleotides in the DNA. The substrates which enter the lymphoid cells were repaired efficiently in vivo by the eukaryotic uracil DNA repair system. Upon plasmid harvest, we distinguished repaired (entered) from unrepaired (not entered) plasmids by cleaving unrepaired molecules with uracil DNA glycoylase and Escherichia coli endonuclease IV in vitro. This method of monitoring DNA entry does not appear to underestimate or overestimate the amount of DNA entry. By using this method, we found no significant quantitative effect of DNA replication on V(D)J recombination efficiency.

Animals↗

PubMed1991

Chromosomal localization of the gene for AA-type platelet-derived growth factor receptor (PDGFRA) in humans and mice.

The full-length cDNA of the receptor for human AA-type platelet-derived growth factor (PDGF) was used to assign the PDGFRA gene to region q11----q21 of human chromosome 4 and to mouse Chromosome 5 by somatic cell hybrid analysis. Since the same region also contains the c-kit oncogene homolog KIT, we carried out pulsed-field gel electrophoresis to determine the physical distance between the two genes in human DNA. The two probes, when successively applied to the same filters, hybridized to a 450-kb EagI-fragment but not to other common restriction fragments. The genes are separated by at least one NotI, one XhoI, and one SalI site.

Animals↗

PubMed1991

Homozygous tyrosinase gene mutation in an American black with tyrosinase-negative (type IA) oculocutaneous albinism.

We have identified a tyrosinase gene mutation in an American black with classic, tyrosinase-negative oculocutaneous albinism. This mutation results in an amino acid substitution (Cys----Arg) at codon 89 of the tyrosinase polypeptide. The proband is homozygous for the substitution, suggesting that this mutation may be frequently associated with tyrosinase-negative oculocutaneous albinism in blacks.

Adult↗

PubMed1990

Isotype switching of an immunoglobulin heavy chain transgene occurs by DNA recombination between different chromosomes.

Transgenic mice carrying an immunoglobulin mu heavy chain transgene exhibit isotype switching of the transgene. We have now characterized the mechanism of transgene switching in these mice. The site of mu transgene insertion in one transgenic line has been localized to chromosome 5 using a series of polymorphic endogenous retroviruses as genetic markers in backcross mice. The endogenous immunoglobulin heavy chain locus resides on mouse chromosome 12, which shows that transgene isotype switching can occur between two different chromosomes even though normal antibody gene switching has generally been thought to occur within one chromosome. We find that transgene isotype switching involves interchromosomal DNA recombination, and our data suggest that the same enzymatic mechanisms mediate both normal isotype switch recombination and interchromosomal transgene switching. Our findings also support the notion that the isotype switching mechanism can induce chromosomal translocations such as observed for the c-myc gene in some B cell tumors.

Animals↗

PubMed1990

Molecular structure, chromosome assignment, and promoter organization of the human matrix Gla protein gene.

Matrix Gla protein (MGP) is an 84-residue vitamin K-dependent protein initially isolated from bovine bone. MGP is also expressed at high levels in heart, kidney, and lung and is up-regulated by vitamin D in bone cells. To characterize the genomic sequences responsible for the regulated expression of this gene, we screened a human genomic library using a MGP cDNA probe and obtained two clones containing the MGP locus. The human MGP gene spans 3.9 kilobases of chromosomal DNA and consists of four exons separated by three large intervening sequences which account for more than 80% of the gene. Southern analysis of total human genomic DNA indicated the presence of a single copy of the MGP gene. Hybridization of the hMGP cDNA to a series of Chinese Hamster x human hybrid clones assigned this gene to the short arm of the human chromosome 12 (12p). The N-terminal sequences of the known vitamin K-dependent vertebrate proteins reveal a transmembrane signal peptide, followed by a putative gamma-carboxylation recognition site and a Gla-containing domain. Each of these regions correspond to a separate exon in MGP. MGP also contains a fourth exon of unknown function which codes for 11 residues and lies between the transmembrane signal peptide and the putative recognition site for the gamma-carboxylase. This four-exon organization is essentially identical to that of bone Gla protein and is quite different from the two exon organization encoding this region in the other known vitamin K-dependent proteins. Analysis of the MGP gene promoter revealed, in addition to the typical TATA and CAT boxes, the presence of a number of putative regulatory sequences homologous to previously identified hormone and transcription factor responsive elements. In particular, two regions of the promoter were delineated containing possible binding sites for retinoic acid and vitamin D receptors.

Amino Acid Sequence↗

PubMed1990

CD14 is a member of the family of leucine-rich proteins and is encoded by a gene syntenic with multiple receptor genes.

We have isolated and characterized genomic and cDNA clones encoding the murine homolog of the human monocyte/granulocyte cell surface glycoprotein, CD14. As in man, the expression of murine CD14 is limited to the myeloid lineage. The murine and human CD14 genes are highly conserved in their intron-exon organization and nucleotide sequence. Their deduced protein sequences show 66% amino acid identity. In both mouse and man, the CD14 protein contains a repeating (10 times) leucine-rich motif (LXXLXLX) that is also found in a group of heterogeneous proteins from phylogenetically distant species. The CD14 gene has been mapped to mouse chromosome 18 which also contains at least five genes encoding receptors (Pdgfr, Adrb2r, li, Grl-1, Fms). Thus CD14 and the receptor genes form a conserved syntenic group localized on mouse chromosome 18 and human chromosome 5. The inclusion of CD14 in the family of leucine-rich proteins, its expression profile and the murine chromosomal localization support the hypothesis that CD14 may function as a receptor.

Amino Acid Sequence↗

PubMed1990

A regular pattern of two types of 100-residue motif in the sequence of titin.

Titin is the largest polypeptide yet described (relative molecular mass approximately 3 x 10(6); refs 1, 2) and an abundant protein of striated muscle. Its molecules are string-like and in vivo span from the M to Z-lines. I-band regions of titin are thought to make elastic connections between the thick filament and the Z-line, thereby forming a third type of sarcomere filament. These would centre the A-band in the sarcomere and provide structural continuity in relaxed myofibrils. The A-band region of titin seems to be bound to the thick filament, where it has been proposed to act as a 'molecular ruler' regulating filament length and assembly. Here, we show that partial titin complementary DNAs encode a regular pattern of two types of 100-residue motif, each of which probably folds into a separate domain type. Such motifs are present in several evolutionarily divergent muscle proteins, all of which are likely to interact with myosin. One or both of the domain types is therefore likely to bind to myosin.

Amino Acid Sequence↗

PubMed1990

Altered structure and expression of the human retinoblastoma susceptibility gene in small cell lung cancer.

Karyotypic and molecular genetic evidence has indicated that deletion or rearrangement of both chromosomes 3 and 13 may be important in the pathology of human small cell lung cancer (SCLC). The retinoblastoma susceptibility gene, RB, on chromosome 13 band q14, has previously been shown to be altered in SCLC [J. W. Harbour et al., Science (Wash. DC), 241: 353-357, 1988; J. Yokota et al., Oncogene, 3: 471-475, 1988]. Our studies of 26 SCLC tumor and normal DNA samples indicate that 6 of 6 patients whose normal cell DNA was heterozygous for an RB restriction fragment length polymorphism have lost one of the two alleles in their tumor DNA. Consistent with other studies, we find 2 of 26 tumors with homozygous deletions within the RB gene. Of 13 SCLC cell lines examined, only 3 expressed greater than trace amounts of RB mRNA. RB protein was detected in 2 of 14 SCLC cell lines examined, unlike the results of Yokota et al. (Oncogene, 3: 471-475, 1988) which showed no RB protein in any of the 9 cell lines they examined. Only unphosphorylated RB protein was detected in SCLC cell line H209, suggesting that the RB protein may be inactivated by a novel mechanism in this cell line. These data suggest that inactivation of the RB gene is a frequent if not universal event in SCLC.

Alleles↗

PubMed1990

Assignment of Amog (adhesion molecule on glia) gene to mouse chromosome 11 near Zfp-3 and Asgr-1,2 and to human chromosome 17.

AMOG, identified as an adhesion molecule that mediates neuron-astrocyte interaction, has structural similarity to the beta-subunit of Na,K ATPase. We have mapped the AMOG gene to human chromosome 17 and mouse chromosome 11 by somatic cell hybrid analysis. Recombinant inbred strain mapping has placed the Amog locus close to genes for zinc finger protein-3 and the asialoglycoprotein receptor in a region of mouse chromosome 11 that is homologous to human 17p.

Adenosine Triphosphatases↗

PubMed1990

Chromosomal localization and cDNA sequence of human ralB, a GTP binding protein.

The human ra1 proteins share more than 50% homology and some properties with the ras proteins. The RALB gene, a new member of the ra1 family, has been isolated, sequenced, and assigned to human chromosome 2 cen-q13. One other member of this family, RALA, has been previously mapped to the short arm of human chromosome 7.

Amino Acid Sequence↗

PubMed1990

The serotonin receptor subtype 2 locus HTR2 is on human chromosome 13 near genes for esterase D and retinoblastoma-1 and on mouse chromosome 14.

Serotonin (5-hydroxytryptamine) functions as a neurotransmitter and a hormone. Its diverse actions are mediated by at least seven distinct cell surface receptor subtypes. The serotonin receptor subtype 2 (gene symbol HTR2) is a G-protein-coupled receptor, expressed primarily in the cerebral cortex, where upon stimulation it stimulates the hydrolysis of inositol phospholipids. We have mapped the HTR2 locus to human chromosome 13 and to mouse chromosome 14 by somatic cell hybrid analysis. Linkage studies in CEPH families, using a PvuII RFLP detected with the HTR2 probe, revealed tight linkage between HTR2 and ESD, the locus for esterase D. The most likely position for HTR2 is between ESD and RB1, the retinoblastoma-1 gene. The homologous loci in mouse, Rb-1 and Esd(Es-10) are on mouse chromosome 14, close to ag, agitans, a recessive neurological mutation. Having mapped Htr-2 to mouse chromosome 14, we predict that it falls into this known conserved gene cluster.

Animals↗

PubMed1990

The gene for the RNA component of the mitochondrial RNA-processing endoribonuclease is located on human chromosome 9p and on mouse chromosome 4.

Mitochondrial RNA-processing endoribonuclease (RNAase MRP) has the capacity to cleave mitochondrial RNA complementary to the light strand of the displacement loop at a unique site. The enzyme is a ribonucleoprotein whose RNA component is a nuclear gene product. The 5' flanking region of the primary transcript has control elements characteristic of RNA polymerase II transcription, and the coding region has features of RNA polymerase III transcription signals. The RNA associated with RNAase MRP is the first known RNA encoded by a single-copy gene in the nucleus and believed to be imported into mitochondria. The gene (RMRP) for this RNA component of RNAase MRP was assigned to human chromosome 9 and mouse chromosome 4 by Southern blot analyses of 11 human X rodent hybrids and 11 mouse X rodent hybrids with probe pHM1.0 and probe pSP270, respectively. In situ hybridization of probe pHSTU300 to normal human chromosomes revealed 29 of 100 cells with label on 9p and 9.6% of 302 silver grains located at 9p21--p12.

Animals↗

PubMed1990

The gene for the ubiquitous octamer-binding protein Oct-1 is on human chromosome 1, region cen-q32, and near Ly-22 and Ltw-4 on mouse chromosome 1.

Oct-1 is a sequence-specific transcription and DNA replication factor that recognizes the octameric sequence ATGCAAAT. This protein shares an extended region of sequence similarity, called the POU domain, with the lymphoid-specific transcription factor Oct-2, the pituitary-specific transcription factor Pit-1, and the Caenorhabditis elegans cell lineage gene product unc-86. Two subdomains, POU-related homeobox and POU-specific box, lie within the POU domain. Unlike other POU or homeodomain proteins, Oct-1 is ubiquitously expressed. A ubiquitous 95- to 100-kDa protein that has the same DNA-binding properties and is variously referred to as OTF-1, NFIII, or OBP100 is probably identical to Oct-1. The human gene was mapped by Southern blot analyses of human x rodent hybrid cell lines to chromosome 1, region cen-q32. In Chinese hamster x mouse hybrid cell lines the mouse locus was also mapped to chromosome 1. With the BXD (from progenitor strains C57BL/6J and DBA/2J) set of recombinant inbred strains of mice, the murine gene was localized on the linkage map of mouse chromosome 1. There were no recombinants among 26 strains with Ly-22 and 1 recombinant among 24 strains with Ltw-4. The gene symbol is OTF1 for humans and Oct-1 for mouse.

Animals↗

PubMed1990

cDNA cloning of human oxysterol-binding protein and localization of the gene to human chromosome 11 and mouse chromosome 19.

Cellular cholesterol metabolism is regulated primarily through sterol-mediated feedback suppression of the activity of the low-density lipoprotein receptor and several enzymes of the cholesterol biosynthetic pathway. We previously described the cloning of a rabbit cDNA for the oxysterol-binding protein (OSBP), a cytosolic protein of 809 amino acids that may participate in these regulatory events. We now use the rabbit OSBP cDNA to clone the human OSBP cDNA and 5' genomic region. Comparison of the human and rabbit OSBP sequences revealed a remarkably high degree of conservation. The cDNA sequence in the coding region showed 94% identity between the two species, and the predicted amino acid sequence showed 98% identity. The human cDNA was used to determine the chromosomal localization of the OSBP gene by Southern blot hybridization to panels of somatic cell hybrid clones containing subsets of human or mouse chromosomes and by RFLP analysis of recombinant inbred mouse strains. The OSBP locus mapped to the long arm of human chromosome 11 and the proximal end of mouse chromosome 19. Along with previously mapped genes including Ly-1 and CD20, OSBP defines a new conserved syntenic group on the long arm of chromosome 11 in the human and the proximal end of chromosome 19 in the mouse.

Amino Acid Sequence↗

PubMed1990

Human embryonic/atrial myosin alkali light chain gene: characterization, sequence, and chromosomal location.

We have isolated and sequenced the gene encoding the human embryonic/atrial myosin alkali light chain isoform (MLC-1emb/A). The gene is split into seven exons by six introns; the last exon, as in all MLC isoform genes sequenced to date, is completely 3' untranslated sequence. Comparison of the MLC-1emb/A isoform gene with the other MLC-1 genes showed that the exon-intron arrangement of the human MLC-1emb/A isoform gene is analogous to that of the other MLC-1 type isoform genes. We have also mapped the human MLC-1emb/A isoform gene to the long arm of chromosome 17; the corresponding mouse gene has been mapped to chromosome 11. This gene, together with a number of others such as the collagen(I) alpha 1, galactokinase, and thymidine kinase genes, is part of the largest syntenic group between mouse and man.

Amino Acid Sequence↗

PubMed1990

Molecular cloning, functional expression, and chromosomal localization of mouse hepatocyte nuclear factor 1.

The homeodomain-containing transcription factor hepatocyte nuclear factor 1 (HNF-1) most likely plays an essential role during liver organogenesis by transactivating a family of greater than 15 predominantly hepatic genes. We have isolated cDNA clones encoding mouse HNF-1 and expressed them in monkey COS cells and in the human T-cell line Jurkat, producing HNF-1 DNA-binding activity as well as transactivation of reporter constructs containing multimerized HNF-1 binding sites. In addition, the HNF-1 gene was assigned by somatic cell hybrids and recombinant inbred strain mapping to mouse chromosome 5 near Bcd-1 and to human chromosome 12 region q22-qter, revealing a homologous chromosome region in these two species. The presence of HNF-1 mRNA in multiple endodermal tissues (liver, stomach, intestine) suggests that HNF-1 may constitute an early marker for endodermal, rather than hepatocyte, differentiation. Further, that HNF-1 DNA-binding and transcriptional activity can be conferred by transfecting the HNF-1 cDNA into several cell lines indicates that it is sufficient to activate transcription in the context of ubiquitously expressed factors.

Amino Acid Sequence↗

PubMed1990

Insulin-degrading enzyme: stable expression of the human complementary DNA, characterization of its protein product, and chromosomal mapping of the human and mouse genes.

We have recently described the isolation of a cDNA encoding an enzyme thought to be involved in the degradation of insulin by insulin-responsive tissues. This enzyme, referred to as insulin-degrading enzyme (IDE), is a cytosolic proteinase of 110,000 mol wt which shares structural and functional homology with bacterial protease III. The enzyme may function in the termination of the insulin response. We report here the mapping of the human and mouse IDE genes to human chromosome 10 and mouse chromosome 19, respectively, and evidence for the existence of a single complex IDE gene. We also describe the stable transfection of Chinese hamster ovary cells with a plasmid containing the IDE cDNA under the transcriptional control of the SR alpha promoter. The recombinant protein synthesized by these cells is indistinguishable from the isolated human enzyme in both its size and immunoreactivity and degrades insulin with a specific activity similar to that of the purified proteinase. Overexpression of IDE using this system should allow for a functional test of the role of IDE in insulin action. In addition, expression of various site-directed mutants of IDE will aid in identifying the residues of IDE and protease III that are essential to the activity of this unique family of proteinases.

Amino Acid Sequence↗

PubMed1990

Genes for two autosomal recessive forms of chronic granulomatous disease assigned to 1q25 (NCF2) and 7q11.23 (NCF1).

Chronic granulomatous disease (CGD) is a heterogeneous group of inherited disorders of impaired superoxide production in phagocytes. The most common X-linked recessive form involves the CYBB locus in band Xp21.1 that encodes the membrane-bound beta subunit of the cytochrome b558 complex. Two autosomal recessive forms of CGD result from defects in cytosolic components of the phagocyte NADPH oxidase system, p47phox (NCF1) and p67phox (NCF2). By using human cDNA probes we have mapped the genes for these proteins to chromosomal sites. The combined data from Southern analysis of somatic cell hybrid lines and chromosomal in situ hybridization localize NCF1 to 7q11.23 and NCF2 to band 1q25. The NCF1 localization corrects an erroneous preliminary assignment to chromosome 10. In the mouse, the locus corresponding to NCF2 (Ncf-2) was mapped with somatic cell hybrid panels and recombinant inbred strains to mouse chromosome 1 near Xmv-21 within a region of conserved homology with human chromosome 1 region q21-q32. A second site, probably a processed pseudogene, was identified on mouse chromosome 13.

Animals↗