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Biomedical subjects

H H Heng

Publications and source records attributed to H H Heng.

At least 91 records · Page 5Linked to original sources
PubMed1995

Differential expression of a basic helix-loop-helix phosphoprotein gene, G0S8, in acute leukemia and localization to human chromosome 1q31.

A basic helix-loop-helix phosphoprotein gene, G0S8, was recently isolated by differential screening of cDNA from human blood mononuclear cells stimulated with a T cell mitogen and cycloheximide. In this study, G0S8 expression was examined in normal and malignant hematopoietic cells by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). G0S8 expression was observed in most fresh samples of acute myelogenous leukemia (AML) (28/30) and most cases of adult acute lymphoblastic leukemia (ALL) (9/11) regardless of clinical classification. G0S8 mRNA was also detected in all cases tested of chronic myelogenous leukemia (CML) in blast crisis. However, G0S8 expression was not detected in CML patients in chronic phase, nor in normal bone marrow or other hematopoietic cells. G0S8 has been mapped using fluorescence in situ hybridization (FISH) to human chromosome 1q31, the same site reported for the B cell homolog BL34/1R20 and within a region implicated in the development of hematological malignancies. The consistent observation of G0S8 mRNA in patient samples of acute leukemia suggests that G0S8 expression may either play a role in leukemogenesis or represent a common consequence of dysregulated growth.

Amino Acid Sequence↗
PubMed1994

Regional localization of 725 human chromosome 7-specific yeast artificial chromosome clones.

Toward the construction of a complete physical map of human chromosome 7, we have localized 725 YAC clones to cytogenetically defined regions using fluorescence in situ hybridization (FISH) and by screening with DNA markers of known chromosomal locations. These chromosome 7-specific YAC clones are part of a library constructed with DNA isolated from monochromosomal 7 human-hamster somatic cell hybrid lines. The FISH mapping for 575 clones was accomplished by using "Alu-PCR" amplified YAC DNA against metaphase chromosome spreads made from a monochromosomal 7 human-mouse somatic cell hybrid line. Hybridization- or PCR-based screening of previously mapped DNA markers was performed for the mapping of 221 YAC clones. There was excellent correlation between the map locations obtained for the 71 YACs localized with both methods. All of the regionally localized YAC clones are valuable reagents for mapping and identification of disease genes on human chromosome 7.

Animals↗
PubMed1994

Cloning of human genes encoding novel G protein-coupled receptors.

We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q21.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1.

Amino Acid Sequence↗
PubMed1994

Organization of heterologous DNA inserts on the mouse meiotic chromosome core.

With simultaneous immunofluorescence and fluorescent in situ hybridization, we have determined the organization of native and heterologous DNA sequences relative to the cores of meiotic prophase chromosomes. The normal chromatin organization is demonstrated with probes of mouse sequences: a cosmid probe that identifies unique sequences and a 720 kb yeast artificial chromosome (YAC) probe that recognizes a specific region of the chromatin domain. The heterologous DNA consists of a 1.8 Mb insertion of 40 tandem head-to-tail phage lambda LIZ vectors and of 11.4 Mb of bacterial/mouse DNA repeats. The lengthy lambda insert is unusual in that it is not contained in the chromatin domain of chromosome 4 and in that it fails to form direct attachments to the chromosome core. The ends are attached indirectly, probably by means of the flanking mouse sequences. At late stages of meiotic prophase, while the terminal attachments remain the same, the lambda DNA becomes highly compacted. Apparently, higher order condensation and core attachment are independent processes. The condensed inserts relax precociously at metaphase I. In the mouse heterozygous for the insert, the two sister inserts are usually merged, as are all four inserts in the homozygous mouse. Evidently chromatin loops with identical sequences can become associated during meiotic prophase. Mouse sequences within a heterologous DNA insert (repeats of bacterial plasmid pBR322 with a mouse beta-globin insert) were observed to restore some degree of core attachment.

Animals↗
PubMed1994

Isolation of P1 bacteriophage clones containing large contiguous segments of the human and mouse loci for the T-cell coreceptor molecule CD8.

The T-lymphocyte coreceptor molecules CD8 (composed of CD8 alpha and CD8 beta chains) and CD4 undergo a complex pattern of regulated expression during T-cell maturation. In the thymus, the most immature cells progress from expressing neither molecule (the double-negative [DN] stage) to an intermediate stage at which both are coexpressed (the double-positive [DP] stage). As a result of thymic selection and further differentiation, DP cells give rise to the most mature thymic cells and peripheral T cells that express either CD8 or CD4 (the single-positive [SP] stage). Our previous studies of the transcriptional regulatory mechanisms controlling CD8 alpha expression during the DN-->DP and DP-->SP transitions suggest the existence of important cis-acting elements located a considerable distance from the CD8 alpha gene and that these elements might serve to regulate both CD8 alpha and CD8 beta. While both genes and intergenic DNA span approximately 60 kb in the mouse, the relevant cis elements could lie either within or beyond this region. As a result, we sought to isolate large contiguous segments of DNA in P1 bacteriophage that covered at least this region from the mouse and human CD8 locus. Our initial physical characterization of these clones demonstrates the value of the P1 system as all isolated clones were found to contain single contiguous 85- to 95-kb segments of DNA that are faithful replicas of the chromosomal locus. The presence of abundant native flanking DNA both upstream and downstream of the intact coding regions will make these clones extremely useful for identifying physiological CD8 cis-active regulatory elements by virtue of their ability to direct appropriate lineage- and stage-specific expression in transfected and transgenic T cells.

Animals↗
PubMed1994

The murine Xe169 gene escapes X-inactivation like its human homologue.

Among a number of genes that escape X-chromosome inactivation in humans, three have been evaluated in mice and unexpectedly all three are subject to X-inactivation. We report here the cloning and expression studies of a novel mouse gene, Xe169, and show that it escapes X-inactivation like its human homologue. Xe169 was assigned to band F2/F3 on the mouse X chromosome by fluorescent in situ hybridization and Southern analysis indicates that the gene is located outside the pseudoautosomal region. Homologous, but divergent, sequences exist on the Y chromosome. In vitro and in vivo studies show that Xe169 is expressed from both the active and the inactive X chromosomes. Xe169 is the first cloned non-pseudoautosomal gene that escapes X-inactivation in mice.

Animals↗
PubMed1994

Genes encoding general initiation factors for RNA polymerase II transcription are dispersed in the human genome.

General transcription factors are required for accurate initiation of transcription by RNA polymerase II. Human cDNAs encoding subunits of these factors have been cloned and sequenced. Using fluorescence in situ hybridization (FISH), we show here that the genes encoding the TATA-box binding protein (TBP), TFIIB, TFIIE alpha, TFIIE beta, RAP30, RAP74 and the 62 kDa subunit, of TFIIH are located at the human chromosomal bands 6q26-27, 1p21-22, 3q21-24, 8p12, 13q14, 19p13.3 and 11p14-15.1, respectively. This dispersed localization of a group of functionally related gene provides insights into the molecular mechanism of human genome evolution and their possible involvement in human diseases.

Cells, Cultured↗
PubMed1994

Refined localization of the asparagine synthetase gene (ASNS) to chromosome 7, region q21.3, and characterization of the somatic cell hybrid line 4AF/106/KO15.

We have mapped the asparagine synthetase gene (ASNS) to 7q21.3 by fluorescence in situ hybridization. While this study refined the localization of the gene, it also revealed a rearrangement in a somatic cell hybrid line which was used in previous ASNS mapping. Using additional probes from other regions of human chromosome 7, we showed that this cell line (4AF/106/KO15) contained a rearranged chromosome 7 in which a segment of the long arm was apparently duplicated and inserted into the short arm. Caution should be used therefore when interpreting data obtained from this cell line for gene mapping studies.

Animals↗
PubMed1994

Receptor-binding, tyrosine phosphorylation and chromosome localization of the mouse SH2-containing phosphotyrosine phosphatase Syp.

The murine phosphotyrosine phosphatase, Syp, is a widely-expressed cytoplasmic enzyme that contains two SH2 domains. Syp is physically associated with activated receptors for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), apparently through its SH2 domains. This phosphatase is rapidly phosphorylated in cells treated with PDGF or EGF, and is constitutively phosphorylated in v-src transformed cells. Here we report that either the N-terminal or C-terminal Syp SH2 domain alone bound to the activated beta PDGF receptor or EGF-receptor in vitro, and that the two SH2 domains linked together exhibited synergistic binding. Substitution of the Tyr1009 autophosphorylation site in the C-terminal tail of activated beta PDGFR with Phe abolished the in vitro binding of either SH2 domain to the activated receptor. A 9 amino acid phosphopeptide corresponding to the Tyr1009 autophosphorylation site of the beta PDGFR inhibited association of the Syp SH2 domains with the receptor. These results indicate that the Syp SH2 domains have an intrinsic specificity for the Tyr1009 autophosphorylation site of the beta PDGFR that dictates binding of the intact Syp phosphatase, and suggest that both SH2 domains have a related binding specificity. Phosphoamino acid analysis of Syp from PDGF-stimulated cells indicated that PDGF primarily induces Syp phosphorylation on tyrosine residues. The mouse Syp gene has been mapped to chromosome 5F region by the fluorescence in situ hybridization. These findings suggest specific functions for Syp in signal transduction downstream of receptor tyrosine kinases.

Animals↗
PubMed1994

MLK-3: identification of a widely-expressed protein kinase bearing an SH3 domain and a leucine zipper-basic region domain.

We have identified a novel protein kinase, designated MLK-3, from human thymus using RT-PCR and cDNA library screening. The deduced open reading frame, derived from sequencing a 3.5 kb MLK-3 cDNA, encodes a protein of 847 amino acids with several interesting structural features. These include an SH3 domain in the absence of an SH2 domain, a region containing two leucine zippers with an adjacent carboxy-terminal basic region, and a proline rich region. This kinase shows homology with the mixed-lineage family of protein kinases (MLK) and shares the unusual leucine zipper-basic motif found in previously identified MLK kinases. By northern analysis, MLK-3 mRNA was detected in a wide variety of normal and transformed human cell lines and tissue specimens. The gene encoding MLK-3 has been mapped using fluorescence in situ hybridization to human chromosome 11 q13.1-13.3, a region frequently altered in human malignancies.

Amino Acid Sequence↗
PubMed1993

A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11.

We report the cloning of a gene, intronless in its coding region, which we have named APJ. This gene was cloned using the polymerase chain reaction (PCR), with a set of primers designed on the basis of the conservation that members of G protein-coupled receptors (GPCR) have in their transmembrane (TM) regions. The putative receptor protein, APJ, shares closest identity to the angiotensin receptor (AT1) ranging from 40 to 50% in the hydrophobic TM regions of these receptors. The transcripts for this gene were detected in many regions of the brain. PCR analysis of somatic cell lines found APJ-related sequences to be only present on chromosome 11, and high-resolution mapping by fluorescence in situ hybridization (FISH) sublocalized APJ on band q12.

Amino Acid Sequence↗
PubMed1993

Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene.

Endothelial nitric oxide (NO) synthase is a unique NO synthase isoform that is expressed constitutively by vascular endothelium both in vivo and in vitro and is believed essential to local vascular homeostasis. This calcium/calmodulin-dependent isoform is distinct from neuronal NO synthase. Genomic clones encoding the human endothelial NO synthase were isolated and the structural organization of the gene was determined. The gene contains 26 exons spanning approximately 21 kilobases of genomic DNA, encodes a messenger RNA of 4052 nucleotides, and is present as a single copy in the haploid human genome. Characterization of 5'-flanking genomic regions indicates that the endothelial NO synthase promoter is "TATA-less" and exhibits proximal promoter elements consistent with a constitutively expressed gene that is found in endothelial cells, namely Sp1 and GATA motifs. The 5'-flanking region contains putative AP-1, AP-2, NF-1, heavy metal, acute-phase response shear stress, and sterol-regulatory cis-elements. The human endothelial NO synthase gene was assigned to the 7q35-->7q36 region of chromosome 7 by Southern blot hybridization of human-rodent somatic cell hybrid lines and fluorescence in situ hybridization, whereas human neuronal NO synthase localized to the 12q24.2 region of chromosome 12.

Amino Acid Oxidoreductases↗
PubMed1993

Refined mapping of the GM2 activator protein (GM2A) locus to 5q31.3-q33.1, distal to the spinal muscular atrophy locus.

The GM2 activator locus (GM2A) had previously been considered as a candidate gene for some forms of spinal muscular atrophy (SMA; mapped to 5q11.2-q13.3). It was eliminated as a possible candidate because PCR-based mapping failed to localize the gene to chromosome 5, as was previously reported using an ELISA-based methodology. However, we demonstrated that the PCR primers used preferentially amplified a processed pseudogene (GM2AP) that we mapped to chromosome 3 and that GM2A was located on chromosome 5. In this report, we reconsider the candidacy of GM2A by refining its localization on chromosome 5 using fluorescence in situ hybridization. We localize GM2A to 5q31.3-q33.1; thus, it is not a candidate gene for SMA.

Cells, Cultured↗
PubMed1993

Chromosomal localization of the human gene encoding the 51-kDa subunit of mitochondrial complex I (NDUFV1) to 11q13.

The 51-kDa flavoprotein subunit of mitochondrial NADH:ubiquinone oxidoreductase (Complex I) [NADH dehydrogenase (ubiquinone), flavoprotein 1 (51 kDa); EC 1.6.5.3] plays an important role in the formation of the NADH-binding site and is believed to be the principal site of entry for electrons donated by NADH into the respiratory chain. Human cDNA fragments of the 51-kDa protein were generated by polymerase chain reaction and used to localize the gene (NDUFV1) for this subunit to 11q13 by two separate techniques. This region of the human genome is strongly implicated in a number of different forms of cancer.

Amino Acid Sequence↗
PubMed1993

Modes of DAPI banding and simultaneous in situ hybridization.

By controlling the degree of chromatin denaturation through formamide incubation, or by heat treatment and/or by high pH, three types of high quality 4',6-diamidino-2-phenylindole (DAPI) bands can be produced sequentially on the same set of 5-bromo-2'-deoxyuridine (BrdU)-incorporated chromosomes: first DAPI multibanding (the equivalent of Q-banding), then partial C-banding including distamycin A (DA)/DAPI banding, and finally C-banding pattern. It is assumed that the different DAPI-chromatin interactions following these treatments reflect the different chromatin structures at the chromosomal sites. Since the DAPI banding protocol is compatible with in situ hybridization, the combination of fluorescent in situ hybridization (FISH) with DAPI banding allows the simultaneous detection of signals from the DNA probes and the identification of the chromosomal band location of the probe. We demonstrate this useful application with the localization of the cystic fibrosis and Duchenne muscular dystrophy gene probes to their appropriate bands.

Cells, Cultured↗