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Biomedical subjects

H H Heng

Publications and source records attributed to H H Heng.

At least 19 recordsLinked to original sources
PubMed2001

Sperm nuclear matrix association of the PRM1-->PRM2-->TNP2 domain is independent of Alu methylation.

Genes or multigenic chromosomal regions are organized by the nuclear matrix into a series of functionally discrete genic domains. Biophysical analysis of the human chromosome 16p13.13 region has shown that the PRM1-->PRM2-->TNP2 protamine containing multigenic locus is bounded by two sperm nuclear matrix attachment regions (MAR). This domain exists in a transcriptionally readied or potentiated (i.e. open) chromatin state when associated with the nuclear matrix. The MAR-bounded PRM1-->PRM2-->TNP2 locus is nestled in an Alu repetitive element dense region. Fluorescence in-situ hybridization, analysis of sperm nuclear matrix/halo preparations showed that the PRM1-->PRM2-->TNP2 domain specifically localizes to the sperm nuclear matrix. This raised the question of whether nuclear matrix association and gene expression in this locus is mediated by Alu methylation. The methylation status of the various Alu elements contained within the human PRM1-->PRM2-->TNP2 locus was therefore assayed. The seven Alu elements tested, including those associated with the matrix attachment regions within the PRM1-->PRM2-->TNP2 locus, were fully methylated in sperm DNA. Conversely, these same Alu repeats were hypomethylated within the erythroleukaemic cell line, K562, which does not express any of the genes from this domain. This study shows that Alu methylation status is independent of attachment of PRM1-->PRM2-->TNP2 locus to the nuclear matrix and that Alu methylation does not play a leading role in the regulation of this domain.

Alu Elements↗
PubMed2001

Spectral karyotyping (SKY) of mouse meiotic chromosomes.

The spectral karyotyping procedure of in situ hybridization with chromosome-specific probes assigns a unique colour code to each of the 21 mouse mitotic chromosomes. We have adapted this procedure to meiotic prophase chromosomes, and the results show that each of the pachytene or metaphase I bivalents can be identified. This technique has the potential to recognize synaptic anomalies and chromosome-specific structural and behavioural characteristics. We confirm these potentials by the recognition of the heterologous synapsis of the X and Y chromosomes and by the variances of synaptonemal complex lengths for each of the colour-coded bivalents in eight prophase nuclei.

Animals↗
PubMed2001

Re-defining the chromatin loop domain.

It is commonly accepted that the loop domain represents the basic structural unit of eukaryotic chromatin associated with DNA replication, gene expression and higher order packaging. However, molecular-cytological information defining the loop domain is lacking. There are gaps in our knowledge of the loop structure and how it regulates gene expression. The combination of new data/reagents from the Human Genome Project plus the use of novel molecular cytological technology will provide answers. Here we briefly review the status of chromatin loop research and pose questions that need to be addressed. New experimental systems are also presented to target some long-standing issues regarding the structure and function of the chromatin loop domain and its relationship with the nuclear matrix. This new knowledge will have a profound impact for modern genetics and molecular medicine.

Animals↗
PubMed2001

The combination of SKY and specific loci detection with FISH or immunostaining.

Spectral karyotyping (SKY) represents an effective tool to detect individual chromosomes and analyze major karyotype abnormalities within an entire genome. We have tested the feasibility of combining SKY and FISH/protein detection in order to combine SKY's unique abilities with specific loci detection. Our experimental results demonstrate that various combined protocols involving SKY, FISH and immunostaining work well when proper procedures are used. This combined approach allows the tracking of key genes or targeted chromosome regions while monitoring changes throughout the whole genome. It is particularly useful when simultaneously monitoring the behavior of both protein complexes and DNA loci within the genome. The details of this methodology are described and systematically tested in this communication.

Animals↗
PubMed2000

Cloning and characterization of additional members of the G protein-coupled receptor family.

A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Spectral karyotyping of mouse cell line WMP2.

We have evaluated the mouse cell line WMP2 using both GTG-banding analysis and spectral karyotyping to verify the reliability of using this established cell line derived from WMP/WMP mice. The WMP cell lines contain easily identifiable metacentric fusion chromosomes and are used extensively for gene mapping. Because of karyotypical changes in the WMP1 cell line, WMP2 was examined. Our results demonstrate that WMP2 is stable during culture, and the karyotype is simple and easy to use. Based on the findings discussed in this paper, we recommend the use of the WMP2 cell line for future prospective gene mapping in the mouse.

Animals↗
PubMed1999

Identification of a human histone acetyltransferase related to monocytic leukemia zinc finger protein.

We describe here the identification and functional characterization of a novel human histone acetyltransferase, termed MORF (monocytic leukemia zinc finger protein-related factor). MORF is a 1781-residue protein displaying significant sequence similarity to MOZ (monocytic leukemia zinc finger protein). MORF is ubiquitously expressed in adult human tissues, and its gene is located at human chromosome band 10q22. MORF has intrinsic histone acetyltransferase activity. In addition to its histone acetyltransferase domain, MORF possesses a strong transcriptional repression domain at its N terminus and a highly potent activation domain at its C terminus. Therefore, MORF is a novel histone acetyltransferase that contains multiple functional domains and may be involved in both positive and negative regulation of transcription.

3T3 Cells↗
PubMed1999

Cloning and expression analysis of a novel WD repeat gene, WDR3, mapping to 1p12-p13.

WD repeat proteins are components of multiprotein complexes that are involved in a wide spectrum of cellular activities, such as cell cycle progression, signal transduction, apoptosis, and gene regulation. These proteins are characterized by repeat units bracketed by Gly-His and Trp-Asp (GH-WD). We report here the isolation of a new member of the WD repeat gene family, WDR3, which encodes a putative 943-amino-acid nuclear protein consisting of 10 WD repeat modules. WDR3 is widely expressed in hematopoietic cell lines and in nonhematopoietic tissues. Fluorescence in situ hybridization mapped WDR3 to human chromosome 1p12-p13, a region that is affected by chromosomal rearrangements in a number of hematologic malignancies and solid tumors.

Amino Acid Sequence↗
PubMed1999

Nontargeted stable integration of recombinant adeno-associated virus into human leukemia and lymphoma cell lines as evaluated by fluorescence in situ hybridization.

A number of studies on human epithelial cells of varying origin have demonstrated integration of recombinant adeno-associated virus (AAV) vectors into a variety of chromosomes compared with the site-specific integration on chromosome 19 predominantly observed for wild-type (wt) AAV. We have constructed a recombinant AAV (rAAV) vector and tested the integration into hematopoietic cells, using the human acute myeloid leukemia cell line AML5 and the human non-Hodgkin's lymphoma cell line OCI-LY18 as targets. The integration sites were visualized by fluorescence in situ hybridization (FISH). Positive signals were observed for chromosomes 1, 2, 3, 8, 14, 15, 19, and Y. The majority of cells demonstrated integration into one specific site. A minority showed simultaneous integration into more than one chromosome. The frequency of observed integrations was not uniformly distributed among chromosomes; for instance, in AML5 chromosome 2 seemed to be favored. Colony-derived AML5 clones bore unique integration patterns indicating successful transduction of clonogenic progenitor cells with high proliferative potential. The integration was stable and observed for more than 12 months after transduction. FISH has been shown to be a powerful tool for detailed analyses of rAAV integration patterns and can be used to evaluate targets and transduction conditions.

Acute Disease↗
PubMed1999

Discovery of three novel orphan G-protein-coupled receptors.

We have discovered three novel human genes, GPR34, GPR44, and GPR45, encoding family A G-protein-coupled receptors (GPCRs). The receptor encoded by GPR34 is most similar to the P2Y receptor subfamily, while the receptor encoded by GPR44 is most similar to chemoattractant receptors. The receptor encoded by GPR45 is the mammalian orthologue of a putative lysophosphatidic acid receptor from Xenopus laevis. Partial sequence of GPR34 was discovered during a search of the GenBank database of expressed sequence tags (ESTs). This sequence information was used both to isolate the full-length translational open reading frame from a human genomic library and to assemble a contig from additional GPR34 EST cDNAs. Northern blot and in situ hybridization analyses revealed GPR34 mRNA transcripts in several human and rat brain regions. Also, we used polymerase chain reaction (PCR) to amplify human genomic DNA using degenerate oligonucleotides designed from sequences encoding transmembrane domains 3 and 7 of opioid and somatostatin receptors. Two PCR products partially encoding novel GPCRs, named GPR44 and GPR45, were discovered and used to isolate the full-length translational open reading frames from a human genomic library. Both GPR44 and GPR45 are expressed in the central nervous system and periphery. For chromosomal localization, fluorescence in situ hybridization analysis was performed to assign GPR34 to chromosomes 4p12 and Xp11. 3, GPR44 to chromosome 11q12-q13.3, and GPR45 to chromosome 2q11. 1-q12.

Amino Acid Sequence↗
PubMed1999

Identification and cloning of three novel human G protein-coupled receptor genes GPR52, PsiGPR53 and GPR55: GPR55 is extensively expressed in human brain.

The G protein-coupled receptor (GPCR) family share a structural motif of seven transmembrane segments with large numbers of conserved residues in those regions. Here, we report the identification and cloning of two novel human intronless GPCR genes, GPR52, GPR55 and a pseudogene PsiGPR53. GPR55 was identified from the expressed sequence tags (EST) database whereas GPR52 and pseudogene PsiGPR53 originated from the high throughput genome (HTG) database. A partial cDNA clone obtained from the IMAGE Consortium of GPR55 was used to screen a human genomic library to acquire the full length gene. GPR52 and PsiGPR53 were amplified from human genomic DNA using primers based on the HTG sequences. GPR55 and GPR52 encode receptors of 319 and 361 amino acids, respectively. GPR55 gene was mapped to chromosome 2q37, using fluorescence in situ hybridization (FISH), and its mRNA transcripts have been detected in the caudate nucleus and putamen, but not in five other brain regions. Human receptors showing the highest amino acid identity to GPR55 include P2Y5 (29%), GPR23 (30%), GPR35 (27%) and CCR4 (23%). GPR52 gene localized to chromosome 1q24 shares the highest identity with GPR21 (71%), histamine H2 (27%) and 5-HT4 (26%) human receptors. PsiGPR53 is a pseudogene mapped to chromosome 6p21 that demonstrates the highest similarity to the MRG (35%), MAS (28%) and C5a (24%) human receptor genes.

Amino Acid Sequence↗
PubMed1999

HDAC4, a human histone deacetylase related to yeast HDA1, is a transcriptional corepressor.

Histone acetylation plays an important role in regulating chromatin structure and thus gene expression. Here we describe the functional characterization of HDAC4, a human histone deacetylase whose C-terminal part displays significant sequence similarity to the deacetylase domain of yeast HDA1. HDAC4 is expressed in various adult human tissues, and its gene is located at chromosome band 2q37. HDAC4 possesses histone deacetylase activity intrinsic to its C-terminal domain. When tethered to a promoter, HDAC4 represses transcription through two independent repression domains, with repression domain 1 consisting of the N-terminal 208 residues and repression domain 2 containing the deacetylase domain. Through a small region located at its N-terminal domain, HDAC4 interacts with the MADS-box transcription factor MEF2C. Furthermore, HDAC4 and MEF2C individually upregulate but together downmodulate c-jun promoter activity. These results suggest that HDAC4 interacts with transcription factors such as MEF2C to negatively regulate gene expression.

Amino Acid Sequence↗
PubMed1999

Identification of the mouse beta'-COP Golgi component as a spermatocyte autoantigen in scleroderma and mapping of its gene Copb2 to mouse chromosome 9.

In an immunological screening of a mouse testicular cDNA library with a human CREST serum we isolated five overlapping cDNA clones encoding the mouse homolog of a Golgi coatomer complex protein (accession number AF043120), designated beta'-COP in bovine and p102 in humans. We generated antibodies against this protein which specifically recognize the Golgi apparatus of mouse spermatocytes. FISH analyses assigned the beta'-COP gene Copb2 to mouse Chromosome 9, region E3-F1. Our results demonstrate that CREST sera can contain antibody components against Golgi proteins as well as against nuclear proteins.

Animals↗
PubMed1998

Structure and mapping of the human beta-defensin HBD-2 gene and its expression at sites of inflammation.

We cloned a second human beta-defensin gene, HBD-2, and determined its gene structure and expression in inflamed tissue sections. The entire gene spanned about 2 kb with two small exons and one intron. Radiation hybrid studies confirmed the location on chromosome 8p, were consistent with the order HNP-1, HBD-1 and HBD-2, and located HBD-2 as the most centromeric of the genes. By three-color fluorescence in situ hybridization on both free chromatin fiber mapping and interphase mapping, HBD-1, HBD-2 and HNP-1 were mapped to chromosome 8p23. HBD-1 was within 40-100kb of HNP-1, while HBD-2 was about 500-600 kb from HBD-1, with the most likely order HNP-1, HBD-1, HBD-2. The expression of HBD-2 was locally regulated by inflammation. HBD-2 mRNA was markedly increased in the epidermis surrounding inflamed regions, but not detectable in adjacent non-inflamed areas, a distribution that was confirmed at the peptide level by immunostaining with HBD-2 antibody. The HBD-2 gene is the first member of the human defensin family that is locally inducible by inflammation.

Base Sequence↗
PubMed1998

Chromosomal mapping and expression of the human B120 gene.

We previously reported a novel human cDNA, designated B120, containing a CAG repeat length polymorphism and many repeat units, loosely identified as YXQQP which is found in several human RNA binding proteins. In the present study, the B120 gene was mapped to human chromosome 1p35-36.1 by fluorescence in situ hybridization (FISH). Several human disorders, including that of Schnyder crystalline corneal dystrophy, have been mapped to this region by genetic linkage. Schnyder crystalline corneal dystrophy is thought to be a primary abnormality of corneal lipid metabolism, resulting in opacification secondary to lipid accumulation. In order to examine the function of B120, we introduced B120 cDNA with an expression vector into various cell lines including Cos1, C3H/10T1/2 and NIH/3T3 cells. These transfected cells exhibited small cytoplasmic spherical bodies. The cytoplasmic bodies appeared to be fat droplets on electron microscopy and histochemical staining. These findings suggested that B120 gene expression is associated with lipid metabolism, and that overexpression of B120 may result in lipid deposition in various cells, including those of fibroblastic cell lines. Since the cornea is composed of fibroblastic cells, overfunction of B120 could be related to the pathogenesis of Schnyder crystalline corneal dystrophy.

3T3 Cells↗