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N C Popescu

Publications and source records attributed to N C Popescu.

At least 55 records · Page 3Linked to original sources
PubMed1998

A chromosome 4 satellite I DNA isolated from SV40-transformed human cells.

Analysis of cellular DNA insert isolated from a free replicative plasmid rescued from human cells transformed with an SV40 vector plasmid revealed the presence of two arrays of repetitive DNA arranged in tandem. One sequence was homologous to the consensus sequence of the human alpha satellite DNA and the adjoining sequence was a satellite DNA sequence which consisted of repetitive units of 42 base pairs (bp) and was designated HR42. The degree of homology between repetitive units was about 92%. By Southern analysis the HR42 sequence was detected in HHW416, a somatic cell hybrid containing human chromosome 4, but not in HDm-5, the somatic cell hybrid which has human chromosome 14. By fluorescence in situ hybridization this repetitive DNA was assigned uniquely to the centromeric region of human chromosome 4. These results show that HR42 belongs to a subfamily of satellite I DNA specific for human chromosome 4.

Base Sequence↗
PubMed1998

BID, a proapoptotic BCL-2 family member, is localized to mouse chromosome 6 and human chromosome 22q11.

BID is a proapoptotic member of the BCL-2 family of cell death regulators. BID shares sequence homology with other members of the family within a single alpha-helical domain, BH3. BH3 is required for BID to interact with BCL-2 and BAX, as well as for its function as a death agonist. We have isolated and characterized mouse Bid and human BID genomic clones. The sequence for BID is encoded within five exons. We used interspecific backcross analysis to localize Bid to the distal region of mouse chromosome 6 near the Atp6e locus. Fluorescence in situ hybridization analysis localized human BID to a syntenic human region, chromosome 22q11, close to the BCR-1 gene.

Animals↗
PubMed1998

Localization of the human stat6 gene to chromosome 12q13.3-q14.1, a region implicated in multiple solid tumors.

Stat6 signaling pathways have been correlated with functional responses induced by IL-4 and PDGF that may play a role in human malignancy. Utilizing fluorescence in situ hybridization, we mapped the human Stat6 gene to chromosome 12q bands 13.3-14.1, a breakpoint region implicated in a wide variety of solid tumors. To understand the genesis of three human Stat6 variant cDNAs, including a naturally occurring dominant negative species, we further characterized the genomic structure and flanking regions of the human Stat6 gene. The human Stat6 gene encompassed over 19 kb and contained 23 exons. For promoter studies, we introduced flanking sequence 5' of Stat6 exon 1 into a promoterless luciferase reporter vector and characterized basal promoter activity by deletion analysis. DNA sequence analysis revealed potential transcriptional regulation of the putative promoter through numerous consensus binding elements. Finally, we conclude that selective exon deletion and utilization of alternative donor/acceptor sites appear to explain best human Stat6 variant mRNAs.

3T3 Cells↗
PubMed1998

Cloning, characterization, and chromosomal localization of a gene frequently deleted in human liver cancer (DLC-1) homologous to rat RhoGAP.

The isolation of genes involved in cancer development is critical for uncovering the molecular basis of cancer. We report here the isolation of the full-length cDNA and chromosomal localization of a new gene frequently deleted in liver cancer (DLC-1) that was identified by representational difference analysis. Loss of heterozygosity was detected for DLC-1 in 7 of 16 primary hepatocellular carcinomas (HCCs) and in 10 of 11 HCC cell lines. Although mRNA for DLC-1 was expressed in all normal human tissues, it was not expressed in 4 of 14 HCC cell lines. Full-length cDNA for DLC-1 of 3800 bp encodes a protein of 1091 amino acids, has 86% homology with rat p122 RhoGAP gene, and was localized by fluorescence in situ hybridization on chromosome 8 at bands p21.3-22. Deletions on the short arm of chromosome 8 are recurrent in liver, breast, lung, and prostate cancers, suggesting the presence of tumor suppressor genes. DLC-1 may be a tumor suppressor gene in liver cancer as well as in other cancers.

Animals↗
PubMed1998

GFRalpha3 is an orphan member of the GDNF/neurturin/persephin receptor family.

GDNF, neurturin, and persephin are transforming growth factor beta-related neurotrophic factors known collectively as the GDNF family (GF). GDNF and neurturin signal through a multicomponent receptor complex containing a signaling component (the Ret receptor tyrosine kinase) and either of two glycosyl-phosphatidylinositol-linked binding components (GDNF family receptor alpha components 1 and 2, GFRalpha1 or GFRalpha2), whereas the receptor for persephin is unknown. Herein we describe a third member of the GF coreceptor family called GFRalpha3 that is encoded by a gene located on human chromosome 5q31.2-32. GFRalpha3 is not expressed in the central nervous system of the developing or adult animal but is highly expressed in several developing and adult sensory and sympathetic ganglia of the peripheral nervous system. GFRalpha3 is also expressed at high levels in developing, but not adult, peripheral nerve. GFRalpha3 is a glycoprotein that is glycosyl-phosphatidylinositol-linked to the cell surface like GFRalpha1 and GFRalpha2. Fibroblasts expressing Ret and GFRalpha3 do not respond to any of the known members of the GDNF family, suggesting that GFRalpha3 interacts with an unknown ligand or requires a different or additional signaling protein to function.

3T3 Cells↗
PubMed1998

Cloning, mapping, expression, function, and mutation analyses of the human ortholog of the hamster putative tumor suppressor gene Doc-1.

doc-1 is a putative tumor suppressor gene isolated and identified from the hamster oral cancer model. Here, we report the molecular cloning and the functional characterization of the human ortholog of the hamster doc-1 gene. Human doc-1 cDNA is 1.6 kilobase pairs in length and encodes for a 115-amino acid polypeptide (12.4 kDa, pI 9. 53). Sequence analysis showed 98% identity between human and hamster doc-1 protein sequences. DOC-1 is expressed in all normal human tissues examined. In oral keratinocytes, expression of DOC-1 is restricted to normal oral keratinocytes. By immunostaining of normal human mucosa, DOC-1 is detected in both the cytoplasm and nuclei of basal oral keratinocytes; while in suprabasilar cells, it is primarily found in the nuclei. Human oral cancers in vivo did not exhibit immunostaining for DOC-1. Like murine DOC-1, human DOC-1 associates with DNA polymerase alpha/primase and mediates the phosphorylation of the large p180 catalytic subunit, suggesting it may be a potential regulator of DNA replication in the S phase of the cell cycle. Using a human doc-1 cosmid as a probe, human doc-1 is mapped to chromosome 12q24. We identified four exons in the entire human doc-1 gene and determined the intron-exon boundaries. By polymerase chain reaction and direct sequencing, we examined premalignant oral lesion and oral cancer cell lines and found no intragenic mutations.

Amino Acid Sequence↗
PubMed1998

Structure and transcriptional regulation of the human mammaglobin gene, a breast cancer associated member of the uteroglobin gene family localized to chromosome 11q13.

The mammaglobin gene encodes a novel secreted protein whose corresponding mRNA is frequently up-regulated in human breast cancer. In non-malignant tissues, expression is also strictly limited to the mammary epithelium. To better understand the mechanisms controlling these patterns of expression, we have isolated the human mammaglobin gene and performed an initial assessment of its promoter activity. Mammaglobin gene architecture is very similar to that of a family of related genes that includes uteroglobin and rat prostatein subunits C1, C2, and C3. However, the mammaglobin gene itself is not well conserved phylogenetically. The human mammaglobin gene is localized by fluorescent in situ hybridization to chromosome 11 band q13, a genomic region frequently amplified in breast neoplasia. The sequence of proximal 1 kb of mammaglobin promoter contains several potential transcriptional control elements and directs high-level expression of a transfected reporter construct in human breast tumor cell lines. However, comparable levels of reporter gene expression are also seen in non-mammary human cell lines. These data suggest that, unlike related gene family members, the striking breast-specific expression and tumor-associated overexpression of mammaglobin is mediated by complex transcriptional control at more distal sequence elements.

Base Sequence↗
PubMed1998

In silico-initiated cloning and molecular characterization of a novel human member of the L1 gene family of neural cell adhesion molecules.

To discover genes contributing to mental retardation in 3p- syndrome patients we have used in silico searches for neural genes in NCBI databases (dbEST and Uni-Gene). An EST with strong homology to the rat CAM L1 gene subsequently mapped to 3p26 was used to isolate a full-length cDNA. Molecular analysis of this cDNA, referred to as CALL (cell adhesion L1-like), showed that it is encoded by a chromosome 3p26 locus and is a novel member of the L1 gene family of neural cell adhesion molecules. Multiple lines of evidence suggest CALL is likely the human ortholog of the murine gene CHL1: it is 84% identical on the protein level, has the same domain structure, same membrane topology, and a similar expression pattern. The orthology of CALL and CHL1 was confirmed by phylogenetic analysis. By in situ hybridization, CALL is shown to be expressed regionally in a timely fashion in the central nervous system, spinal cord, and peripheral nervous system during rat development. Northern analysis and EST representation reveal that it is expressed in the brain and also outside the nervous system in some adult human tissues and tumor cell lines. The cytoplasmic domain of CALL is conserved among other members of the L1 subfamily and features sequence motifs that may involve CALL in signal transduction pathways.

Amino Acid Sequence↗
PubMed1998

Nab1, a corepressor of NGFI-A (Egr-1), contains an active transcriptional repression domain.

Nab proteins constitute an evolutionarily conserved family of corepressors that specifically interact with and repress transcription mediated by three members of the NGFI-A (Egr-1, Krox24, zif/268) family of immediate-early gene transcription factors, which includes NGFI-C, Krox20, and Egr3. We explored the mechanism of Nab1 repression and identified structural domains required for Nab1 function. Nab1 does not act by blocking DNA binding or nuclear localization of NGFI-A. In fact, Nab1 repression is not unique to NGFI-A because multiple types of non-NGFI-A activation domains were repressed, as was a heterologous transcription factor carrying the NGFI-A R1 domain, which is required for Nab1 interaction. Additionally, Nab1 tethered directly to DNA repressed constitutively active promoters. Tethered repression was not dependent on the identity of the basal promoter elements, the presence of a distal enhancer, or the distance separating the binding sites from the promoter. These results suggest that Nab1 repression is not specific to particular activators and that Nab1 is an active repressor that works by a direct mechanism. We identified a bipartite-like nuclear localization sequence and localized the repression function to the Nab conserved domain 2 (NCD2), a region found in the carboxy-terminal half of all Nab proteins. Three small regions of homology between Nab1 and previously characterized corepressors, Dr1 and E1b 55-kDa protein, were identified within NCD2. Replacement mutagenesis of residues conserved between these proteins interfered with Nab1 repression, although Nab1 does not function by the same mechanism as Dr1. The human NAB1 genomic locus was mapped to chromosome 2q32.3-33.

Amino Acid Sequence↗
PubMed1998

Sites of recombinant adeno-associated virus integration.

Adeno-associated virus (AAV), a defective parvovirus, is considered a promising vector for the delivery of therapeutic genes to cells. Both wild-type and recombinant AAV display a wide tropism and integrate into the host genome, in the absence of helper virus, establishing a latent infection. A unique characteristic of wild-type AAV and a potential advantage for use as a delivery system for gene therapy is the site-specific integration of wild-type virus within a small region of chromosome 19, 19q13.3-qter (AAVS1), in up to 85% of cell lines infected with the virus. Although recombinant AAVs, containing only the inverted terminal repeats of wild-type virus, can integrate efficiently into the host genome, specificity for the AAVS1 site appears to be lost. To address this question, the integration characteristics of two recombinant AAVs lacking the rep and cap genes in HeLa cells were examined. Analysis of Southern blots indicated that none of twenty-six cell clones generated after infection with either one of the recombinant AAVs demonstrated integration within the AAVS1 locus on chromo-some 19. Analysis of five of the cell lines by fluorescent chromosome in situ hybridization confirmed the loss of chromosome 19 specificity. Each integration site mapped near a known fragile site and/or location of a proto-oncogene or growth regulatory gene. Retention of site-specific integration of wild-type AAV will require the inclusion of additional AAV-specific sequences within the recombinant vectors.

Dependovirus↗
PubMed1997

Fluorescence in situ hybridization analysis of keratinocyte growth factor gene amplification and dispersion in evolution of great apes and humans.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family. Portions of the gene encoding KGF were amplified during primate evolution and are present in multiple nonprocessed copies in the human genome. Nucleotide analysis of a representative sampling of these KGF-like sequences indicated that they were at least 95% identical to corresponding regions of the KGF gene. To localize these sequences to specific chromosomal sites in human and higher primates, we used fluorescence in situ hybridization. In human, using a cosmid probe encoding KGF exon 1, we assigned the location of the KGF gene to chromosome 15q15-21.1. In addition, copies of KGF-like sequences hybridizing only with a cosmid probe encoding exons 2 and 3 were localized to dispersed sites on chromosome 2q21, 9p11, 9q12-13, 18p11, 18q11, 21q11, and 21q21.1. The distribution of KGF-like sequences suggests a role for alphoid DNA in their amplification and dispersion. In chimpanzee, KGF-like sequences were observed at five chromosomal sites, which were each homologous to sites in human, while in gorilla, a subset of four of these homologous sites was identified; in orangutan two sites were identified, while gibbon exhibited only a single site. The chromosomal localization of KGF sequences in human and great ape genomes indicates that amplification and dispersion occurred in multiple discrete steps, with initial KGF gene duplication and dispersion taking place in gibbon and involving loci corresponding to human chromosomes 15 and 21. These findings support the concept of a closer evolutionary relationship of human and chimpanzee and a possible selective pressure for such dispersion during the evolution of higher primates.

Animals↗
PubMed1997

Mechanism of homophilic binding mediated by ninjurin, a novel widely expressed adhesion molecule.

Ninjurin is a novel protein that is up-regulated after nerve injury both in dorsal root ganglion (DRG) neurons and in Schwann cells. We previously reported that ninjurin demonstrates properties of a homophilic adhesion molecule and promotes neurite outgrowth from primary cultured DRG neurons. We have now found that ninjurin is widely expressed in both adult and embryonic tissues, primarily in those of epithelial origin. Aggregation assays were used to demonstrate that ninjurin-mediated adhesion requires divalent cations and is an energy-dependent process. The critical domain for ninjurin-mediated homophilic adhesion was localized to an 11-residue region (between Pro26 and Asn37) by mutagenesis and by employing synthetic oligopeptides as competitive inhibitors of ninjurin-mediated adhesion. Of particular importance are the Trp residue at position 29 and the 3 arginines in the region. Furthermore, we show that the peptide which inhibits aggregation of Jurkat cells expressing ninjurin is also capable of blocking the ability of ninjurin to promote neurite extension from DRG neurons. Using FISH analysis, the ninjurin gene was localized to human chromosome 9q22. Several genetic diseases of unknown etiology have been mapped to this region, including hereditary sensory neuropathy type 1, self-healing squamous epithelioma, split-hand/foot deformity type 1, and familial dilated cardiomyopathy.

Amino Acid Sequence↗
PubMed1997

Neurturin, a novel neurotrophic factor, is localized to mouse chromosome 17 and human chromosome 19p13.3.

Neurturin is a potent neurotrophic factor closely related to glial cell line-derived neurotrophic factor (GDNF, 40% amino acid sequence identity) and, like GDNF, can promote the survival of numerous neuronal populations including sympathetic, nodose, and dorsal root ganglion sensory neurons. Both neurturin and GDNF signal through the Ret tyrosine kinase and require a glycosylphosphatidylinositol-linked coreceptor. Mutations in Ret and GDNF cause intestinal aganglionosis and renal dysplasia. Activating Ret mutations also cause multiple endocrine neoplasia syndromes (MEN2A and MEN2B). We have isolated mouse and human genomic neurturin clones. The sequence for preproneurturin is encoded by two exons. Mouse and human clones have common intron/exon boundaries. We have used interspecific backcross analysis to localize neurturin to mouse chromosome 17 near the Vav locus and fluorescence in situ hybridization analysis to localize human neurturin to the syntenic region of human chromosome 19p13.3.

Amino Acid Sequence↗
PubMed1997

Purification and molecular cloning of a secreted, Frizzled-related antagonist of Wnt action.

Frizzled polypeptides are integral membrane proteins that recently were shown to function as receptors for Wnt signaling molecules. Here, we report the identification of a novel, secreted 36-kDa protein that contains a region homologous to a putative Wnt-binding domain of Frizzleds. This protein, called Frizzled-related protein (FRP), was first identified as a heparin-binding polypeptide that copurified with hepatocyte growth factor/scatter factor in conditioned medium from a human embryonic lung fibroblast line. Degenerate oligonucleotides, based on the NH2-terminal sequence of the purified protein, were used to isolate corresponding cDNA clones. These encoded a 313-amino acid polypeptide, containing a cysteine-rich domain of approximately 110 residues that was 30-40% identical to the putative ligand-binding domain of Frizzled proteins. A 4.4-kb transcript of the FRP gene is present in many organs, both in the adult and during embryogenesis, and homologs of the gene are detectable in DNA from several vertebrate species. In biosynthetic studies, FRP was secreted but, like Wnts, tended to remain associated with cells. When coexpressed with several Wnt family members in early Xenopus embryos, FRP antagonized Wnt-dependent duplication of the embryonic dorsal axis. These results indicate that FRP may function as an inhibitor of Wnt action during development and in the adult.

Amino Acid Sequence↗
PubMed1997

Molecular cloning, chromosomal localization, and expression of murine dipeptidyl peptidase I.

Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease that catalyzes the sequential removal of dipeptides from the amino termini of various protein substrates. We have isolated a cDNA coding for murine DPPI from mouse thymus and spleen cDNA libraries. The deduced amino acid sequence codes for a protein of 462 amino acid residues; comparison of this deduced sequence with that of rat and human DPPI revealed 90.1% and 77.8% identity, respectively. Using DPPI cDNA, we obtained two BAC (Bacterial Artificial Chromosome) clones that contained the murine DPPI locus. The DPPI gene consists of seven exons and 6 introns, and spans approximately 20 kilobases. Using fluorescence in situ chromosome hybridization, we localized murine DPPI to chromosome 7D3-E1.1. We determined that DPPI protein is widely distributed in mouse tissues, although its relative abundance varies from tissue to tissue. In contrast to previous reports, we show here that DPPI mRNA and protein levels and enzymatic activity are unchanged during in vitro T cell activation, implying that this enzyme is not rate-limiting for granzyme processing.

Amino Acid Sequence↗
PubMed1997

Positions of chromosome 3p14.2 fragile sites (FRA3B) within the FHIT gene.

The FHIT gene spans approximately 1 Mb of DNA at chromosome band 3p14.2, which includes the familial renal cell carcinoma chromosome translocation breakpoint (between FHIT exons 3 and 4), the most frequently expressed human constitutive chromosomal fragile site (FRA3B, telomeric to the t(3;8) translocation), and numerous homozygous deletions in various human cancers, frequently involving FHIT exon 5. The FRA3B has previously been shown to represent more than one specific site, and some specific representatives of FRA3B breaks have been shown to fall in two regions, which we know to be in FHIT introns 4 and intron 5. Because breakage and integration of exogenous DNA in this chromosome region is frequent in aphidicolin-treated somatic cell hybrids, cancer cells, and, presumably, aphidicolin-treated normal lymphocytes that exhibit gaps or breaks, we determined by one- and two color fluorescence in situ hybridization, using cosmids covering specific regions of the FHIT gene, that most of the aphidicolin-induced gaps at FRA3B fall within the FHIT gene, with the highest frequency of gaps falling in intron 5 of the FHIT gene, less than 30 kb telomeric to FHIT exon 5. Gaps also occur in intron 4, where a human papillomavirus 16 integration site has been localized, and in intron 3, where the t(3;8) break point is located. These results suggest that the cancer-specific deletions, which frequently involve introns 4 and 5, originated through breaks in fragile sites.

Acid Anhydride Hydrolases↗
PubMed1997

Molecular cytogenetic characterization of cancer cell alterations.

Chromosomal abnormalities are the hallmark of cancer cells. Recurring and highly consistent structural and numerical alterations have been identified in a large number of leukemias, lymphomas, and solid tumors. The identification of recurrent genetic alterations and the isolation of molecular markers have clinical applications in the diagnosis and prognosis of neoplasia and in the detection of minimal residual disease that are essential for designing the most effective therapeutic approach. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) are powerful techniques for detection of genomic alterations. The battery of FISH methods and DNA probes that are available can resolve virtually any chromosomal alterations regardless of their complexity. Combined chromosome banding, multifluor or spectral karyotype, and comparative genomic hybridization (CGH) allow identification of structural and numerical alterations on a global basis, mapping of the DNA copy number on the entire tumor genome, complete derivation of complex rearrangements, and localization of the breakpoints of translocations and deletions. Regions of recurrent alterations can be microdisected, amplified, microclone libraries constructed and probes localized on extended chromosomes or chromatin fibers for construction of high resolution physical maps that are critical for positional cloning and gene identification. In this review we attempted to cover the current trends in cancer molecular cytogenetics, and to outline the importance of molecular chromosome analysis in the understanding of oncogenesis and its clinical applications.

Chromosome Deletion↗