Biomedical subjects

T W Glover

Publications and source records attributed to T W Glover.

At least 37 records · Page 2Linked to original sources

PubMed1998

Human Na(+)-myo-inositol cotransporter gene: alternate splicing generates diverse transcripts.

Na(+)-myo-inositol cotransport activity generally maintains millimolar intracellular concentrations of myo-inositol and specifically promotes transepithelial myo-inositol transport in kidney, intestine, retina, and choroid plexus. Glucose-induced, tissue-specific myo-inositol depletion and impaired Na(+)-myo-inositol cotransport activity are implicated in the pathogenesis of diabetic complications, a process modeled in vitro in cultured human retinal pigment epithelium (RPE) cells. To explore this process at the molecular level, a human RPE cDNA library was screened with a canine Na(+)-dependent myo-inositol cotransporter (SMIT) cDNA. Overlapping cDNAs spanning 3569 nt were cloned. The resulting cDNA sequence contained a 2154-nt open reading frame, 97% identical to the canine SMIT amino acid sequence. Genomic clones containing SMIT exons suggested that the cDNA is derived from at least five exons. Hypertonic stress induced a time-dependent increase, initially in a 16-kb transcript and subsequently in 11.5-, 9.8-, 8.5-, 3.8-, and approximately 1.2-kb SMIT transcripts, that was ascribed to alternate exon splicing using exon-specific probes and direct cDNA sequencing. The human SMIT gene is a complex multiexon transcriptional unit that by alternate exon splicing generates multiple SMIT transcripts that accumulate differentially in response to hypertonic stress.

Alternative Splicing↗

PubMed1998

Y;16 translocation breakpoint associated with a partial Turner phenotype identifies a foamy virus insertion.

We have previously identified YACs containing the Y breakpoint in a male patient with Turner-like hydrops in the newborn period and a Y;16 translocation (Erickson et al., 1995). We have now subcloned these YACs and built a lambda contig across the Y breakpoint. A subclone near the Y;16 breakpoint of our patient shows great evolutionary conservation by hybridization to a zoo blot. We have sequenced the region responsible for the hybridization and found a foamy (spuma) virus sequence. These viruses are known to be xenotropic and a very similar cross-species hybridization (using the same commercial zoo blot) was recently reported with a human foamy virus clone (Cordonnier et al., 1995). Thus, we have identified a foamy virus insertion near the Y heterochromatin boundary.

Animals↗

PubMed1997

Frequent deletions of FHIT and FRA3B in Barrett's metaplasia and esophageal adenocarcinomas.

The FHIT gene, which spans the common fragile site FRA3B, has been shown to produce aberrant transcripts in a variety of tumor types. Homozygous deletions within the FHIT locus have been detected only in tumor-derived cell lines and LOH has been described in numerous primary tumors. Based on these findings and its location on 3p, FHIT has been proposed as a tumor suppressor gene. To further study the relationship of FRA3B to the findings regarding the FHIT gene and to determine the extent of FHIT mRNA alterations in early stages of tumor development, the status of the FHIT gene was evaluated in the premalignant condition of Barrett's esophagus and associated esophageal adenocarcinomas. FHIT expression was investigated by RT-PCR in normal esophageal, Barrett's metaplasia and adenocarcinoma tissues from 15 patients. Alterations of FHIT transcripts were observed in 12/14 (86%) of Barrett's metaplasia and in 14/15 (93%) of the adenocarcinomas from the same patients. Characterization of the altered transcripts revealed FHIT mRNA lacking one or more exons, with deletion of exons 5-7 being most frequent. Analysis of genomic DNA from 20 patients showed homozygous deletions involving exon 5 of FHIT in 4/20 (20%) esophageal adenocarcinomas, and 7/20 (35%) tumors demonstrated hemizygous loss. Genomic deletions also involved the BE758-6 locus, indicating that a large region is deleted. Fluorescence in situ hybridization (FISH) analysis demonstrated that this region of deletion is localized within FRA3B. Our results extend the range of tumor types in which altered FHIT transcripts have been demonstrated and show that these alterations can be seen in the premalignant stage of esophageal tumor development. These results indicate that the fragility and recombination-prone nature of FRA3B is related to tumor-specific chromosomal instability affecting the FHIT gene in esophageal adenocarcinoma development.

Acid Anhydride Hydrolases↗

PubMed1997

A phosphoglycerate mutase brain isoform (PGAM 1) pseudogene is localized within the human Menkes disease gene (ATP7 A).

We have identified a phosphoglycerate mutase brain isoform (PGAM 1, PGAM B) cDNA that is localized between exons 1 and 2 of the Menkes disease gene (ATP7 A, MNK) at Xq13.3. The cDNA shows 98% identity to the previously identified PGAM 1 cDNA (Sakoda et al., J. Biol. Chem. 263 (1988) 16899-16905) and probably represents a recent retroposition of this parent PGAM 1 mRNA. Although the typical features of a processed pseudogene are present, the open reading frame (ORF) of this PGAM cDNA is potentially expressed. There are 11 bp changes in the 765 bp ORF, none of which are nonsense mutations or deletions. The region upstream from the ORF shows some features of a possible promoter region, although it lacks a CpG island often associated with functional promoters. We analyzed the expression of this PGAM 1 cDNA using RT-PCR followed by restriction enzyme digestion based on a 1 bp missmatch in this cDNA to distinguish it from normal PGAM 1 gene expression. With this sensitive method, we could not find expression in any of the tissues examined. Taken together, we conclude that the PGAM 1 cDNA upstream from exon 2 of the Menkes gene is likely to be a processed pseudogene originating from a very recent retroposition of a PGAM 1 transcript. To our knowledge this is the first report of a pseudogene located within a gene.

Adenosine Triphosphatases↗

PubMed1997

Exclusion of BMP6 as a candidate gene for cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant, generalized skeletal dysplasia in humans that has been mapped to the short arm of chromosome 6. We report linkage of a CCD mutation to 6p21 in a large family and exclude the bone morphogenetic protein 6 gene (BMP6) as a candidate for the disease by cytogenetic localization and genetic recombination. CCD was linked with a maximal two-point LOD score of 7.22 with marker D6S452 at theta = 0. One relative with a recombination between D6S451 and D6S459 and another individual with a recombination between D6S465 and CCD places the mutation within a 7 cM region between D6S451 and D6S465 at 6p21. A phage P1 genomic clone spanning most of the BMP6 gene hybridized to chromosome 6 in band region p23-p24 using FISH analysis, placing this gene cytogenetically more distal than the region of linkage for CCD. We derived a new polymorphic marker from this same P1 clone and found recombinations between the marker and CCD in this family. The results confirm the map position of CCD on 6p21, further refine the CCD genetic interval by identifying a recombination between D6S451 and D6S459, and exclude BMP6 as a candidate gene.

Bone Morphogenetic Protein 6↗

PubMed1997

Aphidicolin-induced FRA3B breakpoints cluster in two distinct regions.

The common fragile site at chromosomal band 3p14.2 (FRA3B) is the most sensitive single site in the human genome to induced chromosomal lesions. This fragile site may predispose chromosome 3p to breakage that is commonly observed in lung, renal, and many other cancers. We previously used aphidicolin induction of FRA3B expression in a chromosome 3-only somatic cell hybrid to generate a series of hybrids with breakpoints in the 3p14.2 region. These breakpoints were localized to two distinct clusters, separated by 200 kb, that lie on either side of a region of frequent breakage within FRA3B as observed by FISH analysis. Seven proximal aphidicolin-induced breakpoints were localized at or near the end of a THE element. The THE-1 element is flanked by LINE and Alu repetitive elements. The eight distal aphidicolin-induced breakpoints clustered in a region capable of forming multiple hairpin-like structures. Thus repetitive elements and hairpin-like structures may be responsible for chromosome fragility in this region.

Aphidicolin↗

PubMed1997

Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans-Golgi network.

We have generated polyclonal antibodies against the amino-terminal third of the Menkes protein (ATP7A; MNK) by immunizing rabbits with a histidine-tagged MNK fusion construct containing metal-binding domains 1-4. The purified antibodies were used in Western analysis of cell lysates and in indirect immunofluorescence experiments on cultured cells. On Western blots, the antibodies recognized the approximately 165 kDa MNK protein in CHO cells and human fibroblasts. No MNK signal could be detected in fibroblasts from a patient with Menkes disease or in Hep3B hepatocellular carcinoma cells, confirming the specificity of the antibodies. Immunocytochemical analysis of CHO cells and human fibroblasts showed a distinct perinuclear signal corresponding to the pattern of the Golgi complex. This staining pattern was similar to that of alpha-mannosidase II which is a known resident enzyme of the Golgi complex. Using brefeldin A, a fungal inhibitor of protein secretion, we further demonstrated that the MNK protein is localized to the trans-Golgi network. This data provides direct evidence for a subcellular localization of the MNK protein which is similar to the proposed vacuolar localization of Ccc2p, the yeast homolog of MNK and WND (ATP7B), the Wilson disease gene product. In light of the proposed role of MNK both in subcellular copper trafficking and in copper efflux, these data suggest a model for how these two processes are linked and represent an important step in the functional analysis of the MNK protein.

Adenosine Triphosphatases↗

PubMed1996

Isolation of human and murine homologues of the Drosophila minibrain gene: human homologue maps to 21q22.2 in the Down syndrome "critical region".

The presence of an extra copy of human chromosome 21 (trisomy 21), especially region 21q22.2, causes many phenotypes in Down syndrome, including mental retardation. To study genes potentially responsible for some of these phenotypes, we cloned a human candidate gene (DYRK) from 21q22.2 and its murine counterpart (Dyrk) that are homologous to the Drosophila minibrain (mnb) gene required for neurogenesis and to the rat Dyrk gene (dual specificity tyrosine phosphorylation regulated kinase). The three mammalian genes are highly conserved, >99% identical at the protein level over their 763-amino-acid (aa) open reading frame; in addition, the mammalian genes are 83% identical over 414 aa to the smaller 542-aa mnb protein. The predicted human DYRK and murine Dyrk proteins both contain a nuclear targeting signal sequence, a protein kinase domain, a putative leucine zipper motif, and a highly conserved 13-consecutive-histidine repeat. Fluorescence in situ hybridization and regional mapping data localize DYRK between markers D21S336 and D21S337 in the 21q22.2 region. Northern blot analysis indicated that both human and murine genes encode approximately 6-kb transcripts. PCR screening of cDNA libraries derived from various human and murine tissues indicated that DYRK and Dyrk are expressed both during development and in the adult. In situ hybridization of Dyrk to mouse embryos (13, 15, and 17 days postcoitus) indicates a differential spatial and temporal pattern of expression, with the most abundant signal localized in brain gray matter, spinal cord, and retina. The observed expression pattern is coincident with many of the clinical findings in trisomy 21. Its chromosomal locus (21q22. 2), its homology to the mnb gene, and the in situ hybridization expression patterns of the murine Dyrk combined with the fact that transgenic mice for a YAC to which DYRK maps are mentally deficient suggest that DYRK may be involved in the abnormal neurogenesis found in Down syndrome.

Adult↗

PubMed1996

Tissue distribution, genomic structure, and chromosome mapping of mouse and human eukaryotic initiation factor 4E-binding proteins 1 and 2.

Two related eukaryotic initiation factor-4E binding proteins (4E-BP1 and 4E-BP2) were recently characterized for their capacity to bind specifically to eIF4E and inhibit its function. Here, we determined the cDNA sequence, tissue distribution, genomic structure, and chromosome localization of murine and human 4E-BP1 and 4E-BP2. Mouse 4E-BP1 and 4E-BP2 consist of 117 and 120 amino acids and exhibit 91. 5 and 95.0% identity, respectively, to their human homologues. 4E-BP1 mRNA is expressed in most tissues, but is most abundant in adipose tissue, pancreas, and skeletal muscle, while 4E-BP2 mRNA is ubiquitously expressed. The structures of the mouse 4E-BP1 and 4E-BP2 were determined. The 4E-BP1 gene consists of three exons and spans approximately 16 kb. In addition, two 4E-BP1 pseudogenes exist in the mouse genome. The 4E-BP2 gene spans approximately 20 kb and exhibits an identical genomic organization to that of 4E-BP1, with the protein coding portion of the gene divided into three exons. There are no pseudogenes for 4E-BP2. The chromosomal locations of 4E-BP1 and 4E-BP2 were determined in both mice and humans by fluorescence in situ hybridization analysis. Mouse 4E-BP1 and 4E-BP2 map to chromosomes 8 (A4-B1) and 10 (B4-B5), respectively, and human 4E-BP1 and 4E-BP2 localize to chromosomes 8p12 and 10q21-q22, respectively.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Frequent breakpoints in the 3p14.2 fragile site, FRA3B, in pancreatic tumors.

FRA3B, at chromosomal band 3p14.2, is the most active common fragile site in the human genome. Homozygous deletions in the region of FRA3B have been observed in many types of solid tumors. Recently, the FHIT gene was reported to span FRA3B and was shown to be homozygously deleted in several gastric and colonic tumor cell lines. Several microsatellite markers that precisely define the 1.0-Mb region surrounding FRA3B and FHIT have been utilized, along with other 3p microsatellites, to analyze the loss of 3p sequences in 25 primary pancreatic adenocarcinomas. The high density of microsatellite markers in the 3pl4.2 region enabled us to both identify losses within and flanking FRA3B in pancreatic cancer and define the breakpoints. We observed loss of het erozygosity of 3pl4.2 markers in 16 of 25 pancreatic tumors and loss of heterozygosity of 3p markers outside of 3pl4.2 in only 2 of 25 tumors of this type. There appears to be a dramatic clustering of chromosomal breakpoints at 3pl4.2 in and immediately distal to FRA3B in pancreatic cancer. We detected no homozygous deletions in this region.

Adenocarcinoma↗

PubMed1996

Transcription elongation factor SII (TCEA) maps to human chromosome 3p22 --> p21.3.

Transcription elongation factors assist RNA polymerase II through transcriptional blockages. The human transcriptional elongation factor SII or Trascription Elongation Factor A (TCEA) releases RNA polymerase II from transcriptional arrest and is encoded by a 2.5-kb intronless gene. Using PCR primers, verified by RT-PCR to amplify the authentic, transcriptionally active SII gene, this locus was mapped to human chromosome 3 by examination of a human/rodent somatic cell hybrid panel. PCR analysis of somatic cell hybrids with chromosome 3 translocations and FISH studies utilizing a human YAC clone containing the SII gene further refine the map position of this locus to human chromosome 3p22 --> p21.3. Since another elongation factor, SIII, has been implicated in human carcinogenesis and since the interval within which the human SII gene maps is frequently deleted in certain cancers, elongation factor SII may therefore be considered a candidate gene for human malignancies involving 3p22 --> p21.3.

Animals↗

PubMed1996

A 350-kb cosmid contig in 3p14.2 that crosses the t(3;8) hereditary renal cell carcinoma translocation breakpoint and 17 aphidicolin-induced FRA3B breakpoints.

The constitutive fragile site at human chromosomal band 3p14.2, FRA3B, has been described as the most active common fragile site in the human genome. FRA3B is cytologically indistinguishable from the chromosome 3 breakpoint observed in the hereditary renal cell carcinoma (hRCC) translocation t(3;8) (p14.2;q24.13). Previous work demonstrated that a 1330-kb YAC clone, YC850A6, spans both the t(3;8) translocation and FRA3B and also encompasses FRA3B-associated breakpoints induced in hamster-human hybrids. This YAC was used to construct a multi-hit cosmid library. Screening of this library resulted in a 350-kb cosmid contig that extends distally from the t(3;8) translocation breakpoint. Seventeen aphidicolin-induced 3p14. 2 breakpoints derived from hamster-human hybrids were mapped within this cosmid contig. These breakpoints were found to localize as two distinct clusters, separated by 200 kb, which lie on either side of a region of frequent breakage within FRA3B as defined by FISH analysis using cosmids from the contigs. The most proximal of the breakpoint clusters lies approximately 100 kb distal to the hRCC t(3;8) breakpoint. The distribution of these breakpoints, together with the region of frequent chromosomal breakage mapped by FISH analysis, further confirms the position of FRA3B and helps to define the extent over which its fragility is exerted. These data indicate that FRA3B comprises several hundred kilobases of DNA sequence within 3p14.2. The 350-kb contig and the cosmid library constructed from YAC YC850A6 will be essential for further characterization of the region surrounding FRA3B and in experiments to determine the molecular basis of the fragility of FRA3B.

Animals↗

PubMed1996

Identification and analysis of the human and murine putative chromatin structure regulator SUPT6H and Supt6h.

We have isolated and sequenced SUPT6H and Supt6h, the human and murine homologues of the Saccharomyces cerevisiae and Caenorhabditis elegans genes SPT6 (P using 1603 aa = 6.7 e-95) and emb-5 (P using 1603 aa = 7.0 e-288), respectively. The human and murine SPT6 homologues are virtually identical, as they share >98% identity and >99% similarity at the protein level. The derived amino acid sequences of these two genes predict a 1603-aa protein (human) and a 1726-bp protein (mouse), respectively. There were several known features, including a highly acidic 5'-region, a degenerate SH2 domain, and a leucine zipper. These features are consistent with a nuclear protein that regulates transcription, whose extreme conservation underscores the likely importance of this gene in mammalian development. Expression of human and murine SPT6 homologues was analyzed by Northern blotting, which revealed a 7. 0-kb transcript that was expressed constitutively. The SPT6 homologue was mapped to chromosome 17q11.2 in human by somatic cell hybrid analysis and in situ hybridization. These data indicate that SUPT6H and Supt6h are functionally analogous to SPT6 and emb-5 and may therefore regulate transcription through establishment or maintenance of chromatin structure.

Amino Acid Sequence↗

PubMed1996

Distinct areas of allelic loss on chromosomal regions 10p and 10q in human prostate cancer.

Utilizing tissue microdissection and PCR techniques, we have examined 35 prostate tumors paired with normal tissues from the same patients for allelic loss at 24 polymorphic loci spanning chromosome 10. Twenty-five tumors (71%) were deleted for at least one chromosome 10 locus. Of the total 35 tumors, 6 (17%) were deleted for 10p loci only, 5 (14%) for 10q loci only, and 14 (40%) were deleted for both 10p and 10q loci. The common region of deletion on 10p included loci D10S211-D10S89-D10S111. Fluorescence in situ hybridization of yeast artificial chromosome probes encompassing these loci demonstrated that the 10p region of deletion maps to 10p11.2. Losses involving 10p loci alone were most common in localized (5/14, 36%) and least common in metastatic (0/8) tumors. The common region of deletion on 10q included loci D10S219-D10S215, consistent with the major region of deletion recently defined for prostate tumors on 10q. Losses involving 10q loci alone were lowest in localized and locally invasive tumors (1/14 and 2/12, respectively) and highest in tumors metastatic to regional lymph nodes (2/8). These results suggest that 10p losses may define less invasive tumors, whereas 10q losses may play a role in the progression to more advanced tumor states in the prostate. Furthermore, this is the first report of allelic loss of a defined region on 10p potentially harboring tumor suppressor gene loci in human prostate cancer.

Alleles↗

PubMed1996

A translocation interrupts the COL5A1 gene in a patient with Ehlers-Danlos syndrome and hypomelanosis of Ito.

Ehlers-Danlos syndrome (EDS) is a genetically and pathogenetically heterogeneous group of disorders of which at least 11 types have been described. All are connective tissue disorders characterized by defects of the skin, ligaments and blood vessels with the clinical spectrum ranging from innocuous findings to lethality. Mutations in the genes encoding the major fibrillar collagen types I and III have been demonstrated in EDS types VII and IV, respectively, while mutations in the lysyl hydroxylase and ATP7A genes, with roles in collagen cross-linking, are responsible for EDS types VI and IX. The biochemical and molecular bases for the most common forms of EDS (types I, II and III) are unknown. Here, we describe a balanced translocation between chromosome 9 and an X chromosome that disrupts the minor fibrillar collagen type V gene COL5A1 in a patient with both EDS type I and hypomelanosis of Ito. The breakpoint occurs at 9q34 within COL5A1 intron 24 and interestingly, within a LINE-1 (L1) element at Xp21.1. A fusion mRNA between COL5A1 and an Alu sequence is produced, but no aberrant protein is detectable. Rather, the amount of type V collagen is reduced in the patient's fibroblasts, suggesting haploinsufficiency as a cuase of the phenotype. This demonstrates that a mutation in a type V collagen gene, COL5A1, results in EDS type I, and shows the involvement of L1 sequences in a constitutional chromosomal translocation. Because collagen type V is a heteromorphic protein in which molecules may be composed of polypeptides encoded by three COL5A genes, this suggests all three genes as candidates for mutations in EDS.

Base Sequence↗

PubMed1996

FRA3B extends over a broad region and contains a spontaneous HPV16 integration site: direct evidence for the coincidence of viral integration sites and fragile sites.

The common fragile site at 3p14.2 (FRA3B) is the most sensitive site on normal human chromosomes for the formation of gaps and breaks when DNA replication is perturbed by aphidicolin or folate stress. Although rare fragile sites are known to arise through the expansion of CCG repeats, the mechanism responsible for common fragile sites is unknown. Beyond being a basic component of chromosome structure, no biological effects of common fragile sites have been convincingly shown, although suggestions have been made that breakage and recombination at these sites may sometimes be mechanistically involved in deletions observed in many tumors and in constitutional deletions. In an observation related to the high rate of recombination at fragile sites, a number of studies have shown a statistical association between the integration of transforming DNA viruses and chromosomal fragile sites. Using FISH analysis we recently identified a 1.3 Mb YAC spanning both FRA3B and the t(3;8) translocation associated with hereditary RCC. Here we report the further localization of FRA3B within this YAC. Using lambda subclones of the YAC as FISH probes, gaps and breaks were found to occur over a broad region of at least 50 kb. Neither CCG nor CAG repeats were found in this region suggesting a different mechanism for fragility than seen with rare fragile sites. We further show that an area of frequent gaps and breaks within FRA3B, defined by a lambda contig, coincides with a previously characterized site of HPV16 integration in a primary cervical carcinoma. The HPV16 integration event gave rise to a short chromosomal deletion limited to the local FRA3B region within 3p14.2. Interestingly, 3p14.2 lies within the smallest commonly deleted region of 3p in cervical cancers, which are often HPV16 associated. To our knowledge this is the first molecular characterization of an in vivo viral integration event within a confirmed fragile site region, supporting previous cytogenetic observations linking viral integration sites and fragile sites.

Bacteriophage lambda↗

PubMed1996

FISH localization of the soluble thymidine kinase gene (TK1) to human 17q25, a region of chromosomal loss in sporadic breast tumors.

Soluble thymidine kinase (TK1) is an important 17q marker in somatic cell genetics. Its activity is increased in many malignancies, including breast cancer. Through somatic cell hybrid and fluorescence in situ hybridization studies, we mapped TK1 to 17q25.2-->25.3, in region demonstrating loss of heterozygosity in primary breast tumors. It lies near D17S836 and is proximal to the avian erythroblastic leukemia viral oncogene homolog 2-like gene (ERBA2L).

Animals↗

PubMed1995

Molecular structure of the Menkes disease gene (ATP7A).

We report a detailed molecular analysis of the genomic structure of the Menkes disease gene (MNK; ATP7A). There are 23 exons in ATP7A covering a genomic region of approximately 140 kb. The size of the individual coding exons varies between 77 and 726 bp, and introns vary in size between 196 bp and approximately 60 kb. All of the splice sites obey the consensus GT-AG rule except the splice donor of intron 9, which is GC instead of GT. The exon following this rare splice donor variant is alternatively spliced. A PGAM pseudogene and two highly polymorphic CA repeats map to introns within the gene. The structure is very similar to that of the closely related Wilson disease gene (WND; ATP7B). From exon 5 (exon 3 in ATP7B) to the end, all of the splice sites occur at exactly the same nucleotide positions as in the WND gene, except for the boundary between exons 17 and 18 (exons 15 and 16 in ATP7B) and a single codon difference at the boundary between exons 4 and 5 of the MNK gene (exons 2 and 3 in ATP7B). In contrast to the WND gene, in which the first four of six metal binding domains are contained in 1 exon, metal binding domains 1 to 4 are divided over 3 exons. The striking similarity of the MNK and WND genes at the genomic level is consistent with their relatively recent divergence from a common ancestral gene.

Adenosine Triphosphatases↗