Biomedical subjects
Publications and source records attributed to S Povey.
We describe our experiences of prenatal diagnosis of alpha-1-antitrypsin deficiency using closely linked polymorphisms detected by Southern blotting and the polymerase chain reaction. Prenatal diagnosis was carried out for 17 pregnancies from 15 couples.
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DNA probes were prepared from the 5'-terminal portion of four cDNA clones encoding human phenol and bilirubin UDP-glucuronosyltransferases (UGTs). An additional sequence common to all four clones was isolated from the 3'-terminal portion of one of the clones (UGT1A1). The four specific and the one common DNA sequences were used as probes on a panel of 16 human--rodent somatic cell hybrid DNAs by Southern-blot analysis. The results obtained indicate that all four cDNA clones are encoded by gene(s) located on human chromosome 2.
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Human brain cDNA clones for the myelin associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) have been isolated and sequenced. The only 5' untranslated region (UTR) sequence found was that of a human CNPII mRNA, with no direct evidence for a CNPI mRNA. Human CNPase cDNAs were used to isolate genomic clones containing the human CNPase gene which is 9 kb long. Four exons were identified, separated by three introns, and the sequence of each exon and intron/exon boundary has been established. The polymerase chain reaction (PCR) was used to detect the presence of the human CNPase gene in DNA from a panel of rodent/human somatic cell hybrids. By this means the human CNPase gene was mapped to chromosome 17. In situ hybridization of a human CNPase genomic clone to metaphase chromosomes further localized this gene to chromosomal band 17q21.
Glutamic acid decarboxylase (GAD) catalyses the conversion of L-glutamic acid to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Two forms of human GAD, GAD65 and GAD67, are encoded by two separate genes. A full length human GAD67 cDNA has been isolated from a human frontal cortex cDNA library and the nucleotide sequence determined. The GAD67 gene has been mapped to chromosome 2 using the polymerase chain reaction to amplify specifically the human sequence in rodent/human somatic cell hybrid DNA. This confirms that human GAD67 is not syntenic with the smaller GAD isoform GAD65 which has been assigned to chromosome 10. Production of polyclonal antiserum to a baculovirus-expressed GAD67 enabled immunocytological detection of GAD in the rat brain.
The mouse genes for cytosolic phosphoenolpyruvate carboxykinase-1 (Pck-1) and neuronal nicotinic acetylcholine receptor alpha 4 subunit (Acra-4) both map to distal chromosome 2 (Siracusa et al. 1989; Bessis et al. 1990). We have utilized Southern blot analysis on human/rodent somatic cell hybrids to map the human homologues of both of these genes, PCK1 and CHRNA4, to human chromosome 20.
To characterize the autoantigen of Goodpasture's (anti-glomerular basement membrane) disease, a molecule of 26-kD reactive with autoantibodies from patients' sera was purified from collagenase digests of sheep glomerular basement membrane. Short internal amino acid sequences were obtained after tryptic or cyanogen bromide cleavage, and used to deduce redundant oligonucleotides for use in the polymerase chain reaction on cDNA derived from sheep renal cortex. Molecules of 175 bp were amplified and found to come from two cDNA sequences. One was identical to that of a type IV collagen chain (alpha 5) cloned from human placenta and shown to be expressed in human kidney. The other was from a type IV collagen chain with close similarities to alpha 1 and alpha 5 chains, and was used to obtain human cDNA sequences by cDNA library screening and by further polymerase chain reaction amplifications. The correspondence of the derived amino acid sequence of the new chain with published protein and cDNA sequences shows it to be the alpha 3 chain of type IV collagen. Its gene, COL4A3, maps to 2q36-2q37. The primary sequence and other characteristics of this chain confirm that it carries the Goodpasture antigen.
The new continuous cell line HOK-1 derived from a grade-III transitional-cell bladder carcinoma with foci of squamous and glandular differentiation was shown to retain this phenotypical heterogeneity for more than 45 passages in vitro. Electron microscopy revealed transitional as well as a considerable proportion of squamous carcinoma and adenocarcinoma cells. PAS-positive mucus was detected in numerous cells. These features were principally maintained when grown as multicellular spheroids and in nude mice. More pronounced signs of differentiation (i.e., expression of cytokeratins 10 and 11, formation of glandular structures) were found in xenograft tumours. Independently, cytokeratins 13, 18 and 19 were detected in vitro and in vivo, reflecting the urothelial origin. The line forms distinct colonies in soft agar, expresses Lewis-x and Lewis-y antigens and reacts with monoclonal antibodies (MAbs) against CEA, beta-HCG and URO-5. Cytogenetic analysis revealed several related clones with a rearrangement at chromosome 1 and loss of one X chromosome as common karyotypic changes in all clones. DNA content, as quantified by image analysis, showed a DNA stemline close to 2c. The new cell line HOK-1 can be used as an in vitro model to study the mechanisms of heterogeneous differentiation patterns in bladder cancer.
cDNA clones that code for a pig and human flavin-containing monooxygenase (FMO) have been isolated. The full-length sequence of the human cDNAs revealed that they encode a polypeptide of 532 amino acid residues containing putative FAD- and NADP-binding sites. The deduced amino acid sequence has 88 and 86% identity, respectively, with the pig and rabbit "hepatic" forms of FMO, but is only 58% similar to the rabbit "pulmonary" FMO, and thus represents the human ortholog of the "hepatic" form of FMO. However, as this FMO is present in low abundance in human adult liver, the general term "hepatic" for this form of the enzyme is misleading, and thus we propose the name FMO1 to describe this human FMO and its mammalian orthologs. Northern blot analysis demonstrated that human FMO1 mRNA is more abundant in fetal than in adult liver, indicating that in man the enzyme is subject to developmental regulation. Southern blot hybridization of human genomic DNA suggests that the protein is encoded by a single gene, which has been designated FMO1 and mapped to chromosome 1.
We have isolated cDNA clones that code for human cytochrome b5. Owing to the high degree of evolutionary conservation of cytochrome b5 sequences and the existence of human and rodent cytochrome b5 processed pseudogenes, we were unable to map unambiguously the chromosomal localization of the human gene(s) by Southern blot hybridization of DNA from human-rodent somatic cell hybrids. An alternative approach, based on restriction enzyme digestion of PCR-amplified DNA, enabled us to map the human cytochrome b5 gene(s) to chromosome 18 and one of its processed pseudogenes to the X chromosome. We propose the designations CYB5 and CYB5P1 for the gene and pseudogene loci, respectively.
The mouse brown locus encodes a tyrosinase-related protein, TRP-1. The human homolog of TRP-1 was recently cloned from a melanoma cDNA library and sequenced. We have made oligonucleotide primers corresponding to the human TRP1 3' untranslated region and used them to map the human TRP1 gene by species-specific PCR in human/rodent somatic cell hybrids. By this means, the human TRP1 gene has been mapped to the short arm of chromosome 9.
The human chromosome complement of somatic cell hybrids must be assessed each time the hybrids are grown in culture. We have developed a panel of human-specific oligonucleotide primers for genes that have been mapped to each of the autosomes and to the X chromosome. These primers enable the human chromosome content of hybrids to be assessed rapidly by PCR. The sequence of the primers is presented together with the appropriate conditions for human DNA-specific amplification for each pair.
We have previously reported the isolation of a genomic clone encoding human liver-specific peroxisomal alanine:glyoxylate aminotransferase (AGT, EC 2.6.1.44), the deficient enzyme in primary hyperoxaluria type 1 (PH1) (P. E. Purdue, Y. Takada, and C. J. Danpure, J. Cell Biol. 111: 2341-2351, 1990). This clone has now been characterized, revealing that the coding sequence is distributed among 11 exons covering 10 kb. The nucleotide sequences of each exon have been determined, confirming that this clone corresponds to previously characterized AGT cDNA (Y. Takada, N. Kaneko, H. Esumi, P. E. Purdue, and C. J. Danpure, Biochem. J. 268: 517-520, 1990). In addition, to provide sequence data for the design of exon-specific PCR primers, the intron sequences immediately flanking each exon have been determined. Furthermore, in an attempt to identify putative transcriptional control sequences we have determined the sequence of 1.25 kb directly upstream of the cDNA 5' end. The results of genomic Southern blotting indicate that human AGT is probably encoded by a single copy gene, and a combination of in situ hybridization and PCR analysis of rodent/human somatic cell hybrids suggests that this gene is located on chromosome 2q36-q37. The gene symbol AGXT has been assigned for this locus.
Ubiquitin carboxy terminal hydrolase 1, UCHL1, is a neurone-specific protein involved in the ubiquitin-mediated proteolytic pathway. The gene for human UCHL1 has been mapped to chromosome 4 using the polymerase chain reaction to amplify specifically the human UCHL1 sequences in rodent/human somatic cell hybrid DNA. A regional assignment of this locus to 4p14 has been made by in situ hybridization to metaphase chromosomes using both tritium and fluorescently labelled probes.
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