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Biomedical subjects

B Wainwright

Publications and source records attributed to B Wainwright.

At least 37 records · Page 2Linked to original sources
PubMed1996

A novel target for the Wilms' tumour suppressor protein (WT1) is bound by a unique combination of zinc fingers.

All isoforms of the Wilms' tumour suppressor protein, WT1, contain four consecutive zinc fingers which facilitate DNA binding. The predominant WT1 transcript contains a 9 base pair insertion resulting in an additional three amino acids, lysine-threonine-serine (KTS), between zinc fingers 3 and 4. WT1 zinc fingers 2, 3 and 4 are highly homologous to the zinc fingers of the early growth response gene, EGR1. However, only WT1--KTS is capable of binding an EGR1 consensus site. In contrast, the previously described genomic fragment, +P5 (D1S3309E), is bound by both WT1--KTS and WT1 + KTS. In this study, the region within + P5 to which both WT1 -- KTS and WT1 + KTS bind was defined as 5'-GGAGAGGGAGGATC-3'. EGR1 did not bind + P5. By creating zinc finger deletions, we demonstrate that zinc finger 1, but not zinc finger 4, is critical for + P5 binding; whereas zinc finger 4, but not 1, is necessary for the binding of WT1 target sites within EGR1, PDGF A chain and IGF2 promoters. Thus, zinc finger usage can vary with target and + P5 may represent a novel type of WT1 binding site, the physiological relevance of which must be investigated.

Binding Sites↗
PubMed1996

Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome.

The nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple basal cell carcinomas (BCCs), pits of the palms and soles, jaw keratocysts, a variety of other tumors, and developmental abnormalities. NBCCS maps to chromosome 9q22.3. Familial and sporadic BCCs display loss of heterozygosity in this region, consistent with the gene being a tumor suppressor. A human sequence (PTC) with strong homology to the Drosophila segment polarity gene, patched, was isolated from a YAC and cosmid contig of the NBCCS region. Mutation analysis revealed alterations of PTC in NBCCS patients and in related tumors. We propose that a reduction in expression of the patched gene can lead to the developmental abnormalities observed in the syndrome and that complete loss of patched function contributes to transformation of certain cell types.

Alleles↗
PubMed1996

A mammalian patched homolog is expressed in target tissues of sonic hedgehog and maps to a region associated with developmental abnormalities.

Drosophila patched is a segment polarity gene required for the correct patterning of larval segments and imaginal discs during fly development and has a close functional relationship with hedgehog. We have isolated a complete human PATCHED cDNA sequence, which encodes a putative protein of 1296 amino acids, and displays 39% identity and 60% similarity to the Drosophila PATCHED protein. Hydropathy analysis suggests that human PATCHED is an integral membrane protein with a pattern of hydrophobic and hydrophilic stretches nearly identical to that of Drosophila patched. In the developing mouse embryo, patched is initially detected within the ventral neural tube and later in the somites and limb buds. Expression in the limb buds is restricted to the posterior ectoderm surrounding the zone of polarizing activity. The results show that patched is expressed in target tissues of sonic hedgehog, a murine homolog of Drosophila hedgehog suggesting that patched/hedgehog interactions have been conserved during evolution. Human PATCHED maps to human chromosome 9q22.3, the candidate region for the nevoid basal cell carcinoma syndrome. Patched expression is compatible with the congenital defects observed in the nevoid basal cell carcinoma syndrome.

Amino Acid Sequence↗
PubMed1995

DNA binding capacity of the WT1 protein is abolished by Denys-Drash syndrome WT1 point mutations.

Constitutional point mutations in the zinc finger (ZF) region of the Wilms' tumour suppressor gene 1 (WT1) lead to Denys-Drash syndrome (DDS). Patients with this syndrome display renal failure, Wilms' tumour (WT) and pseudohermaphroditism. DDS WT1 mutations fall into three major categories: (a) missense mutations altering amino acids which directly interact with the DNA target; (b) substitution of amino acids involved in zinc complexing; and (c) nonsense mutations leading to the removal of at least two zinc fingers. We have expressed the WT1 zinc fingers as glutathione-S-transferase fusion proteins, with the lysine-threonine-serine (KTS) alternate splice between ZF3 and ZF4 either present or absent. WT1 fusion constructs with all three classes of DDS mutation were also created. Wild-type and mutant fusion proteins were assayed for their DNA-binding affinity using four previously identified WT1 DNA targets: an EGR1 consensus site; murine insulin-like growth factor 2 promoter 2 (IGF2P2); a (TCC)n motif from the PDGFA-chain promoter; and +P5, a genomic fragment isolated by its affinity for WT1 + KTS. WT1-KTS bound all four targets, but WT1 + KTS only bound +P5. All three classes of DDS mutation investigated, with or without KTS, abolished binding to all four targets. This provides evidence that DDS mutations act either as dominant-negative antimorphs, or elicit their effect through disturbed isoform dosage balance.

Abnormalities, Multiple↗
PubMed1995

The human growth-arrest-specific gene GAS1 maps outside the candidate region of the gene for nevoid basal cell carcinoma syndrome.

The human growth-arrest specific gene GAS1 maps to chromosome bands 9q21.3-->q22, the region known to contain the tumour suppressor gene responsible for nevoid basal cell carcinoma syndrome (NBCCS). Because of its putative action as a tumour suppressor gene, the GAS1 gene was analysed as a candidate for the NBCCS gene. Using two-colour fluorescence in situ hybridization, the GAS1 gene maps outside the interval which, by genetic analysis, has been shown to contain the NBCCS gene.

Animals↗
PubMed1994

Isolation and genetic mapping of two novel members of the murine Wnt gene family, Wnt11 and Wnt12, and the mapping of Wnt5a and Wnt7a.

The murine Wnt genes are implicated in the control of a variety of developmental processes. Using a PCR-based approach, we have isolated two novel members of the murine Wnt gene family, Wnt11 and Wnt12. These cDNAs display an amino acid sequence identity of between 38 and 49% with all other murine Wnts over the regions that we have isolated. In addition, two previously described Wnt genes, Wnt5a and Wnt7a, were detected in RT-PCR products. Interspecific crosses were used to demonstrate close linkage between Wnt12 and Wnt1 on Chromosome (Chr) 15. Wnt7a was mapped to mouse Chr 6, Wnt5a to the centromeric region of Chr 14, and Wnt11 to Chr7.

Amino Acid Sequence↗
PubMed1994

Fine genetic mapping of the gene for nevoid basal cell carcinoma syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS, or Gorlin syndrome) is a cancer predisposition syndrome characterized by multiple basal cell carcinomas and diverse developmental defects. The gene responsible for NBCCS, which is most likely to be a tumor suppressor gene, has previously been mapped to 9q22.3-q31 in a 12-cM interval between the microsatellite marker loci D9S12.1 and D9S109. Combined multipoint and haplotype analyses of additional polymorphisms in this region in our collection of Australasian pedigrees have further refined the localization of the gene to between the markers D9S196 and D9S180, an interval reported to be approximately 2 cM.

Adult↗
PubMed1991

Toward an animal model of cystic fibrosis: targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells.

A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistant cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possibly passage number, influences the recovery of CFTR-targeted cells.

Amino Acid Sequence↗
PubMed1990

Analysis of the transgenome of MET transfectant cell lines reveals that MET activation is accompanied by an interstitial insertion.

The MET oncogene, present in the MNNG-HOS chemically transformed human cell line, is activated by a gene fusion involving sequences from chromosome 1 and chromosome 7. Activated MET can act as a dominant selectable marker for chromosome-mediated gene transfer, and several transfectant cell lines have been established using this technique. Analysis of the transgenomes within these cell lines indicates that MET activation is not simply due to a chromosome translocation, but may involve an interstitial insertion of DNA from chromosome 1, into chromosome 7, probably associated with other rearrangements. Pulse field gel analysis of two transfectants indicates that, despite the presence of complex rearrangements close to MET, chromosome 7 sequences are grossly intact over a 1-Mb region thought to contain the gene defective in cystic fibrosis.

Animals↗
PubMed1988

Refined linkage map of chromosome 7 in the region of the cystic fibrosis gene.

The genetic map in the region of human chromosome 7 that harbors the gene for cystic fibrosis (CF) has been refined by multilocus linkage studies in an expanded database including a large set of normal families. Six loci known to be linked to CF were examined: MET, an oncogene; COL1A2, collagen, TCRB, T-cell-receptor beta polypeptide; and three arbitrary loci--D7S8, D7S13, and D7S16--defined by probes pJ3.11, pB79a, and p7C22, respectively. The gene order with greatest statistical support is COL1A2-D7S13-D7S16-MET-D7S8-TCRB. Linkage analysis in families segregating for CF suggested that the most likely location of the CF gene on this map is between MET and D7S8.

Chromosome Mapping↗
PubMed1987

Isolation of a polymorphic genomic clone from chromosome 7. Physical and genetic linkage studies to markers around the cystic fibrosis locus.

A peptide prepared from purified factor 13B (F13B) was sequenced, and a single, long oligonucleotide corresponding to its cognate DNA sequence was constructed and used to screen a chromosome 7 specific genomic library. The positive clone isolated, designated pKV13, was only related to F13B at the oligonucleotide region, but has proved to be a valuable chromosome 7 marker. pKV13 maps to 7pter-q22 in hybrid cell lines, and is present in a chromosome-mediated gene transfer (CMGT) cell line that also contains met and other 7q probes. pKV13 defines a common MspI restriction fragment length polymorphism (RFLP), and is genetically linked to two markers on the long arm of chromosome 7, B79a and COLIA2, both themselves linked to the cystic fibrosis locus. Multipoint linkage analysis demonstrates that KV13 maps centromeric to both B79a and COLIA2. pKV13 has been used to demonstrate the existence of rearrangements within CMGT hybrids, and will also prove valuable in multipoint linkage studies of other 7q markers. Finally, pKV13 provides a new polymorphic locus for the characterisation of 7q deletions in myeloid disorders such as myelodysplastic syndrome.

Amino Acid Sequence↗
PubMed1987

The cystic fibrosis locus.

The identification of the cystic fibrosis locus (CF) provides a model for the study of single gene defects where the biochemical lesion is not known. Using families each of which has several affected siblings, it was possible to exclude a number of 'candidate genes' which had previously been proposed as possible sites of the CF mutation. Exclusion mapping of the genome using polymorphic protein and DNA markers showed that CF is on the long arm of human chromosome 7. The most closely linked flanking markers were identified, and human chromosome fragments containing them (and therefore the CF locus) were isolated in rodent cell lines by chromosome-mediated gene transfer. The transgenome was then analysed using cosmid contig mapping, pulse-field gel electrophoresis, HTF island identification and linkage disequilibrium. In this way, a candidate coding sequence has been identified which always segregates with CF.

Animals↗