Katie Morrison

Susceptibility to spina bifida; an association study of five candidate genes.

Clues regarding candidate genes which influence susceptibility to spina bifida and anencephaly come from the identification of folate‐associated risk factors and from studies of mouse mutants showing neural tube anomalies. On this basis we selected five candidate genes; CBS, MS, MTHFR, T (Brachyury) and BRCA1 for genetic analysis in 31 Dutch and 48 British NTD families. Ten polymorphisms, two for each gene, were used in transmission tests for disequilibrium (TDT). In six instances more than 50 transmissions from heterozygous parents could be examined. Using TDT we find evidence for an association between an allele at the T gene and liability to NTD in the embryo. Data from British and Dutch populations showed the same trend and in combination gave a χ2TDT=4.89, P=0.03 (OR 2.39, CI 95% 1.02–5.61). No association, in either population group, was found for CBS, MS and MTHFR, the enzymes most directly associated with the known risk factors in folate metabolism. The possibility of complex genetic interactions was explored; the data show that a Gly919 MS variant occurs more frequently in combination with the MTHFR thermolabile variant in mothers of NTD offspring (OR 3.94, CI 95% 1.0–16.3).

Colon carbonic anhydrase 1: transactivation of gene expression by the homeodomain protein Cdx2

The homeodomain protein, Cdx2, has been implicated in the transcriptional regulation of genes expressed in the small intestine. In vitro studies of the carbonic anhydrase 1 (CA1) colon promoter implied that Cdx2 may also play a role in the regulation of colon‐specific gene expression. The current work follows up this proposal by examining the ability of Cdx2 to transactivate gene expression in cultured cells mediated by CA1 promoter sequences. The results show that Cdx2 exerts a positive regulatory effect by binding to a motif 87 bp upstream of the CA1 TATA box; this motif appears to act as an enhancer since gene activation is independent of its orientation.

A cosmid library specific for human Chromosome regions 6p21.3 and 6q27

We have explored the potential of irradiation-fusion gene transfer (IFGT) hybrids as a source of well-defined human chromosome fragments from which probes can be derived. Extensive characterization of the IFGT hybrid 4J4 with a full panel of markers from Chromosome (Chr) 6 showed that the human DNA content derives largely from 6p21.3 and 6q27. A cosmid library has been constructed from 4J4 DNA, and 370 recombinants containing human DNA have been isolated and overlapping clones ordered into 20 contigs. Regional localization of representative clones from each contig, determined by fluorescent in situ hybridization (FISH), places 13 contigs in 6q27 and 6 in 6p21.3. Preliminary screening of cDNA libraries with selected cosmids has identified two expressed sequences. Since there are a number of medically important genes in both these regions of human Chr 6 with several disease loci linked to the HLA-A region in 6p21.3 and various tumor suppressor genes to 6q27, this library will provide a valuable resource to aid the isolation of candidate genes for these diseases. In addition, unique markers for detailed physical and genetic mapping of these regions of human Chr 6 can be easily obtained.

The caudal type homeobox protein CDX2 binds to the colon promoter of the carbonic anhydrase 1 gene

Carbonic anhydrase 1 (CA1) is an abundant enzyme in colon epithelia. In the gastrointestinal tract, carbonic anhydrase is vital for NaCl resorption, alkalinization of gut contents, and absorption of short-chain fatty acids. The CA1 gene has two promoters, one of which is specifically active in colon epithelia and the other in erythroid cells. We are investigating the factors that regulate CA1 expression from the colon-specific promoter. Colon-specific deoxyribonuclease I hypersensitive sites (DHS) have been mapped close to the colon transcription initiation site (DHS6c) and in the upstream intron (DHS5c). Using electrophoretic mobility-shift assays to search the 650-bp region which contains DHS6c, we have identified sequences that bind a colon-specific factor (COF1) and by deletion analysis we have narrowed down the COF1-binding motif to a 17-bp sequence. A comparison of this motif with a protein-binding motif in the sucrase-isomaltase gene promoter, competition assays, and antibody studies indicate that COF1 is identical to the homeodomain protein Cdx-2. We propose that Cdx-2 plays an important role in the intestine-specific expression of CA1.

Sequence comparisons and functional studies of the proximal promoter of the carbonic anhydrase 3 (CA3) gene

Carbonic anhydrase 3 (CA3) is a member of a gene family encoding proteins which catalyse the hydration of CO2 to generate protons and bicarbonate ions for cellular ion transport and pH homeostasis. In mouse embryos CA3 is expressed at high levels in notochord and skeletal muscle and here we demonstrate that this pattern of expression is the same in the developing human embryo. To investigate mechanisms controlling CA3 transcription, we have isolated and compared 2.8kb of sequence flanking exon 1 from the mouse and human genes. Several segments of high sequence identity >80% have been identified, the longest segments of which represent a proximal promoter region and a putative enhancer element. We have shown previously that in cultured cells the human 2.8kb promoter region imposes high level myogenic specific transcription of a reporter gene. However, we now show that while this promoter region directed muscle-specific expression in transgenic mouse embryos this was subject to position effects.

The identification of novel sequences expressed in the mouse notochord

A large number of human eDNA clones have been sequenced, and over 400,000 expressed sequence tags (ESTs) representing a significant proportion of the coding component of the human genome have been submitted to public databases. However, a number of genes are expressed only at early stages of embryogenesis and, since these stages are inaccessible in humans, are not currently represented in cDNA libraries and sequence databases. For this reason, cDNA libraries prepared from structures of the early developing mouse embryo represent an important supplement to the human gene data resource.