Cloning and characterisation of the human carbonic anhydrase I gene and mapping of the carbonic anhydrase gene cluster on chromosome 8

The carbonic anhydrase I gene (CA1) is part of a multigene family, the protein products of which are enzymes characterised by their ability to catalyse the reversible hydration of CO2. It exhibits a tissue specific pattern of expression, notably being expressed at high levels in erythroid cells in humans. It also shows regulation at a developmental level, with the CA1 protein at very low concentration in the blood of the foetus till a few weeks before birth. cDNA and genomic clones encoding CA1 were isolated and charachterised. Analysis of cDNA clones showed the presence of an occasional exon in the 5' leader of the transcript, while at the 3'-end two polyadenylation sites could be used, while analysis of genomic clones showed that CAI is atypical amongst the carbonic anhydrases in having a large intron of 36 kb separating the erythroid specific promoter from the coding region, making the entire gene some 50 kb in length. Pulsed-field gel electrophoresis was used in the analysis of the physical linkage relationship between CA1 and the CA2 and CA3 genes. Both of these genes lie 5' to CA1 and are transcribed away from it. CA3 lies centrally in this cluster separated from the 5' end of CA1 by 80 kb, while the 5'-end of CA2 lies some 20 kb downstream of the 3' end of CA3. The DNA methylation state of the gene in several erythroid and non-erythroid cell lines was examined. This showed that in the majority of these cell lines, which do not express the CA1 gene, extensive regions around CA1 were largely demethylated. In contrast, DNA from the only CA1 expressing cell line, HEL, appeared highly methylated at all HpaII sites tested apart for one site at the erythroid promoter and another at the 3'-end of the gene. High levels of methylation of the CA1 gene were also found in DNA from untransformed cells. The possible implications of this are discussed