Brady, Hugh Joseph Martin; (1990) Regulation of expression of the human carbonic anhydrase 1 gene. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The HCAI gene is expressed in a developmental stage-specific manner and also exhibits tissue-specific expression patterns. This thesis describes the cloning and characterisation of the 5'-end of the HCAI gene which contains a large intron within its 5' untranslated region. S1 nuclease and primer extension analysis were used to define the transcription start site and the site of 3'-end maturation. Bandshift assays have been used to show that there are at least six DNA sequences, based on the consensus [5 '-TT/AATCA/T-3'] and flanking the HCAI gene, which bind the erythroid-specific factor, GF-1. The presence of GF-1 binding sites is shown to increase expression from a eukaryotic promoter in erythroid cells and not in non-erythroid cells. A transient heterokaryon system was set up by fusing the erythroieukaemic cell lines MEL C88 (expressing MCAI ) and K562 SAI (a human cell line with an embryonic / foetal phenotype, not expressing CAI. RNAase mapping of RNA from the fused cells showed activation of the human CAI gene. This indicates the developmental stage-specific expression of HCAI to be regulated by trans-acti ng factors. Expression of HCAI mRNA in colon tissue was confirmed. Furthermore as in the case of mouse CAI, the HCAI colon mRNA is transcribed from a different promoter to erythroid HCAI mRNA.

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