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Title: Regulation and expression of carbonic anhydrase III : studies in mouse and man
Author: Tweedie, Susan Anne Rodgers
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1990
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The work described in this thesis investigates the regulation of expression of human and mouse carbonic anhydrase 3 (CA3 and Car-3 respectively). A cDNA for mouse Car-3 has been isolated from an expression library, the nucleotide sequence determined and compared with that of mRNAs encoded by other members of the mouse Car gene family and with human CA3 mRNA. The distribution of CAIII protein in mouse tissues has been investigated using IEF and Western blotting and a parallel assessment of mRNA levels made using RNase protection assays and Northern analysis. A polymorphism between inbred strains of mice has been detected using the mouse cDNA as probe which allowed a linkage analysis between Car-3 and other members of the Car gene family on mouse chromosome 3. A cDNA clone isolated during the library screen, but which was not Car-3, was sequenced and identified as a full length cDNA clone encoding mouse heart fatty acid binding protein (H-FABP). The partial characterisation of genomic clones for human CA3 has been completed by restriction mapping and sequence analysis. In particular the 5' promoter region which contains an HTF island has been investigated in detail. A 15kb XhoI fragment containing the entire gene with 3' and 5' flanking sequences was used in attempts to establish mice transgenic for human CA3. A single transgenic mouse was identified which did not express the human gene. Transfection of cultured myogenic cells was investigated as an alternative system for the analysis of CA3 regulation. Three cell lines showing moderate to high levels of CAIII expression were identified and characterised immunocytochemically. These studies showed that CAIII was present at high levels in proliferating myoblasts and was distributed evenly throughout the cytoplasm. Successful conditions were established for electroporation of two of these cell lines and a fibroblast line which did not express CAIII. The human CA3 5' promoter region was subcloned into a promoterless vector containing the bacterial CAT gene and from this two abbreviated constructs were generated. The efficiency of these promoter constructs were tested by transfection into myoblast cells and assay of CAT activity. The results of these experiments suggest that 254bp of sequence 5' of the initiation signal are sufficient for CA3 expression in myoblasts and at least one additional positive regulatory element lies between -2817bp and -722bp.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available