A study on the isolation, localization and regulation of carbonic anhydrase, using the zebrafish and chicken retina as model systems

I am investigating carbonic anhydrase as a gene expressed specifically in Muller glial cells (MC). Carbonic anhydrase (CA) catalyzes the interconversion of CO;Using CA-inhibitor-based affinity chromatography, a single protein was isolated. The protein was characterized by both direct peptide-sequence and enzymatic rate analyses as a high-activity carbonic anhydrase homologue (CAH-Z).;CAH-Z's cellular localization was determined using immunohistochemistry. A polyclonal antiserum, produced against purified CAH-Z, recognized a single band of 29,000 Daltons by Western blot analysis. The antiserum specifically stained the MCs in the adult retina. No CAH-Z staining was present during development until 72 hours post-fertilization (hpf); expression at this time was found only in MCs. Thus, CAH-Z expression in the retina occurred only in the MCs. MC-differentiation was also followed using an additional marker, the HNK-1 carbohydrate epitope. HNK-1 staining was observed as early as 48 hpf, and by 60 hpf was clearly present on radial cells. These same cells express the CAH-Z protein after 72 hpf.;A cDNA sequence was determined for CAH-Z. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone the cDNA. Three sets of overlapping RT-PCR reactions were carried out to clone the cDNA. The clones were sequenced and aligned to determine the cDNA sequence. Translating the cDNA resulted in an open reading frame of 260 amino acids that showed greater than 50% identity to vertebrate CAs. Phylogenetic analysis suggested that CAH-Z was a novel isoform; specifically, the results suggested that CAH-Z shared a common ancestor with the mammalian CA-I, CA-II, and CA-III genes.;A genomic clone containing CAH-Z sequence was isolated using a PAC library. The clone was subcloned and two fragments of 5.0 and 2.3 kb were isolated. The preliminary sequence is presented.;As a preliminary test of the regulatory mechanisms responsible for MC-specific expression, a chicken CA-II promoter was tested for the ability to drive MC-specific expression in a retina culture system. The 1376 basepair proximal 5...