Mouse carbonic anhydrase III: Nucleotide sequence and expression studies

A cDNA for the mouse carbonic anhydrase, CAIII, has been isolated from a gt11 expression library. The cloned cDNA contains all of the coding region (777 bp) and both 5 untranslated (86-bp) and 3 untranslated (217-bp) sequences. The coding sequence shows 87% homology at the nucleotide level and 91% homology, when amino acid residues are compared, with human CAIII. Protein and mRNA analyses show that CAIII is present at low levels in cultured myoblasts and is abundant in adult skeletal muscle and in liver. The marked sex-related differences in CAIII distribution, described for rat liver, are not seen in the mouse. Restriction fragment length polymorphisms usingTaqI andPstI are described which distinguish betweenMus spretus andMus musculus domesticus.S. T. is supported by an MRC postgraduate training award.     

Nucleotide sequence and derived amino acid sequence of a cDNA encoding human muscle carbonic anhydrase

We report the nucleotide (nt) sequence of a full length cDNA clone, pCA15, which encodes the human muscle-specific carbonic anhydrase, CAIII. pCA15 identifies a 1.7-kb mRNA, which is present at high levels in skeletal muscle, at much lower levels in cardiac and smooth muscle and which appears to be developmentally regulated. The CAIII mRNA is distinguished by a 887-nt long 3'-untranslated region, containing two AAUAAA signal sequences and is longer than either of the mRNAs encoding the erythrocyte CAs, CAI and CAII, which each have relatively shorter 3'-untranslated regions, 360 and 670 nt long, respectively. The derived amino acid (aa) sequence for human CAIII shows 85% homology with ox CAIII, 62% homology with human CAII and 54% with human CAI when simple pairwise aa comparisons are made. We describe an allelic variation at a TaqI restriction site for CAIII which occurs at high frequency in the European population.     

The nucleotide sequence and derived amino acid sequence of cDNA coding for mouse carbonic anhydrase II

The nucleotide sequence of a clone containing mouse carbonic anhydrase (CA) cDNA in pBR322 has been determined. The cloned cDNA contains all of the coding region except for nucleotides specifying the first eight amino acids, and all of the 3' noncoding region, which consists of 700 nucleotides. A cDNA clone was identified which contains an additional 54 bp at the 5' end, so that the complete amino acid sequence of mouse CA could be deduced. This sequence showed a 73-81% homology with other mammalian CA form II isozymes, 56-63% with form I isozymes, and 52-56% with form III isozymes. By examination of the amino acids which are unique and invariant for each isozyme, the mouse amino acid sequence was found to contain 16 of the 23 residues that are unique and invariant to mammalian CA form II isozymes, but only one or no residue for forms I and III, respectively.     

Spinach carbonic anhydrase primary structure deduced from the sequence of a cDNA clone

A cDNA clone 1,156 base pairs in length was selected by screening a lambda gt11 library with antibodies directed against spinach chloroplast carbonic anhydrase (carbonate dehydratase, EC 4.2.1.1). Sequence analysis revealed an open reading frame of 957 base pairs encoding a polypeptide containing 319 amino acids with a molecular weight of 34,569. This polypeptide is of sufficient size to represent the precursor of spinach chloroplast carbonic anhydrase. The polypeptide contains a sequence of 19 amino acids identical to the sequence of a cyanogen bromide fragment from spinach carbonic anhydrase. In addition, Escherichia coli was transformed with a plasmid that expresses spinach carbonic anhydrase. Lysates prepared from transformed E. coli contain acetazolamide-inhibitable carbonic anhydrase activity. The amino acid sequence of spinach carbonic anhydrase is distinct from those reported for the mammalian isozymes.     

The sequence of equine muscle carbonic anhydrase

The sequence of equine muscle carbonic anhydrase (CA-III) has been determined. The 2 reactive cysteines of the 5 such residues have been localized. A strong sequence homology to other mammalian carbonic anhydrases exists, and 91% of the residues in the equine and bovine muscle forms are identical.     

Structure and exon to protein domain relationships of the mouse carbonic anhydrase II gene

We have isolated a cosmid clone containing the entire mouse (YBR strain) carbonic anhydrase (CA) II gene in 38 kilobase pairs of genomic DNA. The gene was found to be composed of seven exons and six introns. A TATA box (TATAAAA) and a possible CCAAT box (CCACT) have been located beginning 92 and 142 base pairs, respectively, upstream from the initiation codon ATG. When the regions encoded by exons and protein domains are examined, all but 1 of the 30 putative active site residues are encoded by four exons: exons 2 and 3 mainly code for hydrophilic residues and exons 4 and 6 mostly hydrophobic residues. Two intron splice positions, one between the codons for Glu-116 and Leu-117 and the other interrupting the codon for Gly-143, are located at the bottom of the active site cavity, and the former separates two of the three histidine residues forming ligands to the active site zinc ion. The other four splice sites map to the exterior of the molecule. Thus, except for the possible association of the 29 active site residues encoded by four exons, no obvious correspondence is seen between the regions coded by exons and the functional or secondary structural domains of the mouse CA II molecule. During this study, the possible basis for the two electrophoretic types, CA IIa and CA IIb, of inbred mouse strains was detected as a Gln/His interchange at position 38.     

A non-methylated CpG-rich island associated with the human muscle-specific carbonic anhydrase III gene

A cluster of CpG dinucleotides immediately upstream from exon 1 in the muscle-specific carbonic anhydrase III gene (CAIII) resembles the 'HpaII tiny fragment' (HTF) islands characteristic of mammalian 'housekeeping' genes. Since this CAIII gene shows tissue-specific expression we have carried out a detailed examination of methylation status within the CpG cluster using a polyacrylamide gel/electroblot procedure to extend the range of conventional Southern blotting. None of the clustered CpGs are methylated in DNA from muscle or other somatic tissues or in DNA from spermatozoa although flanking CpGs are methylated. Comparison with a candidate HTF island from the more ubiquitously expressed carbonic anhydrase II gene (CAII) shows that the CAII CpG cluster is markedly more CpG-rich than that from the strictly tissue-specific CAIII gene.