Mouse Carbonic Anhydrase Research Articles

Thyroid hormone drives the expression of mouse carbonic anhydrase Car4 in kidney, lung and brain

Thyroid hormone is a well-known regulator of brain, lung and kidney development and function. However, the molecular mechanisms by which the hormone exerts its function have remained largely enigmatic, and only a limited set of target genes have been identified in these tissues. Using a mouse model with a mutation in thyroid hormone receptor α1 (TRα1), we here demonstrate that the expression of carbonic anhydrase 4 in lung and brain of the adult animal depends on intact TRα1 signaling. In the kidney, carbonic anhydrase 4 mRNA and protein are not affected by the mutant TRα1, but are acutely repressed by thyroid hormone. However, neither lung function - as measured by respiration rate and oxygen saturation - nor urine pH levels were affected by altered carbonic anhydrase 4 levels, suggesting that other carbonic anhydrases are likely to compensate. Taken together, our findings identify a previously unknown marker of TRα1 action in brain and lung, and provide a novel negatively regulated target gene to assess renal thyroid hormone status.

Generation of a Transgenic Mouse for Colorectal Cancer Research with Intestinal Cre Expression Limited to the Large Intestine

Genetically modified mice have been used for colon cancer research, but findings from these models are confounded by expression of cancer in multiple organs. We sought to create a transgenic mouse with Cre recombinase (Cre) expression limited to the epithelial cells of the large intestine and used this model to study colon cancer driven by adenomatosis polyposis coli (APC) gene inactivation. A promoter/enhancer from the mouse carbonic anhydrase I gene was used to generate a Cre-expressing transgenic mouse (CAC). After characterizing transgene expression and distribution, CAC mice were crossed to APC(580S) mice to generate mice with APC inactivation at one (CAC;APC(580S/+)) or both alleles (CAC;APC(580S/580S)). Transgene expression was limited to the epithelial cells of the cecum and colon, extended from the crypt base to the luminal surface, and was expressed in approximately 15% of the crypts. No abnormal gross phenotype was seen in 3- or 6-week-old CAC;APC(580S/+) mice, but CAC;APC(580S/580S) mice had significant mucosal hyperplasia in the colon at 3 weeks, which developed into tumors by 6 weeks. By 10 weeks, 20% of CAC;APC(580S/+) mice developed adenomatous lesions in the distal colon (3.0 +/- 0.4 mm; 1.1 per mouse). Dextran sulfate sodium treatment increased the incidence and number of tumors, and this occurred predominantly in distal colon. Our new model has improved features for colon cancer research, that is, transgene expression is limited to the epithelium of the large bowel with normal cells found next to genetically modified cells.

Hypoxia-inducible expression of the mouse carbonic anhydrase IX demonstrated by new monoclonal antibodies

CA IX is a transmembrane carbonic anhydrase isoenzyme predominantly expressed in human tumors in response to hypoxia and functionally implicated in adaptation of tumor cells to hypoxic stress via control of pH and cell adhesion. Intense investigations of the human CA IX as a hypoxic marker and a therapeutic target have been facilitated by specific monoclonal antibodies. However, no such reagents existed for the mouse CA IX ortholog. We generated five new anti-mouse CA IX monoclonal antibodies AM1-4, AM4-3, AM27-4, AM34-7 and AM35-1 produced using CA IX-deficient mice. The antibodies are suitable for various immunodetection methods including immunoblotting and immunohistochemistry. Using these reagents we show that the mouse CA IX is expressed in three out of nine tested mouse cell lines, namely in L929, MEF and TSA and is regulated by hypoxia and cell density similarly to human CA IX. We also demonstrate that the mouse CA IX exhibits hypoxia-related expression pattern in multicellular spheroids and in tumor xenografts. Our results indicate the use of the mouse model as suitable for further studies of CA IX role in tumor development and for its pre-clinical investigations. The new monoclonal antibodies represent potent tools for accomplishment of these future studies.

Nucleotide sequence and structure of the mouse carbonic anhydrase III gene

At least 14 distinct isozymes of carbonic anhydrase have been identified in mammals. These enzymes catalyze the hydration of carbon dioxide and are essential for regulation of cellular pH and carbon dioxide transport. Carbonic anhydrase III is highly expressed in certain tissues, including muscle and fat where it constitutes up to 25% of the soluble protein. We cloned a cDNA encoding mouse carbonic anhydrase III. This cDNA contains 1653 bp, consisting of 79 bp in the 5′ UTR, a 780 bp open reading frame, and 794 bp of the 3′ UTR, including two potential polyadenylation signals. Fluorescent in situ hybridization confirmed the existence of a single copy of the gene on chromosome 3. We then isolated the genomic DNA for mouse carbonic anhydrase III and analyzed its structure. The gene consists of seven exons and six introns which span 10.5 kb. The 5′ flanking region of the genomic DNA is notable for a pyrimidine rich region consisting of two dinucleotide repeats containing 23 and 20 TC pairs separated by the same 15 bp spacer.

Novel transcripts of carbonic anhydrase II in mouse and human testis.

Intracellular and extracellular sources of bicarbonate are essential for sperm motility, sperm binding to the zona pellucida and the acrosome reaction. Carbonic anhydrase II, catalysing the synthesis of bicarbonate within spermatozoa, must play a significant role in these mechanisms. We report here the expression of carbonic anhydrase II during mouse spermatogenesis and the primary structure of testicular transcripts coding for carbonic anhydrase II isolated from adult mouse and human testes. The mouse carbonic anhydrase II (Car2) mRNA displays a 5' untranslated region (UTR) larger than the corresponding somatic sequence. The additional 5' sequence contains the 'TATA box' used in somatic tissues and other promoter sequences, suggesting the use of testis-specific promoters further upstream with read-through of downstream promoters. The 3'UTR of the Car2 mRNA is shorter in mature testicular cells than in somatic cells. Polysomal gradient analysis of carbonic anhydrase II transcripts isolated from adult mouse testis and kidney revealed different translation potential: most of the testicular transcripts were present in the non-polysomal fractions, whereas a considerable fraction of kidney transcripts were polysome-associated. These results suggest that specific transcriptional and post-transcriptional mechanisms regulate the expression of carbonic anhydrase II during mammalian spermatogenesis.

Transgenic improvement of photosynthetic property in tobacco using E12 .OMEGA. promoter system to express higher level of mouse carbonic anhydrase activity in cytoplasm.

Genetic challenge to express higher carbonic anhydrase (CA) activity in the cytoplasm of tobacco mesophyll cells was carried out by an improved promoter (El2 Ω) with a cDNA of partially modified mouse CA. In leaves of T1 progenies of transgenic tobacco (ffCA plant), the expressed mouse CA peptide was confirmed with 1, 5-3.2 times higher than the transgenic tobacco having foreign mouse CA controlled by simple CaMV 35S promoter (fCA plant). The ffCA plants manifested higher photosynthetic carbon assimilation (PCA) rate under ambient CO2 condition than wild type plants. The chlorophyll fluorescence measurement analysis revealed that the ffCA plant's improved PCA rate is related to the reduction of mesophyll CO2 transfer resistance (Rr) with little change in the stomatal resistance (Rs) or mesophyll CO2 fixation resistance (Rx). Moreover, ffCA plants showed a higher growth rate under high light condition.

Specificity of antibodies against rodent transforming growth factor-alpha protein.

We found that the immunohistochemical distribution of TGF-alpha varied in rodent tissues depending on the antibody used, suggesting that the specificity of anti-TGF-alpha antibodies differs significantly. To address this issue, we compared the specificity of two representative antibodies that have been widely used to detect rodent TGF-alpha. In a competition study, the antibodies were preincubated with an excess of synthetic rat TGF-alpha 34-50 and were used for staining of rat and mouse kidneys and/or uterus. The results revealed that one of the antibodies, anti-rat TGF-alpha polyclonal antibody, was neutralized by the peptide, whereas the other, anti-human TGF-alpha monoclonal antibody, was not absorbed by the peptide up to an excess of 100-fold. Western blotting analysis showed that the anti-rat TGF-alpha polyclonal antibody recognized both human and rat purified TGF-alpha. However, the anti-human TGF-alpha monoclonal antibody did not detect purified rat TGF-alpha, although the antibody reacted with mouse proteins other than TGF-alpha from kidneys and uterus, purified human TGF-alpha, and mouse carbonic anhydrase II. These data indicate that the anti-human TGF-alpha monoclonal antibody does not recognize rodent TGF-alpha under our experimental conditions and suggest that distribution of TGF-alpha in rodent tissues may need to be reexamined.

Expression of Mouse Carbonic Anhydrase VII inE. coliand Demonstration of Its CO2Hydrase Activity

The α-carbonic anhydrase (α-CA) gene family in mammals encodes 10 CA or CA-like proteins (CA I-CA X). Although the gene for human CA VII has been cloned and characterized, the corresponding protein has not previously been purified, and hence, the CO2hydrase activity of its product has not as yet been demonstrated. In this study, we have cloned the mouse CA VII cDNA in anE. coli,glutathione-S-transferase (GST) expression vector. The CO2hydrase activity of the expressed protein is about 4% that of the high-activity CAII isozyme, demonstrating that this evolutionarily highly conserved protein is a catalytically active member of this CA gene family.

Catalytic properties of mouse carbonic anhydrase V.

A cDNA encoding the mouse carbonic anhydrase V gene was isolated by reverse transcription and polymerase chain reaction from BALB/c mouse liver mRNA. Vectors containing the full coding sequence as well as two different NH2-terminal truncated genes expressed enzymatically active protein in Escherichia coli. The carbonic anhydrase V produced by a vector containing the full coding sequence, which includes a possible NH2-terminal mitochondrial targeting signal, was proteolytically processed by E. coli and contained several amino-terminal ends. The two NH2-terminal truncated vectors deleted, respectively, 1) the 29-amino acid putative targeting sequence and 2) 51 amino acids, yielding a protein equivalent to a carbonic anhydrase (CA) V isolated from mouse liver mitochondria; and both vectors produced homogeneous protein fractions. These latter two forms of CA V had identical steady-state constants for the hydration of CO2, with maximal values of kcat/Km at 3 x 10(7) M-1 s-1 and kcat at 3 x 10(5) s-1 with an apparent pKa for catalysis of 7.4 determined from kcat/Km. In catalytic properties, mouse CA V is closest to CA I; however, in inhibition by acetazolamide, ethoxzolamide, and cyanate, CA V is very similar to CA II. Mouse CA V has a tyrosine at position 64, where the highly active isozyme II has histidine serving as a proton shuttle in the catalytic pathway. Investigation of a site-specific mutant of CA V containing the replacement Tyr64-->His showed that the unique kinetic properties of CA V are not due to the presence of tyrosine at position 64.

Carbonic anhydrase in mouse skeletal muscle and its influence on contractility.

Carbonic anhydrase III (EC 4.2.1.1) is the most abundant cytosolic protein in type I skeletal muscle fibers. Investigations of its physiological role have mostly been conducted with rat muscles, which sometimes are unsuitable for in vitro studies. The objective of the present study was to characterize the carbonic anhydrase in the mouse soleus muscle to verify if this muscle can be used as a model to further study the enzyme's function. Total carbonic anhydrase specific activity in the mouse soleus was comparable to the value for rat. However, 60% of the total carbonic anhydrase activity in the mouse was of the sulfonamide-sensitive type and, therefore, not related to carbonic anhydrase III. Electrophoretic analysis revealed the presence of a 29-kDa protein in total and cytosolic extracts of the mouse soleus. Immunoblotting with an antibody developed against rat carbonic anhydrase III showed that it was also specific for this 29-kDa peptide, which presumably is the mouse carbonic anhydrase III. Inhibition of the sulfonamide-sensitive activity had no effect on contractile and fatigue characteristics, whereas inhibition of the sulfonamide-resistant carbonic anhydrase III activity led to a significant increase in resistance to fatigue. We conclude that the mouse soleus may represent an excellent model to understand the contribution of different carbonic anhydrase isoforms to muscle physiology.

Characterization of the mouse carbonic anhydrase II gene promoter.

This work characterizes a 250-base pair (bp) fragment of the mouse carbonic anhydrase II (CAII) gene as an efficient but not cell-specific promoter. This fragment contains multiple consensus cis regulatory regions that interact in a complex fashion to regulate transcriptional activity from the promoter. Truncated fragments of the 250-bp promoter retain transcriptional activity. The 90-bp 5' "GC"-rich portion of the promoter can direct transcription independently from the consensus TATA box and also contains a silencer that diminishes transcription from the 3' 160-bp portion of the CAII promoter as well as from the SV40 promoter. There are two nonconsensus dual functioning Ap2-like elements on the promoter that are essential for core promoter activity and cAMP-mediated increases in transcription of the gene. A nuclear protein of approximately 65 kDa binds to these elements and is present in nuclear extracts of nonstimulated and forskolin-stimulated NIH-3T3 cells. I conclude that these nonconsensus Ap2-like elements and their cognitive binding protein play a major role in the expression of the CAII gene.

A differentiation stage-specific factor interacts with mouse carbonic anhydrase form I gene and a conserved sequence in mammalian β-globin genes

Abstract We have identified in mouse erythroleukemic (MEL) cells a novel factor which binds to the 3′ flanking region of β-globin genes. Upon induction, this DNA binding factor disappears as β-globin gene transcription increases. The factor protects 20–30 base pairs (bp) of a sequence which is tightly conserved in β-globin genes including chicken, human, mouse and rabbit. A very similar sequence binds the factor in the mouse carbonic anhydrase form I gene, whose transcription diminishes upon induction of MEL cells. The factor, or a closely related form, is detected in nonerythroid cells and binding sites have been detected in other genes, including c-myb and immunoglobulin heavy chain-enhancer. We suggest that this factor could play a role in determining the timing of gene expression in several differentiating cell types.

Nucleotide and dervied amino-acid sequence of a cDNA encoding a new mouse carbonic anhydrase

Nucleotide and dervied amino-acid sequence of a cDNA encoding a new mouse carbonic anhydrase

The mouse carbonic anhydrase I gene contains two tissue-specific promoters.

We report the isolation and characterization of the mouse carbonic anhydrase I (CAI) gene. Direct RNA sequence analysis of the 5' nontranslated regions of CAI mRNA from mouse colon and mouse erythroleukemia cells demonstrated tissue specificity in the lengths and sequences of CAI transcripts. Analysis of several mouse CAI genomic clones showed that the transcripts arose from a single CAI gene with two tissue-specific promoters and eight exons. CAI transcripts in the colon were found to initiate just upstream of the erythroid exon 2 of the CAI gene region sequence. Erythroid transcripts originated from a novel promoter upstream of exon 1, which was located more than 10 but less than 250 kilobases upstream of exon 2. Erythroid exon 1 contained only a nontranslated sequence, which was spliced to exon 2 via a cryptic splice acceptor site located in the region that encoded the colon mRNA 5' nontranslated sequence. The remaining exon-intron junctions were conserved in comparison with those of the CAII and CAIII genes.

The Mouse Carbonic Anhydrase I Gene Contains Two Tissue-Specific Promoters

We report the isolation and characterization of the mouse carbonic anhydrase I (CAI) gene. Direct RNA sequence analysis of the 5' nontranslated regions of CAI mRNA from mouse colon and mouse erythroleukemia cells demonstrated tissue specificity in the lengths and sequences of CAI transcripts. Analysis of several mouse CAI genomic clones showed that the transcripts arose from a single CAI gene with two tissue-specific promoters and eight exons. CAI transcripts in the colon were found to initiate just upstream of the erythroid exon 2 of the CAI gene region sequence. Erythroid transcripts originated from a novel promoter upstream of exon 1, which was located more than 10 but less than 250 kilobases upstream of exon 2. Erythroid exon 1 contained only a nontranslated sequence, which was spliced to exon 2 via a cryptic splice acceptor site located in the region that encoded the colon mRNA 5' nontranslated sequence. The remaining exon-intron junctions were conserved in comparison with those of the CAII and CAIII genes.

Expression of carbonic anhydrase II gene in early brain cells as revealed by in situ hybridization and immunohistochemistry

A mouse carbonic anhydrase (CA II) complementary(c) DNA probe was used for in situ hybridization on mouse brain cultured cells in order to follow CA II gene expression during brain development. An improved method was established using biotinated probes that resulted in a high sensitivity and an absence of background; this method could be combined with immunohistochemistry. Hypothalamic cells of embryonic day (ED) 12-14 mice were cultured for various periods. Chronologic appearance of CA II messenger(m)RNA and protein was studied. The CA II gene transcripts are detectable as early as ED 12-13, although the protein they encode is not detectable until ED 17-18. Gene expression is restricted to 0.1% of the total population. Northern blot analysis confirmed the presence of CA II transcripts in embryonic hypothalamus. At postnatal stage, the majority of glial cells express both the CA II mRNA and the protein. Our results favour the early appearance of a glial lineage in a precise area of the developing CNS. The precocity of CA II gene transcription makes in situ hybridization an invaluable approach in defining the onset of nerve cell lineages during embryonic development.

Mouse carbonic anhydrase III: Nucleotide sequence and expression studies

A cDNA for the mouse carbonic anhydrase, CAIII, has been isolated from a lambda 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 using TaqI and PstI are described which distinguish between Mus spretus and Mus musculus domesticus.

Molecular evolution of the carbonic anhydrase genes: calculation of divergence time for mouse carbonic anhydrase I and II.

A cDNA clone in pBR322 that cross-hybridizes with a mouse carbonic anhydrase form II (CAII) probe has been sequenced and identified as mouse carbonic anhydrase form I (CAI). The 1224-base-pair clone encodes the entire 260-amino-acid protein and appears to contain an Alu-like element in the 3' untranslated region. The deduced amino acid sequence exhibits 77% homology to human CAI and contains 17 of the 20 residues that are considered unique to and invariant for all mammalian CAI isozymes. The results of a detailed comparison of the nucleic acid sequences spanning the coding regions of mouse CAI and rabbit CAI have been used to calibrate an evolutionary clock for the carbonic anhydrases (CAs). These data have been applied to a comparison of the mouse CAI and CAII nucleic acid sequences to calculate the divergence time between the two genes. The divergence-time calculation provides the first estimation of the evolutionary relationship between CAs based entirely on nucleotide sequence comparison.

Comparison of the 5′ regions of human and mouse carbonic anhydrase II genes and identification of possible regulatory elements

The nucleotide sequence of the 5′ region of the human carbonic anhydrase II gene has been determined. This sequence begins 643 base pairs upstream from the ATG start site and continues through exon 1, intron 1, exon 2 and the adjoining 125 nucleotides of intron 2. The human sequence is compared with homologous regions of the mouse (YBR strain) carbonic anhydrase II gene by aligning the two sequences for optimal homology. In addition to a TATA box and a putative CCAAT box (CCACC in human and CCACT in mouse), three conserved tandem-repeat elements in mouse and two in human (consensus: cCNGTCACCTCCgC) are located 15 and 22 base pairs upstream, respectively, from the CCAAT boxes in the human and mouse sequences. This repeat element is similar to a tandem repeat sequence located at about the same position in mammalian β-globin genes, and may represent regulatory elements common to both the carbonic anhydrase and β-globin genes. The regions surrounding exon 1 are extremely G + C-rich in both human and mouse genes. In addition, severel CCGCCC or GGGCGG sequences which may be important for transcriptional efficiency are found in the 5′ flanking regions of the human and mouse genes.

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.