Patrick J. Venta

Carbonic anhydrase II is induced in HL-60 cells by 1,25-dihydroxyvitamin D3: A model for osteoclast gene regulation

Carbonic anhydrase II (CA II) generates the H+ required for osteoclast‐mediated bone resorption in humans. We have developed the human promyelocytic cell line HL‐60 as a model system with which to study the osteoclast‐specific expression of the CA II gene. Treatment of the cell line with 1,25‐dihydroxyvitamin D3 resulted in a dramatic de novo induction of CA II at both the protein and mRNA levels. CA II mRNA was also induced to a lesser extent by 12‐O‐tetradecanoyl phorbol 13‐acetate. Treatment with dimethyl sulfoxide did not increase CA II mRNA. These findings indicate that the HL‐60 cell line will be a useful model system to study the osteoclast‐specific expression of the CA II gene.

The gene for human carbonic anhydrase ii (Ca2) is located at chromosome 8q22

The gene CA2 for the human carbonic anhydrase II isozyme is encoded in band q22 of chromosome 8. These data and supporting evidence predict that the genes for carbonic anhydrase I and III are also physically closely linked in this chromosomal region.

Expression of carbonic anhydrase II (CA II) promoter-reporter fusion genes in multiple tissues of transgenic mice does not replicate normal patterns of expression indicating complexity of CA II regulation in vivo

Although the proximal, 5′ 115 bp of the human carbonic anhydrase II (CA II) gene was sufficient for expression of a reporter gene in some transfected cell lines, we found previously that 1100 bp of this promoter (or 500 bp of the mouse CA II promoter) was not sufficient for expression in transgenic mice. We have now studied the expression of linked reporter genes in mice transgenic for either (1) 11 kb of the human 5′ promoter or (2) 8 kb of the human 5′ promoter with mouse sequences from the first exon, part of the first intron (since a CpG island spans this region), and the 3′ sequences of the gene. Expression was found in both cases, but the tissue specificity was not appropriate for CA II. Although there was a difference in the sensitivity of the assays used, the first construct led to expression in many tissues, while the second construct was expressed only in spleen. These findings indicate considerable complexity of DNA control regions for in vivo CA II expression.

DNA polymorphism in the 5′ flanking region of the human carbonic anhydrase II gene on chromosome 8

SummaryA restriction-fragment-length polymorphism (RFLP) is described which is associated with the human carbonic anhydrase II gene (CA2) that codes for one of the three genetically distinct carbonic anhydrase isozymes, CA I, CA II, and CA III. The isolated DNA was cleaved with several restriction enzymes and subjected to Southern blot hybridization analysis using a DNA probe containing the 5′ end of the human CA II gene. A two allele RFLP which was detected with the restriction endonuclease, Taq I, is expressed phenotypically on Southern blots as either a 5.4 kilobase (kb) fragment or as 4.0 and 1.4 kb fragments. These fragments result from the presence or absence of a Taq I recognition site in the 5′ flanking region approximately 1.0kb from the initiation codon of the CA II gene. Segregation analysis showed that the alleles are inherited in a Mendelian fashion, with a frequency of 50%.

Variation in coding exons of two electrophoretic alleles at the pigtail macaque carbonic anhydrase I locus as determined by direct, double-stranded sequencing of polymerase chain reaction (PCR) products

Two, electrophoretically distinct, forms of carbonic anhydrase I (CA Ia and CA Ib) are found at high polymorphic frequencies in red cells of natural populations of pigtail macaques,Macaca nemestrina, from southeast Asia. By use of the polymerase chain reaction, exons of the CA I gene were amplified from homozygous (a/a, b/b) and heterozygous (a/b) animals. Direct sequencing of the amplified DNA from four animals revealed differences between the a and the b electrophoretic alleles ranging from three to six nucleotides, and from one to three differences within each allele. These results indicate a greater genetic variability at the CA I locus in this macaque species than previously realized.

Carbonic Anhydrases in Mammalian Cell Culture and Tumors

This chapter describes some of the work that has been performed with tissue culture cells to understand how carbonic anhydrase (CA) is regulated in various cell types. The advantages of studying genes and gene products in tissue culture are that the treatments of the cells can be completely defined and, in many cases, homogeneous populations of cells can be used without the confounding effects of other cell types. The chapter is not all-inclusive, focusing primarily on the analysis of a number of leukemic cell lines and tissue types that have been studied most frequently or that may be most useful in dissecting the regulation of the CA isozymes and their genes at the molecular level. Many excellent studies using other cell types have not been included.

Features of Gene Structure, Organization, and Expression That Are Providing Unique Insights into Molecular Evolution and Systematics

Over the last 20 or so years, the study of amino acid sequences of proteins derived from different biologic species has greatly enriched our knowledge of both the mechanisms of molecular evolution and the phylogenetic relationships of the species. Increasingly sophisticated computer techniques have been employed in an effort to extract all of the phylogenetic information contained in such data sets. Several problems have arisen along the way, among the most persistent being the uncertainty of whether two sequences are truly orthologous and the species divergence is being examined, or whether they are paralogous and the gene divergences are being examined. Indeed, where gross discrepancies between gene phylogeny and species phylogeny occur, the probability is that paralogous genes are involved (Goodman, 1981; Goodman et al., this volume, Chapter 4).

Characterization of the gene encoding carbonic anhydrase I from the pigtail macaque.

The structure of the gene encoding carbonic anhydrase I (CA I) was determined for the pigtail macaque Macaca nemestrina. When the deduced amino-acid sequence was compared with those of five other primates, four non-primate mammals and a turtle, seven residues were found to be unique and invariant to all of the CA I sequences. A scheme is presented for the probable evolutionary order of the six polymorphic nucleotide changes found in the coding regions of the CA I locus of pigtail macaques.