Structural studies of cDNA and the gene for the beta-subunit of cGMP phosphodiesterase from human retina]

cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase-(PDE) were isolated from a human retinal library. The encoded polypeptide has 854 amino acid residues with calculated molecular mass of 98416 Da. Alignment of the deduced amino acid sequence with the previously analysed alpha-, beta- and alpha'-subunits of the bovine and mouse PDEs demonstrates highly significant similarities. We have also isolated, from a genomic library, two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene. The cloning of the human gene and the knowledge of its genomic organization will allow the rapid assessment of the role of this gene in the causation of human retinopathies.     

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.     

Genomic structure and chromosome location of the murine PDE1B phosphodiesterase gene

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP, thereby participating in regulation of the intracellular concentrations of these second messengers. The PDE1 family is defined by regulation of activity by calcium and calmodulin. We have cloned and characterized the mouse PDE1B gene, which encodes the 63-kDa calcium/calmodulin-dependent PDE (CaM-PDE), an isozyme that is expressed in the CNS in the olfactory tract, dentate gyrus, and striatum and may participate in learning, memory, and regulation of phosphorylation of DARPP-32 in dopaminergic neurons. We screened an I-129/SvJ mouse genomic library and identified exons 2–13 of the PDE1B gene that span 8.4 kb of genomic DNA. Exons range from 67 to 205 nucleotides and introns from 91 to 2250 nucleotides in length. Exon 1 was not present in the 3 kb of genomic DNA 5′ to exon 2 in our clones. The mouse PDE1B gene shares many similar or identical exon boundaries as well as considerable sequence identity with the rat PDE4B and PDE4D genes and the Drosophila dunce cAMP-specific PDE gene dnc, suggesting that these genes all arose from a common ancestor. Using fluorescence in situ hybridization, we localized the PDE1B gene to the distal tip of mouse Chromosome (Chr) 15. Received: 10 November 1997 / Accepted: 12 March 1998     

Assignment of the beta-subunit of rod photoreceptor cGMP phosphodiesterase gene PDEB (homolog of the mouse rd gene) to human chromosome 4p16.

The gene encoding the beta-subunit of rod photoreceptor cGMP phosphodiesterase (gene symbol PDEB, homolog of the mouse rd gene) is mapped to human chromosome 4 using somatic cell hybrids and further localized to the chromosome band 4p16 using in situ hybridization. A mutation in the mouse gene underlies the recessive trait of retinal degeneration in the rd mouse. Thus, the human homolog is a candidate for lesions causing retinal degeneration.     

Genomic structure of murine mitochondrial DNA polymerase-gamma

We have sequenced a genomic clone of the gene encoding the mouse mitochondrial DNA polymerase. The gene consists of 23 exons, which span approximately 13.2 kb, with exons ranging in size from 53 to 768 bp. All intron-exon boundaries conform to the GT-AG rule. By comparison with the human genomic sequence, we found remarkable conservation of the gene structure; the intron-exon borders are in almost identical locations for the 22 introns. The 5' upstream region contains approximately 300 bp of homology between the mouse and human sequences that presumably contain the promoter element. This region lacks any obvious TATA domain and is relatively GC rich, consistent with the housekeeping function of the mitochondrial DNA polymerase. Finally, within the 5' flanking region, both mouse and human genes have a region of 73 bp with high homology to the tRNA-Arg gene.     

Analysis of the regions flanking the human insulin gene and sequence of an Alu family member.

The regions around the human insulin gene have been studied by heteroduplex, hybridization and sequence analysis. These studies indicated that there is a region of heterogeneous length located approximately 700 bp before the 5' end of the gene; and that the 19 kb of cloned DNA which includes the 1430 bp insulin gene as well as 5650 bp before and 11,500 bp after the gene is single copy sequence except for 500 bp located 6000 bp from the 3' end of the gene. This 500 bp segment contains a member of the Alu family of dispersed middle repetitive sequences as well as another less highly repeated homopolymeric segment. The sequence of this region was determined. This Alu repeat is bordered by 19 bp direct repeats and also contains an 83 bp sequence which is present twice. The regions flanking the human and rat I insulin genes were compared by heteroduplex analysis to localize homologous sequences in the flanking regions which could be involved in the regulation of insulin biosynthesis. The homology between the two genes is restricted to the region encoding preproinsulin and a short region of approximately 60 bp flanking the 5' side of the genes.     

Chromosome mapping of the rod photoreceptor cGMP phosphodiesterase beta-subunit gene in mouse and human: tight linkage to the Huntington disease region (4p16.3).

The retinal degeneration mouse (gene symbol, rd) is an animal model for certain forms of human hereditary retinopathies. Recent findings of a nonsense mutation in the rd mouse PDE beta-subunit gene (Pdeb) prompted us to investigate the chromosome locations of the mouse and human genes. We have utilized backcross analysis in mice to verify and define more precisely the location of the Pdeb locus 6.1 +/- 2.3 cM distal of Mgsa on mouse chromosome 5. We have determined that the human gene (PDEB) maps to 4p16.3, very close to the Huntington disease (HD) region. Analysis of the comparative map for mice and humans shows that the mouse homologue of the HD gene will reside on chromosome 5. Linkage of the mouse Pdeb locus with other homologues in the human 4p16.3 region is maintained but gene order is not, suggesting at least three possible sites for the corresponding mouse HD gene.     

Gene transfer, expression, and molecular cloning of the human transferrin receptor gene

We describe the molecular cloning of the human transferrin receptor gene by a gene transfer approach. Mouse Ltk- cells were cotransformed with the herpes simplex thymidine kinase gene and total human DNA. Transformants expressing human transferrin receptor were isolated by selection on hypoxanthine/aminopterin/thymidine (HAT) medium and fluorescence-activated cell sorting of HAT-resistant cells. Thirty-four kilobases of human DNA was isolated by screening a genomic library constructed from the DNA of a secondary transformant. Gene transfer of the cloned DNA established that 31 kb of DNA was sufficient to encode the receptor. A probe from the 5' end of the gene was used to isolate a cDNA clone with an insert of 4.9 kb. Hybridization of the cDNA to the cloned genomic DNA revealed a minimum of 12 exons. They extend over the entire 31 kb of expressing DNA and over 2 kb of adjacent 3' untranslated sequences that are not required for receptor expression in L cells.     

Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene.

Mammalian ribonucleotide reductase consists of two non-identical subunits, proteins M1 and M2. M2-related DNA sequences are present on mouse chromosomes 4, 7, 12 and 13. However, M2-overproducing mouse cells show amplification of a chromosome 12-specific, single 13 kb HindIII fragment, which probably represents the active gene. We have isolated this fragment from parental mouse cell DNA and used it to clone and characterize the functional M2 gene. The 5770 bp transcribed M2 sequence contains ten exons separated by nine 95-917 bp introns. The 501 bp of 5' flanking DNA is G + C rich and contains TTTAAA and CCAAT sequences as well as potential Sp1 binding sites. The M2-related sequence on chromosome 13, which contains only the last six exons and several internal rearrangements, is a pseudogene. Transfection of BALB/3T3 cells with the M2 gene resulted in stable transformants with a 10-fold reduction in sensitivity to hydroxyurea, compared to control cells. This confirmed that the cloned M2 genomic DNA represents the functional gene and conclusively establishes the link between hydroxyurea resistance and M2 expression in mammalian cells. M2 genomic DNA should be a valuable dominant, selectable marker for identifying and isolating stable co-transformants.     

Complete cDNA sequences of mouse rod photoreceptor cGMP phosphodiesterase alpha- and beta-subunits, and identification of beta'-, a putative beta-subunit isozyme produced by alternative splicing of the beta-subunit gene.

We have characterized overlapping cDNA clones encoding cGMP phosphodiesterase (PDE) alpha- and beta-subunits of mouse retinal rod photoreceptors. The open reading frames predict an alpha-subunit of 100 kDa (856 residues), and a beta-subunit of 99 kDa (853 residues). Sequence analysis of two of twelve beta-subunit clones predicts the presence in the retina of an additional PDE, termed beta', which is generated by alternative splicing of the beta-subunit gene. beta' differs from beta only at the C-terminus being 55 residues shorter and lacking the Caax motif found at the C-termini of both the alpha- and beta-subunits. A 300 residue segment thought to contain the active site is present in the C-terminal half of alpha, beta and beta'.     

Characterization of TH1 and CTSZ, two non-imprinted genes downstream of GNAS1 in chromosome 20q13

The clustering and coordinate regulation of many imprinted genes justifies positional searches for imprinted genes adjacent to known ones. We recently characterized a locus on 20q13, containing GNAS1, which has a highly complex imprinted expression pattern. In a search for neighbouring genes, we have now characterized a new gene, TH1, downstream of GNAS1. TH1 and GNAS1 are separated by more than 70 kb consisting largely of interspersed repetitive DNA. TH1 is the homologue of a gene that, in Drosophila, lies adjacent to the DNA repair gene mei-41. We have determined the full-length structures of human, mouse and Drosophila TH1. Though of unknown function, TH1 is highly conserved and widely expressed. Nonetheless, there is no similar Caenorhabditis elegans protein. We have also determined the complete genomic structures of human and Drosophila TH1. The Drosophila gene has five exons spanning 2.6 kb. The last three introns have precise equivalents in the human gene, which has 15 exons spanning 14 kb and is transcribed away from GNAS1. Using a single-nucleotide polymorphism in the 3' untranslated region, we have demonstrated biallelic TH1 expression in human fetal tissues, suggesting that, unlike GNAS1, TH1 is probably not imprinted. Immediately downstream of TH1 lies CTSZ, encoding the recently described cysteine protease, cathepsin Z. We have also elucidated the genomic structure of this gene; it has six exons spanning 12 kb and is oriented tail-to-tail with TH1, only 70 bp separating their polyadenylation sites. A polymorphism was again identified within the CTSZ 3' untranslated region and used to demonstrate biallelic expression in fetal tissues.