Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin

We have isolated cDNA clones generated from the mRNA encoding the opsin apoprotein of bovine rhodopsin and used these cDNAs to isolate genomic DNA clones containing the complete opsin gene. Nucleotide sequence analysis of the cloned DNAs has yielded a complete amino acid sequence for bovine rhodopsin and provided an intron-exon map of its gene. The mRNA homologous sequences in the 6.4 kb gene consist of a 96 bp 5' untranslated region, a 1044 bp coding region, and a surprisingly long approximately 1400 bp 3' untranslated region, and are divided into five exons by four introns that interrupt the coding region. Secondary structure analysis predicts that the bovine rhodopsin chain, like that of bacteriorhodopsin, contains seven transmembrane segments. Interestingly, three of the four introns are immediately distal to the codons for three of these segments, and one of these introns marks the boundary between the C-terminal domain and a transmembrane domain.     

Genomic organization of the gene and a related pseudogene for a human folate binding protein.

An unprocessed pseudogene which is 90% homologous with the cDNA encoding a folate binding protein in KB cells has been cloned from a human genomic library. This pseudogene contains TGA stop codons, base deletions and substitutions and lacks a 5' region. The size of the exons and the intron-exon sites are almost identical to the organization of the gene encoding this protein which has now been characterized from genomic DNA using the polymerase chain reaction with selected primers to the cDNA.     

Exon-intron organization and chromosomal localization of the mouse monoglyceride lipase gene

Monoglyceride lipase (MGL) functions together with hormone-sensitive lipase to hydrolyze intracellular triglyceride stores of adipocytes and other cells to fatty acids and glycerol. In addition, MGL presumably complements lipoprotein lipase in completing the hydrolysis of monoglycerides resulting from degradation of lipoprotein triglycerides. Cosmid clones containing the mouse MGL gene were isolated from a genomic library using the coding region of the mouse MGL cDNA as probe. Characterization of the clones obtained revealed that the mouse gene contains the coding sequence for MGL on seven exons, including a large terminal exon of approximately 2.6 kb containing the stop codon and the complete 3' untranslated region. Two different 5' leader sequences, diverging 21 bp upstream of the predicted translation initiation codon, were isolated from a mouse adipocyte cDNA library. Western blot analysis of different mouse tissues revealed protein size heterogeneities. The amino acid sequence derived from human MGL cDNA clones showed 84% identity with mouse MGL. The mouse MGL gene was mapped to chromosome 6 in a region with known homology to human chromosome 3q21.     

Structure of the gene for human coagulation factor V.

Activated factor V (Va) serves as an essential protein cofactor for the conversion of prothrombin to thrombin by factor Xa. Analysis of the factor V cDNA indicates that the protein contains several types of internal repeats with the following domain structure: A1-A2-B-A3-C1-C2. In this report we describe the isolation and characterization of genomic DNA coding for human factor V. The factor V gene contains 25 exons which range in size from 72 to 2820 bp. The structure of the gene for factor V is similar to the previously characterized gene for factor VIII. Based on the aligned amino acid sequences of the two proteins, 21 of the 24 intron-exon boundaries in the factor V gene occur at the same location as in the factor VIII gene. In both genes, the junctions of the A1-A2 and A2-A3 domains are each encoded by a single exon. In contrast, the boundaries between domains A3-C1 and C1-C2 occur at intron-exon boundaries, which is consistent with evolution through domain duplication and exon shuffling. The connecting region or B domain of factor V is encoded by a single large exon of 2820 bp. The corresponding exon of the factor VIII gene contains 3106 bp. The 5' and 3' ends of both of these exons encode sequences homologous to the carboxyl-terminal end of domain A2 and the amino-terminal end of domain A3 in ceruloplasmin. There is otherwise no homology between the B domain exons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the whey acidic protein genes of the rat and mouse.

Whey acidic protein (WAP), a hormonally-regulated 14,000 dalton cysteine-rich protein, is the principal whey protein found in rodent milk. Genomic clones encompassing both the 2.8 Kb rat and 3.3 Kb mouse WAP genes have been characterized. The genes consist of four exons and three introns. The middle two exons encode the two cysteine-rich regions which probably form separate protein domains. Homology in the 5' flanking DNA of the mouse and rat extends at least 325 bp upstream of the putative CAP site, including a precisely conserved stretch of 50 bp around the unusual TATA and CAAT sites. The homology previously observed between the 3' noncoding sequences of the rat and mouse mRNAs extends at least 20 bp into the 3' flanking region. Several potential glucocorticoid receptor binding sites have been found in the 5' flanking region of the WAP gene. The conservation of the 5' flanking region of the WAP genes may be related to regulation of expression of WAP by peptide and/or steroid hormones.     

The rat casein multigene family. Fine structure and evolution of the beta-casein gene

Eight overlapping phage clones, spanning 34.4 kilobase pairs of genomic DNA, containing the 7.2-kilobase pair rat beta-casein gene have been isolated and characterized. The first 510 base pairs (bp) of 5' flanking, 110 bp of 3' flanking, and all the exon/intron junctions have been sequenced. The beta-casein gene contains 9 exons ranging in size from 21 to 525 bp. We have attempted to identify potential regulatory elements by searching for regions of sequence homology shared between milk protein genes which respond similarly to lactogenic hormones and by searching for previously reported hormone receptor-binding sites. Within the conserved first 200 bp of 5' flanking sequences 3 regions of greater than 70% homology were observed between the rat beta- and gamma-casein genes. One of these contains a region 90% homologous to the chicken progesterone receptor-binding site. The conserved 5' noncoding region, the highly conserved signal peptide, and the hydrophobic carboxyl-terminal region of the protein are each encoded by a separate exon. In contrast the evolutionarily conserved phosphorylation site of beta-casein is formed by an RNA-splicing event. The exons which encode the phosphorylation sites of beta-casein appear to have resulted from an intragenic duplication. Based upon the exon structure of the casein genes, an evolutionary model of intragenic and intergenic exon duplications for this gene family is proposed.     

Structure of the human TNF receptor 1 (p60) gene (TNFR1) and localization to chromosome 12p13 [corrected]

Clones encoding the entire coding and 3' untranslated region of the human type I tumor necrosis factor receptor (p60) gene (TNFR1) were isolated by hybridization using probes derived from TNFR-1 cDNA. The gene was characterized by restriction mapping. DNA blot analysis and sequence analysis. The coding region and the 3' untranslated region are distributed over 10 exons. Each of the four repeats, comprising the extracellular ligand binding domain and characterizing a receptor superfamily, is interrupted by an intron. However, the intron-exon structure is not conserved in the nerve growth factor receptor gene, another member of this superfamily. By PCR analysis of human-mouse somatic cell hybrids and in situ hybridization using biotinylated genomic TNFR1 DNA, we localized the gene to human chromosomal band 12p13. This corresponds to the homologous murine gene localized at the distal region of mouse chromosome 6.     

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.