Gene structure prediction using information on homologous protein sequence

In this paper a new approach for the prediction of protein coding gene structures is described. The principal scheme of prediction is as follows: first, the exons with the best potential are predicted in a sequence with unknown functions and a list of potential amino acid fragments coded by these exons is formed. Second, testing the homology between each amino acid fragment from the list and proteins from the SWISS-PROT database of amino acid sequences. One protein with the best homology is chosen out of all the homologous sequences. Third, reconstruction of the exon-intron structure, basing it on its homology with the chosen protein sequences. The method was tested on an independent control set (20 genes). The results were as follows: 21% of real exons were lost and 3% of non-real exons were found. This system can be used to refine the results of gene prediction systems, especially if highly homologous proteins are found in the amino acid sequence database.     

Immunoreactive endothelin-1, endothelin-2 and big endothelin-1 in follicular fluids of women undergoing ovulation induction for in-vitro fertilization

Atlantic cod (Gadus morhua) transferrin cDNAs were isolated from a liver cDNA library using a cod transferrin-derived polymerase chain reaction product as a hybridization probe. The composite nucleotide sequence of two overlapping clones was 2223 bp in length excluding the poly(A) sequence and was equivalent to 87% of the 3' end of the Atlantic salmon transferrin cDNA sequence. Comparison of the deduced amino acid sequence of cod, salmon, Xenopus and several mammalian transferrins revealed that the two fish sequences are more similar with respect to their amino acid sequence and the position of additions/deletions than to other vertebrate transferrins. Conservation of the iron-binding domains and cysteine residues involved in disulphide bridges indicates that all transferrins share similar tertiary structure and support the hypothesis that extant vertebrate transferrin genes were derived from a gene duplication before the divergence of fish, frogs and mammals. Cod transferrin mRNA was detected in both brain and liver RNA and to a much lesser extent in RNA isolated from kidney and heart in contrast to salmon and several other vertebrates in which the transferrin gene is not expressed in brain.     

Human lysophosphatidic acid acyltransferase. cDNA cloning, expression, and localization to chromosome 9q34.3

Lysophosphatidic acid (1-acyl-sn-glycero-3-phosphate (LPA)) is a phospholipid with diverse biological activities. The mediator serves as an intermediate in membrane phospholipid metabolism but is also produced in acute settings by activated platelets. LPA is converted to phosphatidic acid, itself a lipid mediator, by an LPA acyltransferase (LPAAT). A human expressed sequence tag was identified by homology with a coconut LPAAT and used to isolate a full-length human cDNA from a heart muscle library. The predicted amino acid sequence bears 33% identity with a Caenorhabditis elegans LPAAT homologue and 23-28% identity with plant and prokaryotic LPAATs. Recombinant protein produced in COS 7 cells exhibited LPAAT activity with a preference for LPA as the acceptor phosphoglycerol and arachidonyl coenzyme A as the acyl donor. Northern blotting demonstrated that the mRNA is expressed in most human tissues including a panel of brain subregions; expression is highest in liver and pancreas and lowest in placenta. The human LPAAT gene is contained on six exons that map to chromosome 9, region q34.3.     

Molecular cloning of a full-length cDNA for human type 3 adenylyl cyclase and its expression in human islets

The GK (Goto-Kakizaki) rat is a lean model of type 2 diabetes in which the diabetic state was spontaneously induced. We recently demonstrated the presence in GK rats of two functional point mutations in the promoter region of the type 3 adenylyl cyclase (AC3) gene that resulted in overexpression of AC3 mRNA associated with increased cAMP generation. The AC3 gene promoter mutations are the first molecular changes to be described in any specific gene in the GK rat. Here we report cloning of a full-length cDNA encoding human AC3 from a human fetal brain cDNA library using a PCR-based screening method. This 4142-bp cDNA predicts an open reading frame encoding 1144 amino acids containing putative 12 transmembrane-spanning domains which are typically found in other mammalian AC isoforms. Comparison of the translated amino acid sequence of the AC3 gene between human and rat shows 95% homology. Using RT-PCR, clear AC3 expression was detected in isolated human islets as well as a cDNA panel containing templates from eight different tissues (brain, heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle). This wide distribution of AC3 expression may involve a number of physiological and pathophysiological metabolic processes.     

Isolation, amino acid sequence determination and binding properties of two fatty-acid-binding proteins from axolotl (Ambistoma mexicanum) liver. Evolutionary relationship

Up until now, the primary structure of fatty-acid-binding proteins (FABPs) from the livers of four mammalian (rat, human, cow and pig) and three nonmammalian (chicken, catfish and iguana) species has been determined. Based on amino acid sequence comparisons, it has been suggested that mammalian and nonmammalian liver FABPs may be paralogous proteins that originated by gene duplication, rather than as a consequence of mutations of the same gene. In this paper we report the isolation and amino acid sequence determination of two FABPs from axolotl (Ambistoma mexicanum) liver. One of them is similar to mammalian liver FABPs (L-FABPs) and the other to chicken, catfish and iguana liver FABPs (Lb-FABPs). The finding of both L-FABP and Lb-FABP in a single species, as reported here, indicates that they are paralogous proteins. The time of divergence of these two liver FABP types is estimated to be of approximately 694 million years ago. The ligand-binding properties of axolotl liver FABPs were studied by means of parinaric-acid-binding and parinaric-acid-displacement assays. L-FABP binds two fatty acids per molecule but Lb-FABP displays a fatty-acid-conformation-dependent binding stoichiometry; L-FABP shows a higher affinity for fatty acids, especially oleic acid, while Lb-FABP has a higher affinity for other hydrophobic ligands, especially retinoic acid. In addition, the tissue-expression pattern is different, L-FABP is present in liver and intestinal mucosa while the expression of Lb-FABP is restricted to liver. Data indicate distinct functional properties of both liver FABP types.     

Molecular analysis of the gene cluster involved in cephalosporin biosynthesis from Lysobacter lactamgenus YK90

Determination of the nucleotide sequence downstream from the Lysobacter lactamgenus pcbC gene encoding isopenicillin N synthase revealed that five open-reading frames (ORF) including the pcbC gene were tightly linked in the same orientation. Each ORF has the remarkable feature of the protein-coding frame in the DNA sequence with a high G+C content. Expression in Escherichia coli and a comparison of the deduced amino acid sequences with published sequences showed that the gene cluster contained a deacetoxycephalosporin C synthetase (DAOCS) gene (cefE), an ORF having homology with the Cephalosporium acremonium DAOCS/deacetylcephalosporin C synthetase gene (cefEF), an isopenicillin N epimerase gene(cefD), and a beta-lactamase gene. The gene order was pcbC-cefE-ORF3-cefD-beta-lactamase.     

Molecular cloning and nucleotide sequence of the genomic DNA for 1,2-alpha-D-mannosidase gene, msdC from Penicillium citrinum

A gene encoding 1,2-alpha-D-mannosidase (EC 3.2.1.113) was cloned from Penicillium citrinum genomic DNA using the polymerase chain reaction (PCR). The coding region of the gene, msdC, occupied 1737 bp and was separated into four exons by three introns. The predicted protein consisted of 511 amino acid residues with M(r) 56,569. Penicillium enzyme had a hydrophobic signal peptide at the N-terminal region as did mammalian membrane-bound alpha-mannosidases, but in this case a proteolytic cleavage occurred at Lys-35-Ser-36 to remove the signal sequence during cell growth. Parts of amino acid sequences were similar to those of mammalian Golgi alpha-mannosidase IA and IB, but the sequence around the aspartic acid residue which interacted with 1-deoxymannojirimycin (Yoshida et al. (1994) Biochem. J. 303, 97-103) was unique in Penicillium enzyme.     

The e subunit gene of murine F1F0-ATP synthase. Genomic sequence, chromosomal mapping, and diet regulation

Genomic sequences encoding murine Lfm1, whose predicted protein sequence is 96% and 98% similar to bovine and rat F1F0-ATP synthase e subunits (respectively), have been amplified from BALB/cByJ DNA, cloned, and sequenced. The 1.1-kilobase gene has 3 introns and 4 exons, and its coding sequence differs by two nucleotides compared to the previously published BALB/cHnn Lfm1 cDNA sequence. A PstI restriction site polymorphism in intron 2 between C57BL/6J and Mus spretus was used to map this gene to Chromosome 5 near D5Mit9. Related sequences were mapped on Chromosomes 8, 11, and 2 unlinked loci on Chromosome 2 using Southern blot analyses with the 1. 1-kilobase gene as probe. Previous studies from this laboratory indicated that the Lfm1/e subunit was regulated by the level of dietary fat and carbohydrate. Northern hybridization analyses demonstrated that e subunit mRNA abundance showed statistically significant differences (p < 0.025) between hearts of BALB/c mice fed 3% and those fed 20% corn oil for 2 weeks and in liver (p < 0. 05) from the same animals. Significant differences were also observed in hepatic and heart mRNA expression at different times after eating in animals subjected to a fast/refeed regimen. The implications of the high degree of sequence similarity to the e subunit for rat and bovine F1F0-ATP synthase and its regulation by diet are discussed.     

Determinants of cholesterol screening and treatment patterns. Insights for decision-makers

We isolated rat UCP2 cDNA, which has been proposed to play an important role in mammalian thermogenesis and body weight regulation. The nucleotide sequence of the cDNA revealed that the rat UCP2 protein is composed of 309 amino acid residues, and is 99% and 95% identical to the mouse and human proteins, respectively. The molecular weight of rat UCP2, calculated from the predicted amino acid sequence, was 33,369, and the UCP2 protein of this size was detected when the cDNA was expressed in vitro. Northern blot analysis revealed that the corresponding mRNA is approximately 1.7 kb in size, and is expressed in a variety of rat organs, with predominant expression in the heart, lung and spleen. UCP2 mRNA levels in the heart, liver, muscle and epididymal adipose tissue of Zucker fatty (fa/fa) rats were comparable to those in the lean littermates, while ob mRNA level markedly increased in the epididymal adipose tissue of Zucker (fa/fa) rats.     

Cloning and expression of human deoxyguanosine kinase cDNA

A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.