Cloning and sequencing of a novel metallothionein gene in Mytilus galloprovincialis Lam

Metallothionein (MT) is a ubiquitous, metal-inducible protein with an important role in the homeostasis and in the detoxification of heavy metals. This work reports the cloning and sequencing of a MT gene encoding a MT isoform (MT20-IIIa) in the mussel Mytilus galloprovincialis Lam, a lamellibranch mollusc known to accumulate and to detoxify large amounts of metal. The MT gene, lacking the 5' promoter region, is 1865 bp long and has a tripartite structure consisting of three exons and two introns. The putative open reading frame (ORF) encodes a polypeptide of 72 amino acids, which corresponds to the MT-I class, type 2 family (http://www.unizh.ch/~mtpage/classif.html). The structure of the gene and the putative MT20-III protein have been compared with those of other species. The putative biological significance of the differences at the amino acid level among the different MTs is discussed.     

Structure of the horseradish peroxidase isozyme C genes

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.     

A single-base deletion in soybean flavonol synthase gene is associated with magenta flower color

The Wm locus of soybean [Glycine max (L.) Merr.] controls flower color. Dominant Wm and recessive wm allele of the locus produce purple and magenta flower, respectively. A putative full-length cDNA of flavonol synthase (FLS), gmfls1 was isolated by 5′ RACE and end-to-end PCR from a cultivar Harosoy with purple flower (WmWm). Sequence analysis revealed that gmfls1 consisted of 1,208 nucleotides encoding 334 amino acids. It had 59–72% homology with FLS proteins of other plant species. Conserved dioxygenase domains A and B were found in the deduced polypeptide. Sequence comparison between Harosoy and Harosoy-wm (magenta flower mutant of Harosoy; wmwm) revealed that they differed by a single G deletion in the coding region of Harosoy-wm. The deletion changed the subsequent reading frame resulting in a truncated polypeptide consisting of 37 amino acids that lacked the dioxygenase domains A and B. Extracts of E. coli cells expressing gmfls1 of Harosoy catalyzed the formation of quercetin from dihydroquercetin, whereas cell extracts expressing gmfls1 of Harosoy-wm had no FLS activity. Genomic Southern analysis suggested the existence of three to four copies of the FLS gene in the soybean genome. CAPS analysis was performed to detect the single-base deletion. Harosoy and Clark (WmWm) exhibited longer fragments, while Harosoy-wm had shorter fragments due to the single-base deletion. The CAPS marker co-segregated with genotypes at Wm locus in a F₂ population segregating for the locus. Linkage mapping using SSR markers revealed that the Wm and gmfls1 were mapped at similar position in the molecular linkage group F. The above results strongly suggest that gmfls1 represents the Wm gene and that the single-base deletion may be responsible for magenta flower color.

---

     

Molecular Characterization of Duplicate Cytosolic Phosphoglucose Isomerase Genes in Clarkia and Comparison to the Single Gene in Arabidopsis

The nucleotide sequence of PgiC1-a which encodes a cytosolic isozyme of phosphoglucose isomerase (PGIC; EC 5.3.1.9) in Clarkia lewisii, a wildflower native to California, is described and compared to the previously published sequence of the duplicate PgiC2-a from the same genome. Both genes have the same structure of 23 exons and 22 introns located in identical positions, and they encode proteins of 569 amino acids. Exon and inferred protein sequences of the two genes are 96.4% and 97.2% identical, respectively. Intron sequences are 88.2% identical. The high nucleotide similarity of the two genes is consistent with previous genetic and biosystematic findings that suggest the duplication arose within Clarkia. A partial sequence of PgiC2-b was also obtained. It is 99.5% identical to PgiC2-a in exons and 99.7% in introns. The nucleotide sequence of the single PgiC from Arabidopsis thaliana was also determined for comparison to the Clarkia genes. The A. thaliana PgiC has 21 introns located at positions identical to those in Clarkia PgiC1 and PgiC2, but lacks the intron that divides Clarkia exons 21 and 22. The A. thaliana PGIC protein is shorter, with 560 amino acids, and differs by about 17% from the Clarkia PGICs. The PgiC in A. thaliana was mapped to a site 20 cM from restriction fragment length polymorphism marker 331 on chromosome 5.     

Foldability and the amino acid compositions of exons and introns

Various procedures are employed to relate the structural tendencies of polypeptide chain fragments to amino acid residues that in general have low background frequencies. A numerical evaluation of the content of these amino acids, named amino acid diversity, is defined. Distributions of the amino acid diversity parameter in databases containing exons, introns, and randomized exons show that there is a small difference between exons and shuffled exons, a detectable difference between exons and introns, and a large difference between exons and totally randomized exons.     

Positional cloning of rice semidwarfing gene, sd-1: rice "green revolution gene" encodes a mutant enzyme involved in gibberellin synthesis.

A rice semidwarfing gene, sd-1, known as the "green revolution gene," was isolated by positional cloning and revealed to encode gibberellin 20-oxidase, the key enzyme in the gibberellin biosynthesis pathway. Analysis of 3477 segregants using several PCR-based marker technologies, including cleaved amplified polymorphic sequence, derived-CAPS, and single nucleotide polymorphisms revealed 1 ORF in a 6-kb candidate interval. Normal-type rice cultivars have an identical sequence in this region, consisting of 3 exons (558, 318, and 291 bp) and 2 introns (105 and 1471 bp). Dee-Geo-Woo-Gen-type sd-1 mutants have a 383-bp deletion from the genome (278-bp deletion from the expressed sequence), from the middle of exon 1 to upstream of exon 2, including a 105-bp intron, resulting in a frame-shift that produces a termination codon after the deletion site. The radiation-induced sd-1 mutant Calrose 76 has a 1-bp substitution in exon 2, causing an amino acid substitution (Leu [CTC] to Phe [TTC]). Expression analysis suggests the existence of at least one more locus of gibberellin 20-oxidase which may prevent severe dwarfism from developing in sd-1 mutants.     

Gene structure of human cytochrome P-450(SCC), cholesterol desmolase

Four independent clones containing a part of the P-450(SCC), cholesterol desmolase, gene were isolated from human genomic libraries using bovine P-450(SCC) cDNA as a probe. These clones covered the entire P-450(SCC) gene except for a part of the 1st intron. The gene is at least 20 kb long and is split into 9 exons by 8 introns. The sequence analysis revealed that the nine separated exons code for a primary structure consisting of 521 amino acids which shows 72% homology with that of bovine P-450(SCC). A CATT sequence and a TATAAT sequence, which are possibly a "CAT" box, and a "TATA" box, respectively, are present 129 and 91 bp upstream from the initiation codon. An unusual exon/intron junctional sequence that begins with GC was found in the 6th intron of the gene. A putative extension peptide consisting of 39 amino acids was found in the sequence of human P-450(SCC) by comparison with that of the bovine counterpart. Two conserved regions were found in the extension peptide of these two forms of P-450(SCC), suggesting a functional role of the portions in the mitochondrial localization and processing of P-450(SCC) precursor. The mature form of human P-450(SCC) has only one cysteine residue, which was located in the center of the HR2 region (Gotoh et al. (1983) J. Biochem. 97, 807-817). This observation established beyond doubt that the sole cysteine residue in the HR2 region is the 5th ligand to the heme.     

Nucleotide sequence of a chicken delta-crystallin gene.

We have determined the complete nucleotide sequence of one of the two non-allelic delta-crystallin genes in the chicken, arbitrarily designated delta-gene 1, using a genomic clone (lambda g delta 106) containing the entire gene sequence. By comparison of the genomic sequence and the delta-crystallin cDNA sequence previously determined, we have identified exon sequences in the genomic sequence. Thus, the presence of 17 exons and 16 introns in the gene has been clarified. The delta-crystallin polypeptide deduced from the exon sequences consists of 465 amino acids which is larger, by 19 amino acid residues, than the polypeptide deduced from the cDNA sequence previously reported. Re-examination of the cDNA sequence using the same cDNA clone previously used shows that the present exon sequences are correct and the molecular weight of the deduced delta-crystallin polypeptide is 50,615 daltons instead of the previously reported value of 48,447 daltons. In addition, some structural features of the delta-crystallin gene including putative expression signals are discussed.     

Molecular cloning and nucleotide sequence of a gene encoding a cotton palmitoyl-acyl carrier protein thioesterase.

A cotton genomic clone containing a 17.4-kb DNA segment was found to encompass a palmitoyl-acyl carrier protein (ACP) thioesterase (Fat B1) gene. The gene spans 3.6 kb with six exons and five introns, and is apparently the first plant FatB acyl-ACP thioesterase gene to be completely sequenced. The six exons are identical in nucleotide sequence to the open reading frame of the corresponding cDNA, and would encode a preprotein of 413 amino acids. The preprotein can clearly be identified as a FatB acyl-ACP thioesterase from its similarity to the deduced amino acid sequences of other FatB thioesterase preproteins. A 5'-flanking region of 914 bp was sequenced, with the potential TATA basal promoter 324 bp upstream from the ATG initiation codon. The 5'-flanking sequence also has a putative CAAT box and two presumptive basic region helixloop-helix (bHLH) elements with the consensus motif CANNTG (termed an E box), implicated as being a positive regulatory element in seed-specific gene expression.     

Structural features of a gene encoding the vacuolar H+-ATPase c subunit from a marine red alga, Porphyra yezoensis.

We report the nucleotide sequence of a gene encoding the c ('16 kDa') subunit of the vacuolar-type H+-ATPase (V-ATPase) from a marine red alga, Porphyra yezoensis. A cDNA clone was isolated from a leafy gametophyte cDNA library and analyzed for the sequence. The genomic DNA sequence was directly determined by nested PCR. The structural gene contained four introns within a coding sequence of 483 base pairs which encodes a polypeptide of 161-amino acids with four hydrophobic transmembrane-spanning regions. Comparison of the deduced amino acid sequences showed higher similarity to the land plant Oryza sativa (69.1%) than to the Ulvophyceae Acetabularia acetabulum (64.1%). The mRNA was detected both in the leafy gametophytes and filamentous sporophytes.     

Genomic organization and tissue-specific expression of splice variants of mouse organic anion transporting polypeptide 2

cDNAs that code for mouse organic anion transporting polypeptide 2 (oatp2) have been cloned. At least three forms of mouse oatp2 cDNAs containing the same coding sequence were isolated. The common coding sequence is for a protein of 670 amino acids with 12 putative transmembrane domains. The deduced amino acid sequence of the mouse oatp2 shares 89% identity with the reported rat oatp2. Cloning and analysis of mouse oatp2 gene indicates that these isoforms are alternatively spliced products from the same gene. Heterogeneity was observed in the 5'-untranslated region of the cDNAs. Two of the three isoforms lacked the noncoding exon 3 sequence. Northern-blot hybridization analysis using the exon 3-specific probes demonstrated that mouse oatp2 mRNA containing exon 3 sequence is expressed in heart and lung, whereas exon 1-, 2-, and 17-specific probes detected mRNA only in brain and liver. The mouse oatp2 gene consists of 17 exons, including three noncoding exons, and 16 introns. All of the introns are flanked by GT-AG splice sequences except for intron 10 that is flanked by GC-AG splice sequence.     

Cloning, characterization and expression analysis of interleukin-10 from the common carp, Cyprinus carpio L.

Interleukin (IL)-10 was cloned from the common carp (Cyprinus carpio L.) using IL-10 primers from carp head kidney following stimulation with concanavalin A and lipopolysaccharide. The cDNA consisted of a 1096 bp sequence containing a 55 bp 5' untranslated region and a 498 bp 3' untranslated region. An open reading frame of 543 bp encoded a putative 180 amino acid protein with a putative signal peptide of 22 amino acids. The signature motif of IL-10 is conserved in carp sequence. A 2083 bp genomic sequence of carp IL-10 was found to contain five exons interrupted by four introns. With the exception of much more compact introns, the genomic structure was similar to that of mammalian IL-10. By homology, phylogeny and genomic analyses, the carp gene cloned was designated as IL-10. Carp IL-10 was expressed in head, kidney, liver, spleen and intestine during the resting phase. The gene was also expressed in head kidney and liver following in vitro stimulation with lipopolysaccharide.