A new gene coding for <Emphasis Type="Italic">p</Emphasis>-coumarate 3-hydroxylase from <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

p-Coumarate 3-hydroxylase (C3H) is a rate-limiting enzyme involved in monolignol biosynthesis. The full-length cDNA from Ginkgo biloba and genomic DNA sequence encoding C3H (designated as GbC3H) were cloned and characterized for the first time by rapid amplification of cDNA ends technique. The full-length cDNA of GbC3H was of 1860 bp containing a 1527 bp open reading frame encoding a cytochrome P450 protein of 508 amino acids with a calculated mol wt of 57.46 kD and an isoelectric point of 7.09. Two introns were present in the GbC3H gene. Comparative and bioinformatic analyses revealed that GbC3H had close similarity with C3Hs from other species and contained a conserved cytochrome P450 cysteine heme-iron ligand signature. Phylogenetic analysis indicated that GbC3H shared a common evolutionary origin based on sequence and had the closest relationship to C3H from gymnosperm species. Southern blot analysis indicated that GbC3H belonged to a small-gene family. Tissue expression pattern analysis revealed the highest expression of GbC3H in roots followed by leaves, and no expression was detected in stems. Only a few proteins of this class have been found, so the cloning and characterization of GbC3H will be useful in understanding the role of C3Hs in the lignin biosynthesis at the molecular level. This text was submitted by the authors in English.     

Hydrolysis of the 5'-p-nitrophenyl ester of TMP by oligoribonucleases (ORN) from Escherichia coli, Mycobacterium smegmatis, and human

Escherichia coli oligoribonuclease (EcoORN), encoded by the orn gene, is a 3'-5' exonuclease that degrades short single-stranded oligoribonucleotides to rNMPs in the final step of RNA degradation. The orn gene is essential in E. coli, but not in higher organisms, and close homologues are present in other genomes from the beta and gamma subdivisions of the Protobacteriaceae, including many pathogenic species. We report here the expression in E. coli of orn and homologues from Mycobacterium smegmatis and human, and large-scale purification of the three enzymes. All three were found to promote the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of Michaelis-Menten parameters (k(cat)=100-650 min(-1), K(M)=0.4-2.0 mM, at pH 8.00 and 25 degrees C, with 1 mM Mn(2+)). Hydrolysis by pNP-TMP by all three enzymes depended on a divalent metal ion, with Mn(2+) being preferred over Mg(2+) as cofactor, and was inhibited by Ni(2+). The concentration dependency of Mn(2+) was examined, giving K(Mn) values of 0.2-0.6 mM. The availability of large amounts of the purified enzymes and a simple spectrophotometric assay for ORN activity should facilitate large-scale screening for new inhibitors of bacterial oligoribonucleases.     

MCM3AP is transcribed from a promoter within an intron of the overlapping gene for GANP

MCM3 acetylase (MCM3AP) and germinal-centre associated nuclear protein (GANP) are transcribed from the same locus and are therefore confused in databases because the MCM3 acetylase DNA sequence is contained entirely within the much larger GANP sequence and the entire MCM3AP sequence is identical to the carboxy terminus of GANP. Thus, the MCM3AP and GANP genes are read in the same reading frame and MCM3AP is an N-terminally truncated region of GANP. However, we show here that MCM3AP and GANP are different proteins, occupying different locations in the cell and transcribed from different promoters. Intriguingly, a promoter for MCM3AP lies within an intron of GANP. This report is an interesting example in nature of two separate gene products from the same locus that perform two entirely different functions in the cell. Therefore, to avoid further confusion, they should now be referred to as separate but overlapping genes.     

The Drosophila homologue of the dystrophin gene - introns containing promoters are the major contributors to the large size of the gene

We show that the drosophila gene encoding the dystrophin-like protein (DLP) is as complex as the mammalian dystrophin gene. Three 5' promoters and three internal promoters regulate the expression of three full-length and three truncated products, respectively. The existence of this complex gene structure in such evolutionary remote organisms suggests that both types of products have diverse important functions. The promoters of both the DLP gene and the mammalian dystrophin gene are located in very large introns. These introns contribute significantly to the large size of the genes. The possible relevance of the conservation of the large size of introns containing promoters to the regulation of promoter activity is discussed.     

Identification, mRNA expression and characterization of a novel ANK-like gene from Chinese mitten crab Eriocheir japonica sinensis

Ankyrins are a family of adapter molecules mediating linkages between integral membrane and cytoskeletal proteins. Ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. A novel ANK-like gene of Chinese mitten crab Eriocheir japonica sinensis (denoted as EjsANK) was identified and cloned by expressed sequence tag and rapid amplification of cDNA end approaches. The full-length cDNA of EjsANK is 4375 bp and contains an open reading frame of 1095 bp which encodes a 364 amino acids polypeptide (40.23 kD) bearing seven ankyrin repeats. EjsANK cDNA has a 3073 bp uniquely long 3′ untranslated region with three K-box elements, one GY-like box domain and one Brd-like box domain. Sequence alignment and three-dimensional structural analyses revealed that EjsANK should be a novel cytosolic member of the ankyrin family. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsANK mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that the relative level of EjsANK mRNA expression was significantly higher in the abdomen at the first crab stage. Functional bioinformatics prediction analyses indicated that EjsANK has an analogical effect like IκB which is a key component of IκB/NF-κB complex in mammalian cells playing very important roles in the development process. Results suggest that EjsANK gene is involved in the early developmental regulation of Chinese mitten crab, especially brachyurization regulation.     

Crystal structure of the protein from Arabidopsis thaliana gene At5g06450, a putative DnaQ‐like exonuclease domain‐containing protein with homohexameric assembly

Arabidopsis thaliana gene At5g06450 encodes a putative DnaQ‐like 3′‐5′ exonuclease domain‐containing protein (AtDECP). The DnaQ‐like 3′‐5′ exonuclease domain is often found as a proofreading domain of DNA polymerases. The overall structure of AtDECP adopts an RNase H fold that consists of a mixed β‐sheet flanked by α‐helices. Interestingly, AtDECP forms a homohexameric assembly with a central six fold symmetry, generating a central cavity. The ring‐shaped structure and comparison with WRN‐exo, the best structural homologue of AtDECP, suggest a possible mechanism for implementing its exonuclease activity using positively charged patch on the N‐terminal side of the homohexameric assembly. The homohexameric structure of AtDECP provides unique information about the interaction between the DnaQ‐like 3′‐5′ exonuclease and its substrate nucleic acids.Proteins 2013. © 2013 Wiley Periodicals, Inc.     

Functional diversification of kir7.1 in cichlids accelerated by gene duplication

Mutation in the inward rectifier potassium channel gene, kir7.1, was previously identified as being responsible for the broader stripe zebrafish skin pattern mutant, jaguar/obelix. An amino acid substitution in this channel causes a broader stripe pattern than that of wild type zebrafish. In this study we analyzed cichlid homologs of the zebrafish kir7.1 gene. We identified two kinds of homologous genes in cichlids and named them cikir7.1 and cikir7.2. Southern hybridization using cichlid genome revealed that cichlids from the African Great Lakes, South America and Madagascar have two copies of the gene. Cichlids from Sri Lanka, however, showed only one band in this experiment. Database analysis revealed that only one copy of the kir7.1 gene exists in the genomes of the teleosts zebrafish, tetraodon, takifugu, medaka and stickleback. The deduced amino acid sequence of cikir7.1 is highly conserved among African cichlids, whereas that of cikir7.2 has several amino acid substitutions even in conserved transmembrane domains. Gene expression analysis revealed that cikir7.1 is expressed specifically in brain and eye, and cikir7.2 in testis and ovary; zebrafish kir7.1, however, is expressed in brain, eye, skin, caudal fin, testis and ovary. These results suggest that gene duplication of the cichlid kir7.1 occurred in a common ancestor of the family Cichlidae, that the function of parental kir7.1 was then divided into two genes, cikir7.1 and cikir7.2, and that the evolutionary rate of cikir7.2 might have been accelerated, thereby effecting functional diversification in the cichlid lineage. Thus, the evolution of kir7.1 genes in cichlids provides a typical example of gene duplication--one gene is conserved while the other becomes specialized for a novel function.