Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.     

Characterization and identification of the integrin family in silkworm, Bombyx mori

As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. Integrins are evolutionarily conserved from sponges to humans, and play vital roles in many physiological and pathological processes. To explore their diverse functions of integrins in insect, eleven integrins including six α and five β subunits were cloned and characterized from silkworm. Our results showed that integrins from silkworm own more family members compared to other invertebrates. Among those α subunits, integrins α1, α2, and the other four subunits belong to PS1, PS2, and PS3 groups, respectively. The β subunits mainly gather in the insect βν group except the β1 subunit which belongs to the insect β group. Expression profiles demonstrated that the integrins exhibited distinct patterns, but were mainly expressed in hemocytes. α1 and β2 subunits are the predominant ones either in the embryogenesis or larva stages. Interestingly, integrins were significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo. These results indicate that integrins perform diverse functions in hemocytes of silkworm. Overall, our results provide a new insight into the functional and evolutionary features of integrins.     

A novel silk-like shell matrix gene is expressed in the mantle edge of the Pacific oyster prior to shell regeneration

During shell formation, little is known about the functions of organic matrices, especially about the biomineralization of shell prismatic layer. We identified a novel gene, shelk2, from the Pacific oyster presumed to be involved in the shell biosynthesis. The Pacific oyster has multiple copies of shelk2. Shelk2 mRNA is specifically expressed on the mantle edge and is induced during shell regeneration, thereby suggesting that Shelk2 is involved in shell biosynthesis. To our surprise, the database search revealed that it encodes a spider silk-like alanine-rich protein. Interestingly, most of the Shelk2 primary structure is composed of two kinds of poly-alanine motifs-GXNA(n)(S) and GSA(n)(S)-where X denotes Gln, Arg or no amino acid. Occurrence of common motifs of Shelk2 and spider silk led us to the assumption that shell and silk are constructed under similar strategies despite of their living environments.     

两株希木龙假丝酵母14α-去甲基化酶基因(ERG11)的克隆及其功能初步验证

为探讨希木龙假丝酵母(假丝酵母又称念珠菌)的耐药机制,首先克隆出两株希木龙念珠菌ERG11基因,初步验证其功能,从而为后续研究奠定基础。从美国国家生物技术信息中心(National Center of Biotechnology Information,NCBI)基因数据库中获取白念珠菌、热带念珠菌、近平滑念珠菌和光滑念珠菌Erg11蛋白的保守序列,设计简并引物,聚合酶链反应(polymerase chain reaction,PCR)扩增获得希木龙念珠菌ERG11cDNA部分片段;用快速cDNA末端扩增法(rapid amplification of cDNA ends,RACE)分别扩增其5′和3′端,获得完整的ERG11编码序列(coding sequence,CDS);将CDS克隆到pYES2表达载体中,在尿嘧啶营养缺陷型酿酒酵母中过表达ERG11;用微量液基稀释法检测转化后的酿酒酵母对氟康唑的敏感性,初步验证其功能。结果显示,简并PCR扩增获得预期708bp片段,5′RACE和3′RACE分别获得385bp和1 336bp片段,经纯化、克隆、测序、比对分析,获得两株菌的ERG11CDS;比对其编码的蛋白,与其他念珠菌的Erg11蛋白高度同源;分别检测克隆了这两株希木龙念珠菌ERG11CDS表达载体的酿酒酵母对氟康唑的敏感性,发现过表达ERG11明显降低其对氟康唑的敏感性。结果提示,简并PCR联合RACE能准确有效地克隆出希木龙念珠菌ERG11基因,用pYES2酿酒酵母表达系统能初步验证其功能。     

Two newly identified exons in human GRM1 express a novel splice variant of metabotropic glutamate 1 receptor

To date, five human metabotropic glutamate (mGlu) 1 receptor splice variants (1a, 1b, 1d, 1f, and 1g) have been described, all of which involve alternative C-terminal splicing. mGlu1a receptor contains a long C-terminal domain (341 amino acids), which has been shown to scaffold with several proteins and contribute to the structure of the post-synaptic density. However, several shorter mGlu1 receptor splice variants lack the sequence required for these interactions, and no major functional differences between these short splice variants have been described. By using RT-PCR we have shown that two human melanoma cell lines express both mGlu1a and mGlu1b receptors. In addition, using 3′RACE, we identified three previously unknown mGlu1 receptor mRNAs. Two differ in the length of their 3′ untranslated region (UTR), and encode the same predicted protein as mGlu1g receptor—the shortest of all mGlu1 receptor splice variants. The third mRNA, named mGlu1h, encodes a predicted C-terminal splice variant of 10 additional amino acids. mGlu1h mRNA was observed in two different melanoma cell lines and is overexpressed, compared with melanoma precursor cells, melanocytes. Most importantly, this new splice variant, mGlu1h receptor, is encoded by two previously unidentified exons located within the human GRM1 gene. Additionally, these new exons are found exclusively within the GRM1 genes of higher primates and are highly conserved. Therefore, we hypothesize that mGlu1h receptors play a distinct role in primate glutamatergic signaling.     

Pulmonary cytochrome P450 enzymes belonging to the CYP4B subfamily from an Australian marsupial,the tammar wallaby (Macropus eugenii)

Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. We have previously reported the cloning and characterisation of the koala CYP4A15, the first reported member of the CYP4 family from marsupials, and have demonstrated important species differences in CYP4A activity and tissue expression. In the present study, the cloning of CYP4B1 in the wallaby (Macropus eugenii) and their expression across marsupials is described. Rabbit anti-mouse CYP4B1 antibody detected immunoreactive proteins in lung and liver microsomes from all test marsupials, with relative weak signal detected from the koala, suggesting a species-specific expression. Microsomal 2-aminofluorene bio-activation (a CYP4B1 marker) in wallaby lung was comparable to that of rabbit, with significant higher activities detected in wallaby liver and kidneys compared to rabbit. A 1548 bp wallaby lung CYP4B complete cDNA, designated CYP4B1, which encodes a protein of 510 amino acids and shares 72% nucleotide and 69% amino acid sequence identity to human CYP4B1, was cloned by polymerase chain reaction approaches. The results demonstrate the presence of wallaby CYP4B1 that shares several common features with other published CYP4Bs; however the wallaby CYP4B1 cDNA contains four extra amino acid residues at the NH₂-terminal, a fundamentally conserved transmembrane anchor of all eukaryote CYPs.     

Discovery of a disused desaturase gene from the pheromone gland of the moth Ascotis selenaria, which secretes an epoxyalkenyl sex pheromone

Female Ascotis selenaria (Geometridae) moths use 3,4-epoxy-(Z,Z)-6,9-nonadecadiene, which is synthesized from linolenic acid, as the main component of their sex pheromone. While the use of dietary linolenic or linoleic fatty acid derivatives as sex pheromone components has been observed in moth species belonging to a few families including Geometridae, the majority of moths use derivatives of a common saturated fatty acid, palmitic acid, as their sex pheromone components. We attempted to gain insight into the differentiation of pheromone biosynthetic pathways in geometrids by analyzing the desaturase genes expressed in the pheromone gland of A. selenaria. We demonstrated that a Δ11-desaturase-like gene (Asdesat1) was specifically expressed in the pheromone gland of A. selenaria in spite of the absence of a desaturation step in the pheromone biosynthetic pathway in this species. Further analysis revealed that the presumed transmembrane domains were degenerated in Asdesat1. Phylogenetic analysis demonstrated that Asdesat1 anciently diverged from the lineage of Δ11-desaturases, which are currently widely used in the biosynthesis of sex pheromones by moths. These results suggest that an ancestral Δ11-desaturase became dysfunctional in A. selenaria after a shift in pheromone biosynthetic pathways.     

Molecular cloning and characterization of an Hsp90/70 organizing protein gene from Frankliniella occidentalis (Insecta: Thysanoptera,Thripidae)

The heat shock 90/70 organizing protein (Hop), also known as Sti-1 (stress-induced protein-1), is a co-chaperone that usually mediates the interaction of Hsp90 and Hsp70 and has been extensively characterized in mammals and plants. However, its role in insects remains unknown. In the present study, we isolated and characterized a Hop homologue gene from Frankliniella occidentalis (Fohop). The Fohop contains a 1659 bp ORF encoding a protein of 552 amino acids with a caculated molecular mass of approximately 62.25 kDa, which displays a reasonable degree of identity with the known Hops and shares several canonical motifs, including three tetratricopeptide repeated motif domains (TPR1, TPR2A and TPR2B) and two aspartic acid–proline (DP) repeat motifs (DP1 and DP2). As in other hops, Fohop contains introns, but the number and the position are quite variable. The mRNA expression patterns indicated that Fohop was constitutively expressed throughout the developmental stages, but was obviously upregulated by heat stress both in larvae and adults. Our studies imply that Hop, as in other Hsps, may play an important role in heat shock response of F. occidentalis.     

Molecular cloning and characterization of the sheep α-TTP gene and its expression in response to different vitamin E status

The α-tocopherol transfer protein (α-TTP) is a ~ 32 kDa protein that exhibits a marked ligand specificity and selectively recognizes of α-tocopherol, which is the most active form of vitamin E. The α-TTP gene has been cloned and its physiological functions have been studied in numbers of species, however, the understanding of sheep α-TTP is still in his infancy. In this study, the full-length cDNA of sheep α-TTP gene was cloned from sheep liver by using of rapid amplification of complementary DNA ends (RACE). As a result, the sheep α-TTP gene was 1098 bp in nucleotide which contained 23 bp 5'-untranslated region (UTR), 226 bp 3'-UTR and 849 bp open reading frame (ORF) that encoded a basic protein of 282 amino acids. Further bioinformatic analysis indicated that the sheep α-TTP gene had a high homologous of both nucleotide and amino acid sequences compared with that of other species and had a Sec14p-like lipid-binding domain which called the CRAL-TRIO domain. Moreover, the expression of sheep α-TTP mRNA and protein in response to different vitamin E supplemented levels were observed according to quantitative real-time PCR (qRT-PCR) and Western blotting analysis. The results showed that dietary vitamin E levels did not affect α-TTP mRNA expression significantly while the low vitamin E supplemented level groups of sheep had significantly higher α-TTP protein compared to high-vitamin E groups.     

Molecular cloning and expression in vitro of a carboxylesterase gene from the Glanville fritillary butterfly (Melitaea cinxia)

Carboxylesterase (EC 3.1.1.1) is a member of the carboxyl/cholinesterase (CCE) superfamily, which is widely distributed in animals, plants and microorganisms. This enzyme has been known to be associated with insecticide resistance and detoxification. Although CCEs have been extensively studied in insects, including lepidopterans, the research on butterflies, a major subgroup in Lepidoptera, is still poor. In the present study, we cloned a CCE gene (McCCE1) from the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The full-length cDNA encoding McCCE1 was 1786 bp, containing a 1641 bp open reading frame encoding 546 amino acids, a 38 bp 5′-untranslated region (5′-UTR), and a 107 bp 3′-UTR with a poly(A) tail. The functionally conserved amino acids in McCCE1 shared the 55% identity with the cytoplasmic esterase CCE017a in Helicoverpa armigera (Lepidoptera: Noctuidae), which has been associated with detoxification. Assays in vitro showed that the recombinant McCCE1 could hydrolyze α- and β-naphthyl acetate. Thus, the present study adds to the body of knowledge concerning the detoxification of pesticides by lepidopterans.     

Molecular cloning and mRNA tissue expression of thyroid hormone receptors in yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta

Thyroid hormones (THs) play a pivotal role in many physiological functions in vertebrates, including fish. Their effects are mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. In this study, full-length cDNA sequences of TRs from yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta were cloned and their mRNA tissue expression profiles were determined. In P. fulvidraco, the validated cDNAs encoding for TRα and TRβ were 1789 and 1848 bp in length, encoding peptides of 401 and 378 amino acid residues, respectively. In addition, a TRβ spliced variant (named P. fulvidraco-TRβv), containing a 60-bp insertion, was detected. In S. hasta, cDNAs encoding for TRαA, TRαB and TRβ were 1827, 2295 and 2258 bp in length, encoding peptides of 401, 409 and 393 amino acid residues, respectively. The phylogenetic analysis revealed that TRα and TRβ cDNAs grouped into two separate clusters with other vertebrate counterparts and two TRα sequences grouped separately, suggesting that the two TRαs derived from paralogous genes that might arise during a teleost-specific genome duplication event. All TR mRNAs were detected in various tissues sampled. The mRNA levels of both TRα and TRβ from P. fulvidraco were the highest in brain, followed by liver, and lowest in heart, intestine, muscle, gill and spleen. However, in S. hasta, TRαA, TRαB and TRβ showed the highest mRNA levels in brain and lowest in muscle. Identification and mRNA tissue expression of TR genes from P. fulvidraco and S. hasta provide an initial step towards understanding their biological roles in the two fish species.     

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.