Molecular Cloning and Characterization of the Myostatin Gene in Croceine Croaker, Pseudosciaena crocea

Myostatin (MSTN) is a negative regulator of skeletal muscle mass and has a potential application in aquaculture. We reported the characterization of the myostatin gene and its expression in the croceine croaker, Pseudosciaena crocea. The myostatin gene had three exons encoding 376 amino acids. The cDNA was 1,906 bp long with a 5′-UTR and 3′-UTR of 108 bp and 667 bp, respectively. A microsatellite sequence, CA₃₀ and CA₂₆ separated by TA, existed in the 3′-UTR. Intron I and II were 343 bp and 758 bp in length, respectively. The deduced amino acid sequence was highly conserved, and had more than 90% identical to shi drum, gilthead seabream, striped sea-bass, white perch, and white bass proteins. The myostatin of croceine croaker had a putative amino terminal signal sequence (residues 1–22), a transforming growth factor-beta (TGF-β) propeptide domain (residues 41–256), a RXXR proteolytic processing site (RARR, residues 264–267, matching the RXXR consensus site), and a TGF-β domain (residues 282–376). There were 13 conserved cysteine residues in croceine croaker myostatin, nine of which are common to all TGF-β superfamily members. The most conserved region of vertebrate myostatins is the TGF-β domain, which was the mature bioactive domain of the myostatin protein. The myostatin gene was expressed not only in the skeletal muscle, but also in the other tissues.     

Molecular cloning of a dehydration-responsive protein gene (MRD22) from mulberry,and determination of abiotic stress patterns of MRD22 gene expression

A full-length cDNA sequence coding for dehydration-responsive protein gene of mulberry tree, which we designated was MRD22 (GenBank accession number: JQ804833) was cloned based on mulberry expressed sequence tags (ESTs). MRD22 is 1503 bp long, contains a 334 bp 5′-UTR (untranslated region) and a 563 bp 3′-UTR, encodes 201 amino acids with a predicted molecular weight of 54.28 kDa and an isoelectric point of 9.35. Phylogenetic analysis based on MRD22 sequences from different species showed that mulberry has close relationship with Populus trichocarpa, Ricinus communis, Camellia sinensis, Gossypium hirsutum, Gossypium barbadense and so on. The expression level of the MRD22 gene under conditions of drought, low temperature and salt stresses was quantified by qRT-PCR. The results show that the expression level changed significantly under the stress conditions compared to the normal growth environment. It helps us to get a better understanding of the molecular basis for signal transduction mechanisms underlying the stress response in mulberry.     

Characterization of a pollen-preferential gene, BAN102, from Chinese cabbage

We isolated and characterized a pollen-preferential gene, BAN102, from Chinese cabbage and analyzed the activity of its promoter. There were three or four copies of the BAN102 gene in the Chinese cabbage genome that specifically expressed in pollen and pollen tube. There were 2137 bp of BAN102 genomic clone comprising 186 bp of protein coding region, and 1178 bp of 5′ and 773 bp of 3′ non-coding regions. TATA box were located at 1071 nt of the promoter region while the polyadenylation signal and polyadenylation site were at 1470 and 1486 nt of the 3′ non-coding region. BLAST search of BAN102 sequence showed that coding region of BAN102 gene was the greatest percent similarity with arabinogalactan protein (AGP23) gene from Arabidopsis thaliana. Promoter analysis using GUS gene as a reporter showed that the pollen-specificity of BAN102 resided within the −112 to −44 bp of proximal promoter from the transient expression in tobacco and Chinese cabbage plants.     

A new β-glucosidase gene from the zygomycete fungus Rhizomucor miehei

In this study, a β-glucosidase coding gene (bgl) of the zygomycete fungus Rhizomucor miehei has been cloned and characterized. The gene comprises a total of 2,826 bp including the coding sequence of a 717 amino acids length putative protein and 10 introns dispersed in the whole coding region. The putative N-and C-terminal catalytic domains (aa 68 to aa 274 and aa 358–601, respectively) were identified; the two domains are connected with a 84-amino-acids linker. The catalytic region showed an extensive sequence homology with other fungal β-glucosidases classified as family 3 glycoside hydrolases. The isolated Rhizomucor gene was expressed in the related fungus Mucor circinelloides. Transformant Mucor strains maintained the introduced plasmid in an autoreplicative manner and showed significantly higher cellobiase activity than the recipient strain.     

<Emphasis Type="Italic">Arthroderma benhamiae</Emphasis> (The Teleomorph of <Emphasis Type="Italic">Trichophyton mentagrophytes</Emphasis>) Mating Type-Specific Genes

This study first report to identify the mating type (−)-specific gene of alpha-box and the mating type (+)-specific gene of the high-mobility-group (HMG) DNA-binding domain in zoophilic dermatophytes of Arthroderma benhamiae in an effort to understand the epidemiological characteristics of Trichophyton mentagrophytes. The sequence of the alpha-box gene (1,387 bp) was found to contain two exons, from 184 to 475 bp and from 525 to 1,387 bp, coding a protein of 384 amino acids, beginning with a putative initiating methionine (ATG). The sequence of the HMG gene (1,910 bp) contained two exons, from 234 to 415 bp and from 479 to 1,457 bp, coding a protein of 386 amino acids, beginning with a putative initiating methionine (ATG).     

Sequence and expression pattern of the Drosophila melanogaster mitochondrial porin gene: evidence of a conserved protein domain between fly and mouse

We have recently cloned a cDNA encoding mitochondrial porin in Drosophila melanogaster and shown its chromosomal localization (Messina et al., FEBS Lett. (1996) 384, 9–13). Such cDNA was used as a probe for screening a genomic library. We thus cloned and sequenced a 4494-bp genomic region which contained the whole gene for the mitochondrial porin or VDAC. It was found that this D. melanogaster porin gene contains five exons, numbered IA (115 bp), IB (123 bp), II (320 bp), III (228 bp) and IV (752 bp). The exons II, III and IV contain the protein coding sequence and the 3′ untranslated sequence (3′-UTR). The first base in exon II precisely corresponds to the first base of the starting ATG codon. Exon IA corresponds to the 5′-UTR sequence reported in the published cDNA sequence. Exon IB corresponds to an alternative 5′-UTR sequence, demonstrated to be transcribed by 5′-RACE experiments. The exon-intron splicing borders and the length of the exon III perfectly match a homologous internal exon detected in the mouse genes. Such exon encodes a protein domain predicted by sequence transmembrane arrangement models to contain major hydrophilic loops and it is thus suspected to have a conserved distinct function. In situ hybridization experiments confirmed the localization of the genomic clone on the chromosome 2L at region 32B3-4. Together with genomic Southern blotting at various stringencies, the same experiment did not confirm the presence of a second genetic locus on D. melanogaster chromosomes. Northern blots demonstrated that the porin gene is a housekeeping one: three messages of approx. 1.2–1.6 kbp are transcribed in every fly developmental stage that was studied. They were shown to derive by an alternative usage of different promoters and polyadenylation sites.     

Complete mitochondrial genome sequence of Catla catla and its phylogenetic consideration

Complete nucleotide sequence of mitochondrial genome (mitogenome) of the Catla catla (Ostariophysi: Cypriniformes: Cyprinidae) was determined in the present study. Its length is 16,594 bp and contains 13 protein coding genes, 22 transfer RNAs, two ribosomal RNAs and one non-coding control region. Most of the genes were encoded on the H-strand, while the ND6 and eight tRNA (Gln, Ala, Asn, Cys, Tyr, Ser (UCN), Glu and Pro) genes were encoded on the L-strand. The reading frames of two pair of genes overlapped: ATPase 8 with 6 and ND4L with ND4 by seven nucleotides each. The main non-coding region was 929 bp, with three conserved sequence blocks (CSB-I, CSB-II, and CSB-III) and an unusual simple sequence repeat, (TA)₇. Phylogenetic analyses based on complete mitochondrial genome sequences were in favor of the traditional taxonomy of family Cyprinidae. In conclusion present mitogenome of Catla catla adds more information to our understanding of diversity and evolution of mitogenome in fishes.     

中华鲟促性腺激素释放激素受体基因cDNA序列的克隆及表达分析

为了研究中华鲟(Acipenser sinensis)促性腺激素释放激素受体(Gonadotropin-releasing hormone receptor, GnRH-R)基因在中华鲟中的组织表达特征, 为中华鲟生长发育调控研究提供基础数据, 通过构建中华鲟(Acipenser sinensis)垂体的SMART cDNA质粒文库, 采用cDNA末端快速扩增(RACE), 克隆得到了中华鲟GnRH-R基因的cDNA全长序列。该序列全长1530 bp, 有478 bp的5′非翻译区, 579 bp的开放阅读框和473 bp的3′非翻译区, 共编码192个氨基酸, 其成熟多肽含有5个Ｎ连糖基化位点。通过和已知其他鱼类的GnRH-R基因进行氨基酸序列多重比对, 发现其与真鲷(Pagrus major)的同源性最高, 为76%, 与米氏叶吻银鲛(Callorhinchus milii)的同源性最低, 为39%。采用实时荧光定量PCR (Real time PCR)方法, 检测了GnRH-R的mRNA在中华鲟心、肝、脾、肾、肠道、精巢、肌肉及脑组织中的表达状况, 发现其在精巢中大量转录, 而在其他组织中则表达微弱。以上结果表明中华鲟GnRH-R基因在性腺发育特别是精子发生过程中可能起重要作用。     

Identification and characterization of interferon regulatory factor-1 from orange-spotted grouper (<Emphasis Type="Italic">Epinephelus coioides</Emphasis>)

Interferon-regulatory factor 1 (IRF-1) is the first member of IRF family, which is involved in many biological processes such as immune response, antiviral defense, cell growth regulation, and apoptosis. In this study, an IRF-1 gene, EcIRF-1, was isolated and characterized from orange-spotted grouper (Epinephelus coioides). The full-length cDNA of EcIRF-1 is 1,730 bp, including an open reading frame of 906 bp, a 5′-terminal untranslated region (5′-UTR) of 153 bp, and a 3′-UTR of 671 bp. The EcIRF-1 gene consists of 10 exons and 9 introns, spanning over approximate 4.3 kb of genomic sequence. The 5′-UTR sequence contains an exon and an intron, and the 3′-UTR sequence is included in the last exon. Expression analysis by real-time PCR reveals that the EcIRF-1 gene is ubiquitously expressed in various healthy fish tissues, whereas its expression is upregulated in vivo in response to polyinosinic–polycytidylic acid or lipopolysaccharide stimulation. Subcellular localization analysis shows the EcIRF-1 is an intranuclearly localized and immobile protein in the cultured fish cells. Data presented in this paper provide an important base to further understand EcIRF-1 gene function and its regulation associated with interferon immune system in orange-spotted grouper.     

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked. Received: 4 December 1996 / Accepted: 21 January 1997     

Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (<Emphasis Type="Italic">Penaeus monodon</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5′ untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 3′ UTR of 288 bp. The sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence. The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37°C. In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage.     

Identification of a transforming growth factor-β type I receptor transcript in Eriocheir sinensis and its molting-related expression in muscle tissues

The transforming growth factor-β (TGF-β) signaling pathway is conserved across animals, and knowledge of its roles during the molt cycle in crustaceans is presently very limited. This study investigates the roles of the TGF-β receptor in molting-related muscle growth in Eriocheir sinensis. Using the RT-PCR and RACE techniques, we obtained a 1722 bp cDNA sequence encoding a transforming growth factor-β type I receptor in Eriocheir sinensis, designated EsTGFBRI, which contains a 124 bp 5′-untranslated region, a 20 bp partial 3′-untranslated region and a 1578 bp open reading frame encoding 525 amino acids. The deduced EsTGFBRI contains an N-terminal 24 amino acid signal peptide, an activin type I and II receptor domain, a transmembrane helix region, a glycine-serine-rich motif, and a conserved serine/threonine kinase catalytic domain including an activation loop. The qRT-PCR results showed that EsTGFBRI gene was highly expressed in the intermolt testis and ovary in mature crabs. In juvenile crabs, the mRNA levels of EsTGFBRI in claw and abdominal muscles in the later premolt D_3–4 stage were significantly higher than those in the intermolt C and postmolt A–B stages. There was no significant change in EsTGFBRI mRNA levels in walking leg muscles during the molt cycle. The results suggest that EsTGFBRI is probably play roles in molting-related muscle growth in E. sinensis. This study provides a necessary basis for elucidating the functions of TGF-β-like signaling mediated by TGFBRI in molting-related muscle growth in crustaceans.

     

Investigations into the taxonomy of the mushroom pathogen <Emphasis Type="Italic">Verticillium fungicola</Emphasis> and its relatives based on sequence analysis of nitrate reductase and ITS regions

The full sequence of the nitrate reductase gene was obtained from a type isolate of Verticillium fungicola var. fungicola and used for phylogenetic analysis against other ascomycete fungi. Sequencing obtained 2749 bp of coding region, 668 bp of 5′ flanking sequence and 731 bp of 3′ flanking sequence. In silico analysis indicated that the coding region contains a single intron and translates into an 893 amino acid protein, with BLAST analysis identifying five conserved nitrate reductase domains within the protein. The 5′ flanking sequence contains numerous conserved sites putatively involved in binding nitrogen regulatory proteins, indicating that the regulation of the gene is likely to be subject to the same regulation as that of model fungi such as Aspergillus nidulans. The central portion of this gene was amplified and sequenced from a number of V. fungicola isolates and related fungi and the resulting phylogenies compared to those obtained from analysis of the rDNA internal transcribed spacer regions for these fungi. Both nitrate reductase and ITS analyses provide additional evidence that reinforces previous findings that suggest the mushroom pathogenic Verticillium species are more related to other chitinolytic fungi such as the insect pathogens Verticillium lecanii and Beauveria bassiana than to the plant pathogenic Verticillia.