Molecular characterization of monoamine oxidase in zebrafish (Danio rerio)

Monoamine oxidase (MAO) is responsible for the degradation of a number of neurotransmitters and other biogenic amines. In terrestrial vertebrates, two forms of the enzyme, named MAO A and B, were found in which mammals are coded by two similar but distinct genes. In teleosts, the biochemical data obtained so far indicate that enzyme activity is due to a single form, whose sequence, obtained for trout, displays 70% identity with mammal MAO A and B. In this paper, we carried out an investigation of zebrafish MAO (Z-MAO) to shed further light on the nature of the MAO form present in aquatic vertebrates. Sequencing studies have revealed an open reading frame 522-amino-acids long with MW 58.7 kDa, displaying 84% identity with trout MAO and about 70% identity with mammal MAO A and MAO B. Analysis of the sequence and of the predicted secondary structure shows that also in Z-MAO principal domains characterizing the MAOs are present. The domain linking the FAD is very well conserved, while the transmembrane domain sequence linking the enzyme to the external mitochondrial membrane does not appear to be conserved even with respect to trout MAO. Comparison with the amino acids which, according to the human MAO B and rat MAO A models, line the substrate-binding site shows that in Z-MAO, several residues (V172, N173, F200, L327) differ from MAO B but are similar or identical to the corresponding ones present in rat MAO A, as well as in trout MAO. A three-dimensional model is reported of the substrate-binding site of Z-MAO obtained by comparative modeling. Our observations support the hypothesis that the MAO form present in aquatic vertebrates is a MAO A-like form. Experiments performed to test the effect of selective MAO A (clorgyline) and MAO B (deprenyl) inhibitors on the enzyme's activity in liver and brain confirm the presence of a single form of MAO in zebrafish.     

Molecular cloning and functional characterization of zebrafish ATM

Ataxia-telangiectasia mutated (ATM) is the gene product mutated in ataxia-telangiectasia (A-T), which is an autosomal recessive disorder with symptoms including neurodegeneration, cancer predisposition and premature aging. ATM is thought to play a pivotal role in signal transduction in response to genotoxic DNA damage. To study the physiological and developmental functions of ATM using the zebrafish model system, we cloned the zebrafish homolog cDNA of human ATM (hATM), zebrafish ATM (zATM), analyzed the expression pattern of zATM during early development, and further developed the system to study loss of zATM function in zebrafish embryos. Employing information available from the zebrafish genomic database, we utilized a PCR-based approach to isolate zATM cDNA clones. Sequence analysis of zATM showed a high level homology in the functional domains of hATM. The putative FAT, phosphoinositide 3-kinase-like, and FATC domains of zATM, which regulate ATM kinase activity and functions, were the most highly conserved regions, exhibiting 64-94% amino acid identity to the corresponding domains in hATM, while exhibiting approximately 50% amino acid identity outside these domains. The zATM gene is expected to consist of 62 coding exons, and we have identified at least 55 exons encompassing more than 100kb of nucleotide sequence, which encodes about 9 kb of cDNA. By in situ hybridization, zATM mRNA was detected ubiquitously with a dramatic increase at the 18-somite stage, then more specifically in the eye, brain, trunk, and tail at later stages. To inhibit zATM expression and function, we designed and synthesized splice-blocking antisense-morpholino oligonucleotides targeting the phosphoinositide 3-kinase-like domain. We demonstrated that this knockdown of zATM caused abnormal development upon ionizing radiation-induced DNA damage. Our data suggest that the ATM gene is structurally and functionally conserved in vertebrates from zebrafish to human.     

Molecular characterization and SNPs analysis of the porcine Deleted in AZoospermia Like (pDAZL) gene

The Deleted in AZoospermia Like (DAZL) gene is expressed in prenatal and postnatal germ cells. In this study, we cloned and characterized the porcine Deleted in AZoospermia Like (pDAZL) gene. We found the full-length coding sequence of the pDAZL encoded a protein of 295 amino acids with a RNA recognition motif (amino acids 41-111) and a DAZ repeat (amino acids 167-120). The deduced protein sequence of pDAZL is 92.5% and 91.5% similar to those of human and bovine, respectively. PCR-MspI-RFLP and PCR-TaqI-RFLP were established to detect an A/G mutation in intron 7 and a C/A mutation in intron 9, respectively. Associations of two SNPs with litter size traits were assessed in Large White (n=275) and DIV (n=128) pig populations, and the statistical analysis demonstrated that CC produced 0.716 more (P<0.05) piglets born alive than CD genotypes in Large White pigs at TaqI locus (C/A mutation in intron 9), and the dominance effect was 0.304 pig per litter (P<0.05). This result suggests that the pDAZL gene might be a good candidate gene of litter size trait and provides some marker information for marker-assisted selection (MAS).     

棉铃虫酪氨酸羟化酶基因的分子特性及功能分析

【目的】酪氨酸羟化酶(tyrosine hydroxylase, TH)是黑色素形成的关键酶,在昆虫表皮骨化过程中扮演重要角色。本研究旨在获得棉铃虫Helicoverpa armigera TH基因序列,并研究其分子特性、表达模式和功能,为更深入探析该基因作用机理奠定基础。【方法】通过生物信息学和分子生物学技术获得了棉铃虫TH基因序列,利用qRT-PCR分析该基因在棉铃虫不同生长发育阶段的表达模式;利用qRT-PCR技术,分别测定了蜕皮激素20E(400 ng/头)处理不同时间和RNAi成功干扰蜕皮激素受体基因(EcR)前提下再用20E(400 ng/头)处理后,棉铃虫5龄幼虫TH表达量变化;采用生物化学方法检测鞣化激素(30 μg/mg组织)和环腺苷酸(cAMP, 200 ng/mg 组织)处理后棉铃虫幼虫脂肪体中TH活性。【结果】获得了棉铃虫酪氨酸羟化酶基因TH (GenBank登录号: MF440319) cDNA片段,长2 270 bp,开放阅读框1 686 bp,编码561个氨基酸残基。该基因在棉铃虫整个发育期均表达,其中在卵期第3天、2龄幼虫第1天、3-5龄蜕皮期、预蛹期和成虫羽化第1天表达量相对较高。研究还发现,400 ng/头 20E注射能够促进TH的转录;在成功干扰并调低幼虫EcR转录水平的前提条件下(对照仅注射dsGFP)再注射20E,对TH表达量无明显影响;而鞣化激素(30 μg/mg组织)和cAMP(200 ng/mg组织)均显著提高了TH的酶活性。【结论】20E在转录水平参与了TH的表达;鞣化激素和cAMP均能够提高TH活性,在蛋白水平上对TH进行调控。     

Molecular characterization and gene functional analysis of Dicer‐2 gene from Nilaparvata lugens (Hemiptera: Geometroidea)

Abstract Nilaparvata lugens (Stål) (Hemiptera: Geometroidea), a serious rice pest in many countries of Asia, causes a great loss in rice production every year. RNA interference (RNAi) is a powerful technology for gene function study in insects and a potential tool for pest control. As a core component of RNAi pathway, Dicer‐2 (Dcr‐2) protein determines the production of small interfering RNA (siRNA) and is crucial for the efficiency of RNAi. In this study, the full‐length complementary DNA (cDNA) of N. lugens Dcr‐2 (NlDcr‐2) was first cloned and analyzed, and then the RNAi experiment was conducted to explore the function of NlDcr‐2 gene. The complete Dcr‐2 cDNA of N. lugens was 4 971 bp in length with an open reading frame (ORF) of 1,656 amino acids. Phylogenetic and protein domain analysis showed that the predicted NlDcr‐2 protein was similar to Tribolium castaneum. In the RNAi experiment, the messenger RNA level of NlDcr‐2 was significantly reduced by NlDcr‐2 double‐stranded RNA (dsRNA) (dsDcr‐2). Fifty‐five per cent decrease of NlDcr‐2 was found after 4 days of unremitting feeding. No significant effect was observed on the development of N. lugens after dsRNA ingestion.     

Molecular cloning and characterization of the catalytic domain of zebrafish homologue of the ataxia-telangiectasia mutated gene

Inherited biallelic mutations of the ATM (ataxia-telangiectasia mutated) gene in humans cause ataxia-telangiectasia, a rare autosomal recessive disorder associated with progressive neuro-degeneration, cancer predisposition, immunodeficiency, and hypersensitivity to ionizing radiation. The ATM gene is highly conserved across a wide range of species. In an attempt to establish a zebrafish (Danio rerio) model of ataxia-telangiectasia, we cloned the coding sequence of the catalytic domain of the zebrafish homologue of ATM and found it to contain an open reading frame encoding 907 amino acids at the carboxyl terminus of the zebrafish ATM (zATM). The catalytic domain of zATM shares 67% and 66% homology with human ATM (hATM) and mouse ATM (mATM), respectively. The full-length mRNA encoding zATM is found to be approximately 11 kb by Northern hybridization, and the expression of zATM is observed in different adult and embryonic tissues. Overexpression of a kinase-inactive zATM domain in human cells has a dominant-negative effect against hATM function. Expression of the altered zATM in ZF4 cells leads to an A-T-like phenotype in response to ionizing radiation. These results taken together indicate that zATM is the homologue of hATM. Furthermore, using the kinase-inactive form of zATM should allow manipulation of zATM function in fish cells.     

中华稻蝗几丁质脱乙酰基酶1基因的分子特性及功能

【目的】通过研究中华稻蝗Oxya chinensis几丁质脱乙酰基酶1 (chitin deacetylase 1, CDA1)基因的分子特性和生物学功能, 为新型农药靶标筛选提供理论基础。【方法】在中华稻蝗转录组数据库搜索获得几丁质脱乙酰基酶基因片段, 用生物信息学方法对其进行同源比对分析并作聚类关系图; 利用cDNA末端快速扩增（rapid-amplification of cDNA ends, RACE）获得该基因cDNA全长序列; 通过Real-time quantitative PCR(qPCR)方法检测OcCDA1在5龄若虫不同组织部位和不同发育时期的表达情况, 并采用RNAi技术研究OcCDA1在飞蝗生长发育中的功能。【结果】获得中华稻蝗几丁质脱乙酰基酶1基因全长cDNA,命名为OcCDA1（GenBank登录号：KP271171）。OcCDA1在前肠、体壁、后肠和脂肪体组织中高表达, 在5龄第1天表达量显著高于以后几天。RNAi结果显示, 注射该基因的dsRNA后, OcCDA1基因的表达量降低了93.3%。OcCDA1基因沉默后虫体出现进食减少、发育迟缓现象, 总死亡率达到95.2%,其中38.1%的试虫在蜕皮前死亡, 57.1%出现因蜕皮困难而死亡的表型。【结论】OcCDA1在中华稻蝗的发育中起着重要作用, 该基因沉默引起虫体进食减少和蜕皮异常从而导致死亡。     

Molecular structure, expression analysis and functional characterization of APRIL (TNFSF13) gene in bat (Vespertilio superans Thomas)

A proliferation-inducing ligand (APRIL) is a novel member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the full-length cDNA of APRIL (designated bAPRIL) from bat was cloned using RT-PCR and its biological activities have been characterized. The open reading frame (ORF) of this cDNA consists of 753 bases, encoding a protein of 250 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other, known APRIL homologs. Real-time quantitative PCR (qPCR) analysis indicated that bAPRIL mRNA was predominantly expressed in bat lymphoid tissue spleen. The SUMO-bsAPRIL was efficiently expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that bsAPRIL could bind to its receptors on B cells. In vitro, MTT assays indicated that bsAPRIL could promote the survival/proliferation of mouse splenic B cells grown with anti-mouse IgM. These findings indicate that bsAPRIL plays an important role in the survival and proliferation of B cells and has functional cross-reactivity among mammalians. The present findings may provide valuable information for research into the immune system of the bat.     

Molecular cloning and characterization of the chicken pro-opiomelanocortin (POMC) gene.

The gene for pro-opiomelanocortin (POMC), a common precursor of melanocortins, lipotropins and beta-endorphin, was isolated in the chicken first among avian species. The chicken POMC gene was found to be a single copy gene and appeared to show the same structural organization as that of other species of different classes. The predicted POMC displayed the highest identity to Xenopus POMC(A) (60. 1%), and consisted of 251 amino acid residues with nine proteolytic cleavage sites, suggesting that it could be processed to give rise to all members of the melanocortin family, including adrenocorticotropic hormone and alpha-, beta- and gamma-melanocyte-stimulating hormones, as well as the other POMC-derived peptides. RT-PCR analysis detected the POMC mRNA in the brain, adrenal gland, gonads, kidney, uropygial gland and adipose tissues, each of which has been demonstrated to express melanocortin receptors. These results suggest that melanocortins act in a paracrine and/or autocrine manner to control a variety of functions both in the brain and in the peripheral tissues in the chicken.