Sequence and organization of the Neodiprion lecontei nucleopolyhedrovirus genome

All fully sequenced baculovirus genomes, with the exception of the dipteran Culex nigripalpus nucleopolyhedrovirus (CuniNPV), have previously been from Lepidoptera. This study reports the sequencing and characterization of a hymenopteran baculovirus, Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), from the redheaded pine sawfly. NeleNPV has the smallest genome so far published (81,755 bp) and has a GC content of only 33.3%. It contains 89 potential open reading frames, 43 with baculovirus homologues, 6 identified by conserved domains, and 1 with homology to a densovirus structural protein. Average amino acid identity of homologues ranged from 19.7% with CuniNPV to 24.9% with Spodoptera exigua nucleopolyhedrovirus. The conserved set of baculovirus genes has dropped to 29, since NeleNPV lacks an F protein homologue (ac23/ld130). NeleNPV contains 12 conserved lepidopteran baculovirus genes, including that for DNA binding protein, late expression factor 11 (lef-11), polyhedrin, occlusion derived virus envelope protein-18 (odv-e18), p40, and p45, but lacks 21 others, including lef-3, me53, immediate early gene-1, lef-6, pp31, odv-e66, few polyhedra 25k, odv-e25, protein kinase-1, fibroblast growth factor, and ubiquitin. The lack of identified baculovirus homologues may be due to difficulties in identification, differences in host-virus interactions, or other genes performing similar functions. Gene parity plots showed limited colinearity of NeleNPV with other baculoviruses, and phylogenetic analysis indicates that NeleNPV may have existed before the lepidopteran nucleopolyhedrovirus and granulovirus divergence. The creation of two new Baculoviridae genera to fit hymenopteran and dipteran baculoviruses may be necessary.     

Proteins associated with Culex nigripalpus nucleopolyhedrovirus occluded virions

Occlusion-derived virions (ODVs) of the nucleopolyhedrovirus of Culex nigripalpus (CuniNPV) were purified by Ludox density gradient ultracentrifugation, and the proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were identified by using Edman sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry, nanoelectrospray quadrupole time-of-flight mass spectrometry, or a combination of these methods. Half of the 44 polypeptide sequences identified in this analysis were unique open reading frames (ORFs) encoded by the CuniNPV genome and did not show similarity to any other sequences present in protein databases. Of the 22 polypeptides that showed similarities to other baculovirus-encoded proteins, only 17 sequences have previously been identified as structural proteins. The newly identified CuniNPV structural proteins cun058, cun059, cun087, cun106, and cun109 are homologues of Autographa californica nucleopolyhedrovirus (AcMNPV) ORFs 68, 62, 98, 81, and 2, respectively. The products of four genes, namely, lef-1 (cun045), alkaline exonuclease (cun054), helicase (cun089), and DNA polymerase (cun091), were not detected in the CuniNPV ODV preparations. These four genes are conserved among all annotated baculovirus genomes, and their homologues have been detected in the ODV of AcMNPV.     

小麦叶绿体信号识别颗粒54基因的克隆与分析

根据LWSRC336(GenBank登录号为EV254029)的cDNA序列设计引物,从受条锈菌(Puccinia striifor-misf.sp.tritici)诱导的小麦幼叶中提取总RNA,采用RACE与RT-PCR相结合的技术对该基因克隆.测序结果表明,扩增片段长度为1 725 bp,其中包含一个编码481个氨基酸的开放阅读框,与水稻的叶绿体信号识别颗粒54蛋白高度同源.实时荧光定量PCR分析结果显示,该基因在受条锈菌诱导下,在亲和组合中表达趋势明显下调,而在非亲合组合中的表达在24 h之前呈下降趋势,到24 h最低,在72 h又恢复到正常水平,随后又下降.推测条锈菌侵染小麦后,影响了叶绿体蛋白的运输,进而影响了小麦的光合作用.     

A root acyl carrier protein-II from spinach is also expressed in leaves and seeds

During the synthesis of fatty acids and their utilization in plastids, fatty acyl moieties are linked to acyl carrier protein (ACP). In contrast to previously cloned organ-specific ACP isoforms, we have now isolated a cDNA clone for a potentially constitutive ACP isoform from a spinach root library. Identity between the amino acid sequence encoded by this cDNA and N-terminal sequence data for ACP-II protein from spinach leaf indicates that the root cDNA encodes ACP-II. The deduced amino acid sequence for ACP-II shows 62% identity with spinach leaf ACP-I. Southern analysis suggests that multiple ACP genes or pseudogenes occur in the spinach genome. High-stringency northern blot analysis and RNase protection studies confirm that, within the region encoding the mature ACP-II, the cloned ACP sequence is expressed in leaves and seeds as well as in roots. Quantitative RNase protection data indicate that the ratio of ACP-I and ACP-II mRNA sequences in leaf is similar to the ratio of the two proteins.     

The RNase PD2 gene of almond (Prunus dulcis) represents an evolutionarily distinct class of S-like RNase genes

A cDNA for an S-like RNase (RNase PD2) has been isolated from a pistil cDNA library of Prunus dulcis cv. Ferragnés. The cDNA encodes an acidic protein of 226 amino acid residues with a molecular weight of 25 kDa. A potential N-glycosylation site is present at the N-terminus in RNase PD2. A signal peptide of 23 amino acid residues and a transmembrane domain are predicted. The two active-site histidines present in enzymes of the T2/S RNase superfamily were detected in RNase PD2. Its amino acid sequence shows 71.2% similarity to RNS1 of Arabidopsis and RNase T2 of chickpea, respectively. Northern blotting and RT-PCR analyses indicate that PD2 is expressed predominantly in petals, pistils of open flowers and leaves of the almond tree. Analyses of shoots cultured in vitro suggested that the expression of RNase PD2 is associated with phosphate starvation. Southern analysis detected two sequences related to RNase PD2 in the P. dulcis genome. RFLP analysis showed that S-like RNase genes are polymorphic in different almond cultivars. The PD2 gene sequence was amplified by PCR and two introns were shown to interrupt the coding region. Based on sequence analysis, we have defined three classes of S-like RNase genes, with the PD2 RNase gene representing a distinct class. The significance of the structural divergence of S-like RNase genes is further discussed. Received: 24 January 2000 / Accepted: 17 March 2000     

Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri

Catalase is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by degradation of hydrogen peroxide to oxygen and water. The full-length catalase cDNA of Zhikong scallop Chlamys farreri (denoted as CfCAT) was identified from hemocytes by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of CfCAT cDNA consisted of 3146bp with a 5' UTR of 103bp, an unusually long 3' UTR of 1519bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1521bp encoding a polypeptide of 507 amino acids with predicted molecular weight of 57.5kDa. The deduced amino acid sequence of CfCAT has significant homology to catalases from animals, plants and bacteria. Several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of His(72), Asn(145) and Tyr(355) were identified in the deduced amino acid sequence of CfCAT. The CfCAT was demonstrated to be a peroxisomal glycoprotein with two potential glycosylation sites and a peroxisome targeting signal of ANL that was consistent with human, mouse and rat catalases. The time-course expression of CfCAT in hemocytes was measured by quantitative real-time PCR. The expression of CfCAT increased gradually and reached the highest point at 12h post-Vibrio infection, then recovered to the original level at 24h. All these results indicate that CfCAT, a constitutive and inducible protein, is a member of the catalase family and is involved in the process against ROS in scallop.     

肺形侧耳变温结实相关基因Ppcsl-1的克隆及功能预测

CSL（CBF1/RBP-Jκ/suppressor of hairless/LAG-1）转录因子家族在真菌发育和细胞分化过程中扮演重要角色。前期研究已构建了肺形侧耳变温结实相关消减杂交文库,并从中筛选到一个代表csl基因部分序列的EST。通过TAIL PCR（thermal asymmetric interlaced PCR）技术克隆了该基因（Pleurotus pulmonarius csl-1,简写为 Ppcsl-1）,并利用RACE（rapid-amplification of cDNA ends）技术获得该基因的cDNA全长。Ppcsl-1 cDNA全长2 991bp,编码一个996个氨基酸组成的蛋白（命名为PpCSL-1）。进化分析显示在担子菌CSL中,PpCSL-1与糙皮侧耳Pleurotus ostreatus CSL（PoCSL）亲缘关系最近。荧光定量检测结果表明Ppcsl-1在菌丝经过5℃ 12h冷处理之后的表达量最高,表明其有可能被冷刺激诱导表达并在开启子实体形成的过程中起重要作用。