Sequence and genetic organization of the 19.3-kb erythromycin- and dalfopristin-resistance plasmid pLME300 from Lactobacillus fermentum ROT1

Lactobacillus fermentum ROT1 was isolated from a raw milk dairy product. It is resistant to novobiocin, tetracycline, erythromycin and dalfopristin. A chromosomal tetracycline-resistance determinant was identified as tetM. A 19,398-bp plasmid (pLME300), present in several erythromycin-resistant strains of Lb. fermentum, was isolated from strain ROT1 and completely sequenced. Based on putative open reading frames, pLME300 contains at least four different functional regions. In region I, ORF1 shows high homologies to replication proteins of different theta-replicating plasmids. In addition, a tandem repeat of a 22-bp sequence appears 4.5 times. In region II, ORF3 may code for a methylase, and ORF4 has homologies to Mrr restriction system proteins of Deinococcus radiodurans and Escherichia coli suggesting a restriction-modification system. Region III harbours antibiotic-resistance genes, coding for a macrolide-lincosamide-streptogramin B (MLS) methylase Erm(LF) and the streptogramin A acetyltransferase Vat(E), which is identical to Vat(E) from Enterococcus faecium. Furthermore, region III shows a 91% nucleotide sequence identity to an erm-vat linkage of E. faecium. Region IV carries ORFs that appear to be involved in plasmid mobilization as characterized by a putative origin of transfer and a mobilization protein. pLME300 is the largest completely sequenced multi-resistance plasmid isolated from any Lactobacillus strain so far.     

小麦胁迫相关基因W1的克隆及表达模式分析

应用噬菌体原位杂交技术从干旱胁迫诱导的小麦cDNA文库中克隆到一个胁迫诱导的基因片段别。删全长cDNA为901bp,其中,编码区长498bp,编码166个氨基酸。Southern杂交表明,W1是一个低拷贝基因。RT—PCR结果表明,W1受干旱、低温的诱导,但不受高盐的诱导。氨基酸序列分析发现W1有一个USP保守区（pfam00582）。同源性分析发现W1与一个水稻胁迫诱导蛋白（NM_001061239）的同源性为83％,但该类蛋白的功能尚无报道。肼是小麦第1个被克隆的胁迫相关蛋白基因,该基因的克隆有助于阐明小麦的抗逆机制,并为今后培育抗逆性小麦品种提供候选基因。     

A diurnally regulated dehydrin from Avicennia marina that shows nucleo-cytoplasmic localization and is phosphorylated by Casein kinase II in vitro

Dehydrins have a key role in protecting plants from dehydration stress. We report here the isolation of two cDNAs coding for the same dehydrin, AmDHN1 and AmDHN1a from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening. AmDHN1 was found to contain a retained intron that was absent in AmDHN1a. AmDHN1 expression in the context of various environmental stresses was investigated. In leaves, AmDHN1 shows a diurnal pattern of regulation and is induced only by mannitol application. In roots, AmDHN1 is rapidly induced by salinity (NaCl) and dehydration stress (PEG and mannitol). A fragment of 795 bp corresponding to the 5′ upstream region of AmDHN1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative stress regulatory elements (ABRE, DRE, MYB and MYC binding sequences). Putative phosphorylation sites for Casein kinase II were identified in the AmDHN1a ORF. In vitro phosphorylation of Escherichia coli expressed Trx-AmDHN1a by Casein kinase II was observed that was reversed by Shrimp Alkaline Phosphatase treatment. A putative nuclear targeting domain was identified in the translated AmDHN1a ORF and stably transformed AmDHNIa-GFP was found to show nucleo-cytoplasmic localization in tobacco guard cells. As observed for maize Rab17, the phosphorylation of AmDHN1a may contribute to its nuclear localization.     

高山离子芥CBF/DREB1转录因子基因的分离与表达

以冷胁迫和脱水处理的高山离子芥幼叶为材料,采用RT-PCR技术获得1条新的C重复/脱水应答元件结合因子(CBF/DREB1)基因(CbCBF,登录号AY994127).该基因含651 bp的开放阅读框(ORF),编码216个氨基酸的蛋白质,预测该蛋白具有一个AP2 DAN结合域,一个核定位信号和碳端酸性激活域.多序列比对结果表明,CbCBF蛋白与拟南芥及其他植物CBF具有较高的相似性.Northern杂交结果显示,CbCBF基因不能被冷处理诱导表达,但可被脱水和ABA处理快速诱导;同时发现CbCBF基因也能被紫外辐射和机械刺激诱导表达.表明高山离子芥CbCBF基因可能参与应答多种非生物胁迫过程.     

Complete Sequence of Plasmid pMP1 from the Marine Environmental Vibrio vulnificus and Location of Its Replication Origin

A novel cryptic plasmid, pMP1, from an environmental Vibrio vulnificus MP-4 isolated from Mai Po Nature Reserve in Hong Kong, has been characterized. The 7.6-kb plasmid had guanine–cytosine content of 40.03% and encoded four open reading frames (ORFs) with >100 amino acids. The predicted protein of ORF1 contained 478 amino acids showing 29% identity and 50% similarity over 309 amino acids to the integrase of Vibrio cholerae phage VP2. ORF2 encoded a putative protein of 596 amino acids, which were 23% identity and 42% similarity over 455 amino acids to the tail tape measure protein TP901 of Chromohalobacter salexigens phage. ORF3 and ORF4 encoded putative proteins of 103 and 287 amino acids, respectively, but showed no homologies to any known proteins. Further experiments indicated that a 3.2-kb fragment from EcoRI digestion could self-replicate. Analysis indicated that a sequence upstream of ORF4 had the features characteristic of theta-type replicons: AT-rich region, six potential direct repeats (iterons) spaced approximately two DNA helical turn apart (about 23 bp), two copies of 9 bp dnaA boxes, three Dam methylation sites, and five inverted repeats. Complementation experiments confirmed that the protein encoded by ORF4 was required for plasmid replication. We propose that ORF4 encode a new type of Rep protein and pMP1 is a new type of theta plasmid.     

The marker SCK13<Subscript>603</Subscript> associated with resistance to ascochyta blight in chickpea is located in a region of a putative retrotransposon

The sequence characterized amplified region (SCAR) marker SCK13(603), associated with ascochyta blight resistance in a chickpea recombinant inbred line (RIL) population, was used as anchored sequence for genome walking. The PCRs performed in the walking steps to walk in the same direction produced eight bands in 5' direction and five bands in 3' direction with a length ranking from 530 to 2,871 bp. The assembly of the bands sequences along with the sequence of SCK13(603) resulted in 7,815 bp contig. Blastn analyses showed stretches of DNA sequence mainly distributed from the nucleotides 1,500 to 4,500 significantly similar to Medicago truncatula genomic DNA. Three open reading frames (ORFs) were identified and blastp analysis of predicted amino acids sequences revealed that ORF1, ORF2 and ORF3 had significant similarity to a CCHC zinc finger protein, to an integrase, and to a precursor of the glucoamylase s1/s2, respectively, from M. truncatula. The high homology of the putative proteins derived from ORF1 and ORF2 with retrotransposon proteins and the prediction of the existence of conserved domains usually present in retrotransposon proteins indicate that the marker SCK13(603) is located in a region of a putative retrotransposon. The information generated in this study has contributed to increase the knowledge of this important region for blight resistance in chickpea.     

Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains.

The replicase open reading frame lb (ORF1b) protein of equine arteritis virus (EAV) is expressed from the viral genome as an ORF1ab fusion protein (345 kDa) by ribosomal frameshifting. Processing of the ORF1b polyprotein was predicted to be mediated by the nsp4 serine protease, the main EAV protease. Several putative cleavage sites for this protease were detected in the ORF1b polyprotein. On the basis of this tentative processing scheme, peptides were selected to raise rabbit antisera that were used to study the processing of the EAV replicase ORF1b polyprotein (158 kDa). In immunoprecipitation and immunoblotting experiments, processing products of 80, 50, 26, and 12 kDa were detected. Of these, the 80-kDa and the 50-kDa proteins contain the putative viral polymerase and helicase domains, respectively. Together, the four cleavage products probably cover the entire ORF1b-encoded region of the EAV replicase, thereby representing the first complete processing scheme of a coronaviruslike ORF1b polyprotein. Pulse-chase analysis revealed that processing of the ORF1b polyprotein is slow and that several large precursor proteins containing both ORF1a- and ORF1b-encoded regions are generated. The localization of ORF1b-specific proteins in the infected cell was studied by immunofluorescence. A perinuclear staining was observed, which suggests association with a membranous compartment.     

小麦盐胁迫相关基因的克隆与表达分析

采用RT-PCR方法,从小麦中克隆获得1个盐诱导小麦MYB类转录因子基因TaSIM（Triticum aestivum salt-induced MYB）,该基因cDNA全长1 213bp,具有1个831bp的开放阅读框,编码276个氨基酸,预测分子量约为29.903kD,等电点为10.12,推测的氨基酸序列中含有2个高度保守的SANT结构域。系统发生树分析表明,TaSIM与二穗短柄草XP₀03576185亲缘关系最近。半定量RT-PCR检测结果显示,TaSIM基因受盐胁迫诱导表达。亚细胞定位结果显示,TaSIM-hGFP融合蛋白定位于细胞核中。研究结果表明,小麦TaSIM基因编码的蛋白可能在细胞核内参与小麦对盐胁迫的应答反应。     

Nucleotide sequence of a putative peroxisomal protein from the yeast Lipomyces kononenkoae

Abstract A cDNA corresponding to a putative peroxisomal protein from the yeast Lopomyces kononenkoae has been isolated. It contains an ORF of 498 bp that predicts a polypeptide of 166 amino acids with an apparent molecular mass of 17365 Da. The protein shares a high degree of homology with proteins associated to the peroximal membrane of Candida boidinii .     

Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.