Comparison of the immunogenicities of HIV-1 mutants based on structural modification of env

Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around NAb epitopes and deletions of variable regions in env. The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γ ELISPOT. Overall, five mutants (dWt, M2, M5-2, M5-1 and dM7) induced higher neutralization activities for both pseudoviruses than plasmid Wt, while only two of the mutants (dWt and M5-2) showed significant differences (P<0.05). Two mutants (M2 and dM2) induced more Env-specific T cells than plasmid Wt. Statistically however, significance was only reached for mutant M2. Thus, properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.      

Silencing of HIV-1 Gene Expression by Sirnas in Transduced Cells

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (≥90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490–7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.     

Specific truncations of an acetolactate synthase gene from Brassica napus efficiently complement ilvB/ilvG mutants of Salmonella typhimurium

Summary The expression of an acetolactate synthase (ALS) gene isolated from the cruciferous plant Brassica napus was investigated in Salmonella typhimurium. Using an expression plasmid containing the highly active trc (trp-lac) promoter, several plant ALS constructs were made containing successive in-frame truncations from the 5 end of the coding region. Functional complementation by these plant ALS constructs of a S. typhimurium mutant devoid of ALS enzymic activity was assayed on minimal medium. Truncations which eliminated a large portion of the transit peptide coding sequence proved to act as efficient ALS genes in the bacterial host. Truncations close to the putative processing site of the plant protein were inactive in the complementation test. A full length copy of the gene, including the entire transit peptide coding region, was also inactive. The efficiency of the complementation, estimated by comparison to the growth rate of wild-type S. typhimurium, was found to correlate with levels of ALS activity in the transformed bacteria. Specific mutations, known to produce herbicide resistance in plants, were introduced into the truncated ALS coding sequence by site-directed mutagenesis. When expressed in bacteria these constructs conferred a herbicide resistance phenotype on the host. The potential of this system for mutagenesis and enzymological studies of plant proteins is discussed.     

一种适用于链霉菌异源合成基因簇同框缺失的高效方法

【背景】对抗生素生物合成途径的阐明有助于提高目标化合物的产量并开发具有更高活性的新化合物。基因的同框缺失是天然产物生物合成研究的常规手段,通过分析突变菌株积累的中间产物,可以帮助推导天然产物的合成途径及相关基因的功能。天然产物生物合成基因簇的大小一般在20 kb以上,对每个基因进行同框缺失耗时耗力,因此,优化链霉菌来源的基因同框缺失的方法有重要的意义。【目的】基于PCR-targeting重新设计了一套在链霉菌柯斯文库质粒上进行基因同框缺失的方法,实现链霉菌基因在大肠杆菌中快速、高效的基因同框缺失的技术体系。【方法】使用氨苄青霉素抗性基因bla作为PCR-targeting DNA片段的筛选标记,同时使用体外的Pac I酶切和酶连系统代替体内的Flp/FRT系统来介导同框缺失的构建。【结果】利用这种方法,在6 d内完成了米多霉素生物合成基因簇中14个基因的同框缺失。【结论】此方法与传统的PCR-targeting方法相比,构建同框缺失载体的效率明显提高;Pac I识别序列在链霉菌基因组上的稀有性使得此方法在构建抗生素生物合成基因簇必需基因的同框缺失载体上具有普适性。     

Expression of human immunodeficiency virus genes using baculovirus expression system

The structural protein genes of HIV-1 and HIV-2 have been expressed inSpodoptera frugiperda (SF) cells using baculovirus expression system. The noncoding flanking sequences of HIV structural genes were removed and a putative ribosome binding site was placed in front of the open reading frame of each gene by using crossover linker mutagenesis. The coding sequences of thegag, pol, env, andvif proteins were inserted intoAutographa californica nuclear polyhedrosis virus (AcNPV) so that HIV genes were under the control of the AcNPV polyhedrin promoter. All recombinant AcNPV-infected SF cells express high levels of HIV structural proteins. Detailed strategies of recombinant AcNPV construction for high level protein expression are presented.     

Identification of <Emphasis Type="Italic">Ganoderma</Emphasis>, the causal agent of basal stem rot disease in oil palm using a molecular method

From comparison of the alignments of the internally transcribed spacers (ITS) of ribosomal DNA from Ganoderma associated with oil palm basal stem rot (BSR) and other Ganoderma species, two specific primer pairs were selected to provide a specific DNA amplification of pathogenic Ganoderma in oil palm. Each primer pair produced a single PCR product of about 450 bp (for primer pair IT1–IT2) and 334 bp (for primer pair IT1–IT3) when oil palm Ganoderma DNA was used. No PCR amplification product was observed when other Ganoderma species DNA was used in PCR amplification with these primer pairs. Three specific restriction enzyme sites were identified in the ITS and intergenic spacer (IGS1) regions. The restriction enzymes MluI, SacI and HinfI were used to digest the ITS-PCR product and restriction enzymes TfiI, ScaI and HincII were used to digest the IGS1-PCR product. Of the three restriction enzymes used in each rDNA region, MluI specifically digested the ITS regions, and TfiI specifically digested the IGS1 region of oil palm Ganoderma. Analysis of the published ITS nucleotide sequences of 31 Ganoderma species showed that the MluI restriction site was not present in other Ganoderma species. The use of both specific primers and restriction enzyme analysis can be applied as a standard protocol to identify pathogenic Ganoderma in oil palm. In this study, the use of specific primers and PCR-RFLP analyses of the rDNA gave consistent results for the characterisation of pathogenic Ganoderma, and indicated that Ganoderma strains associated with BSR disease in oil palms belong to a single species.     

Genes for phaseolotoxin synthesis are located on the chromosome ofPseudomonas syringae pv.phaseolicola

Transposon mutagenesis was used to locate the genes for phaseolotoxin biosynthesis inPseudomonas syringae pathovar.phaseolicola. Mutants unable to produce toxin were obtained that carried Tn5 on different chromosomal restriction fragments. None of the Tn5-induced nontoxigenic mutants carried the transposon in plasmid DNA. The insertion of Tn5 intotox DNA was confirmed by site-directed mutagenesis. The results reported here suggest the involvement of at least five chromosomal genes in phaseolotoxin biosynthesis. All of the toxinminus mutants retained both full pathogenicity on beans and resistance to the toxin.     

The paradox of viable <Emphasis Type="Italic">sup45</Emphasis> STOP mutations: a necessary equilibrium between translational readthrough,activity and stability of the protein

The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q → TAA, 242R → TGA, 317L → TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells.     

Genetic inactivation of the psaB gene in Synechocystis sp. PCC 6803 disrupts assembly of photosystem I

The reaction center of photosystem (PS) I is comprised of a heterodimer of homologous polypeptides, PsaA and PsaB. In order to investigate the biogenesis of PS I, the psaB gene was inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis 6803. This mutation resulted in disruption of stable PS I assembly, but PS II assembled normally. Expression of the psaA gene was not affected by the mutation, but PsaA protein was not detected, indicating that stable PsaA homodimers did not form. The ability to inactivate psaB makes it a viable target for site-directed mutagenesis.     

Key Features of Cereal Genome Organization as Revealed by the Use of Cytosine Methylation-Sensitive Restriction Endonucleases

Unlike mammalian genomes, cereal (Gramineae) genomes exhibit little suppression of CpG dinucleotides. In cereal genomes, however, most of the numerous potential recognition sites for CpG methylation-sensitive restriction enzymes are methylated. Analysis of cereal genomic libraries and of regions flanking genes indicates that unmethylated NotI sites are useful landmarks for regions containing genes/single-copy sequences. Studies of a rye chromosome arm indicate that its pericentromeric region has a reduced density of unmethylated NotI (and MluI) sites and therefore of genes. Unmethylated MluI and NruI sites are distributed nonrandomly in the genomes of wheat, barley, and rice. Analysis of the genomic blocks defined by these sites in wheat and barley indicates that they are most likely to have arisen by amplification. These observations form the basis of a proposed model for the organization and evolution of the wheat, barley, and rice genomes.     

野生鸟类分离的多重耐药肺炎克雷伯氏菌(Klebsiella pneumoniae)耐药基因分子进化特征研究

【目的】调查野生鸟类携带菌的耐药状况,探索其在细菌耐药性传播过程中的作用。【方法】从野生鸟类石鸡、绯胸鹦鹉、太阳锥尾鹦鹉和黑领椋鸟的新鲜粪便分离4株Klebsiella pneumoniae,采用微量肉汤稀释法评估其多重耐药表型,并利用全基因组测序技术和细菌全因组关联分析、比较基因组学方法对分离株进行分子溯源,系统解析其携带的多重耐药质粒或基因与其宿主、同源质粒间的关联。【结果】4株肺炎克雷伯菌的耐药谱各不相同,来自石鸡样本的分离株S90-2对9种药物耐受,绯胸鹦鹉样本分离株S141对3种药物耐受,太阳锥尾鹦鹉分离株M911-1仅耐受氨苄西林,黑领椋鸟的样本分离株S130-1对所使用的14种药物完全敏感。S90-2属于ST629型,携带bla_CTX-M-14、fosA6、aac(3)-Iid和bla_SHV-11为主的30个耐药基因和携带1个耐药性质粒pS90-2.3 (IncR型)。S141属于ST1662型,携带fosA5、bla_SHV-217等27个耐药基因,1个质粒pS141.1 [IncFIB(K)(pCAV1099-114)/repB型]仅携带耐药基因adeF。M911-1为新ST类型,携带bla_SHV-1、fosA6等共计27个耐药基因,其质粒pM911-1.1携带了3个耐药基因。S130-1属于ST3753型,携带bla_SHV-11、fosA6等27个耐药基因,pS130-1 [IncFIB(K)型]则仅携带一个耐药基因tet(A)。质粒比对表明,质粒pS90-2.3携带的耐药基因片段源自不同的肠杆菌科菌株染色体或质粒。pS90-2.3的同源质粒主要来自人类宿主菌,且主要在中国分布,这些质粒主要细菌宿主为K. pneumoniae和Escherichia coli,且ST11型K. pneumoniae分离株为重要宿主菌。【结论】本研究中来自野生鸟类的多重耐药K. pneumoniae,其耐药基因主要来自质粒,质粒耐药基因主要由转座子、插入序列、整合子和前噬菌体等可移动元件介导,这些多重耐药质粒与人类的宿主菌密切相关。     

Isolation and physical characterization of streptomycete plasmids

Summary Covalently closed circular DNA was isolated from a strain of Streptomyces coelicolor ATCC 10147 and from a strain of Streptomyces coelicolor subspecies flavus ATCC 19894, using two different methods. The two plasmids were of uniform monomer size: 8.9 kb for pS 10147, the plasmid from S. coelicolor ATCC 10147, and around 125 kb for the plasmid from S. coelicolor ATCC 19894.A restriction enzyme map was constructed for pS 10147, using seven enzymes. Four of the enzymes, (BamHI, Bgl,II, PvuII, and XhoI) cut pS 10147 once while PstI made two cuts. The GC content of this plasmid was calculated to be 72%. The possible utilisation of pS 10147 as a cloning vector in Streptomyces is discussed.     

Organization and regulation of the conjugation genes of IncI1 plasmid ColIb-P9

The IncI₁ plasmid ColIb-P9 is among a group of related plasmids that encode the I₁ type of conjugation system. The I₁ system is known to include two morphologically distinct types of pilus, a DNA primase gene (sog) and an exclusion determinant (exc). Transposon mutagenesis and analysis of cloned fragments of ColIb were used to identify the location of these determinants with respect to an EcoRI restriction map. Also identified were the location of the origin of transfer (oriT) and a gene determining an EDTA-resistant nuclease, which is coordinately regulated with the transfer genes. The results indicate that the ColIb tra genes are separated into at least three Tra regions. The pleiotropic nature of transposon insertion mutations in two of these regions suggests that two positive regulators are required for expression of the transfer genes and evidence is also found for a trans-acting repressor. It is suggested that the I₁ conjugation system may have evolved following fusion of two distinct types of conjugative plasmid.