Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the M_r-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.     

Mass treatment with ivermectin for filariasis control in Papua New Guinea: impact on mosquito survival

Field studies were carried out to determine the impact of mass human treatment with ivermectin on the survival of anthropophagic mosquitoes of the Anopheles punctulatus complex (Diptera: Culicidae), the vectors of lymphatic filariasis and malaria in Papua New Guinea. In a village where mass treatment had been given, using 400 microg/kg ivermectin plus 6 mg/kg diethylcarbamazine citrate (DEC), we performed pre- and post-treatment collections of freshly blood-engorged mosquitoes from the same nine bedrooms. All blood-fed mosquitoes collected less than 4 days after mass treatment died within 9 days, whereas 67% of those collected before treatment survived for >9 days. Comparison (using the log-rank test) of the survival curves for mosquitoes collected (i) before treatment, (ii)<4 days after treatment, and (iii) 28 days after treatment, showed the survival rate of group (ii) to be significantly lower than the other two (chi2=176, df=2, P<0.0001). Pre- and post-treatment all-night landing catches showed no reduction in human biting rates in the experimental village. In another village, where people were mass treated with ivermectin (400 microg/kg) only, the survival rates of freshly blood-engorged An. punctulatus collected from bedroom resting-sites less than 1 day after treatment, were compared to similar collections carried out at the same time in a nearby village where people were not treated with ivermectin. The 48-h survival rate for the ivermectin-treated village was 31% compared to 94% for the other; this difference was highly significant (chi2=32.42, df=1, P<0.0001). Mosquitoes fed 2 months post-treatment with DEC or collected 38 days post-treatment with ivermectin had normal survival rates. We conclude that the duration of the systemic lethal effect of ivermectin on mosquitoes is insufficient to be of epidemiological significance in filariasis control programmes that are based on biannual and annual single-dose treatments, but might reduce vectorial capacity sufficiently to block epidemics of dengue or even malaria.     

Dolichos yellow mosaic virus belongs to a distinct lineage of Old World begomoviruses; its biological and molecular properties

Dolichos yellow mosaic disease (DYMD) affects the production of dolichos in South Asia. Diseased plants produce characteristic bright yellow mosaic patches on the leaves and early infections cause reductions in yield. The putative dolichos yellow mosaic virus (DoYMV) was transmitted poorly (maximum 18.3% transmission) by the whitefly, Bemisia tabaci. DoYMV has a narrow host range and infected only Lablab purpureus and L. purpureus var. typicum out of the 36 species tested. Virus was detected using monoclonal antibodies in a triple‐antibody sandwich enzyme‐linked immunosorbent assay and by PCR. Complete DNA‐A components of DoYMV isolates from Mysore and Bangalore, South India, were sequenced, but several attempts to identify DNA‐B and DNA‐β were unsuccessful. DoYMV isolates shared DNA‐A nucleotide identities of 92.5–95.3% with previously described isolates from North India and Bangladesh. They were most similar to mungbean‐infecting begomoviruses at 61.6–64.4% of DNA‐A nucleotide identities. Phylogenetic analyses of DNA‐A sequences grouped the dolichos‐infecting and mungbean‐infecting begomoviruses into a distinct cluster away from begomoviruses infecting non‐leguminous plants in the Indian subcontinent. Antigenically, legume‐infecting begomoviruses were most similar to each other compared with non‐legume viruses. Collectively, these results indicate that legume‐infecting begomoviruses in the Indian subcontinent belonged to a distinct lineage of Old World begomoviruses.     

Escherichia coli engineered to produce eicosapentaenoic acid becomes resistant against oxidative damages

The colony-forming ability of Escherichia coli genetically engineered to produce eicosapentaenoic acid (EPA) grown in 3mM hydrogen peroxide (H(2)O(2)) was similar to that of untreated cells. It was rapidly lost in the absence of EPA. H(2)O(2)-induced protein carbonylation was enhanced in cells lacking EPA. The fatty acid composition of the transformants was unaffected by H(2)O(2) treatment, but the amount of fatty acids decreased in cultures of cells lacking EPA and increased in cultures of cells producing EPA, suggesting that cellular EPA is stable in the presence of H(2)O(2) in vivo and may protect cells directly against oxidative damage. We discuss the possible role of EPA in partially blocking the penetration of H(2)O(2) into cells through membranes containing EPA.     

VPMCD: variable interaction modeling approach for class discrimination in biological systems

Data classification algorithms applied for class prediction in computational biology literature are data specific and have shown varying degrees of performance. Different classes cannot be distinguished solely based on interclass distances or decision boundaries. We propose that inter-relations among the features be exploited for separating observations into specific classes. A new variable predictive model based class discrimination (VPMCD) method is described here. Three well established and proven data sets of varying statistical and biological significance are utilized as benchmark. The performance of the new method is compared with advanced classification algorithms. The new method performs better during different tests and shows higher stability and robustness. The VPMCD is observed to be a potentially strong classification approach and can be effectively extended to other data mining applications involving biological systems.     

逆转录病毒载体介导的RNA干扰稳定抑制肌肉生长抑制素GDF-8的表达

为将肌肉生长抑制素的干扰序列表达盒hU6-siGDF-8有效导入肌成纤维细胞C2C12中, 以pAV-hU6 + 27载体为基础构建逆转录病毒载体pXSN-hU6-siGDF-8, 并使之与pVSV-G质粒共转染GP-293细胞, 用包装出的病毒粒子感染宿主细胞C2C12, G418筛选稳定整合逆转录病毒的抗性细胞库。2周后, Western Blotting和Real-Time PCR分析结果显示, 细胞内源性的GDF-8基因的表达得到了有效的抑制; MTT法和细胞流式仪分析表明, G418抗性细胞得到了更有效的增殖, 并且G0/G1期细胞数量减少了13.7%, S期细胞数量增加了14.9%。因此, 逆转录病毒载体的RNA干扰系统可以稳定抑制 GDF-8基因表达, 它将成为治疗肌肉萎缩疾病的一个强有力的工具。     

小麦叶绿体基因组定点整合表达载体的构建

从小麦中分别克隆了包括rbcL基因3′端部分和完整的psaI、ycf4基因的DNA片段.利用克隆到的DNA片段作为同源重组片段、烟草叶绿体16S rRNA基因的启动子Prrn和PsbA基因的终止子PsbA3′控制筛选标记基因aadA和报告基因gfp的转录,构建了小麦叶绿体基因组定点整合表达载体pRAGY. 用该载体转化大肠杆菌,在激光扫描共聚焦显微镜下,检测到了gfp基因成功表达的产物被激发出的强烈的绿色荧光.     

A transgenic plant cell‐suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles

We describe a novel strategy to produce vaccine antigens using a plant cell‐suspension culture system in lieu of the conventional bacterial or animal cell‐culture systems. We generated transgenic cell‐suspension cultures from Nicotiana benthamiana leaves carrying wild‐type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot‐and‐mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co‐expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large‐scale production of immunopeptide vaccines in a cost‐effective manner using a plant cell‐suspension culture system.     

鲑鱼生长激素基因分泌型表达质粒的构建

生长激素(GH)是动物垂体前叶分泌的一种多肽类激素.应用分子重组及PCR等技术,构建了一种鲑鱼生长激素基因分泌型表达质粒pOsGH153,使编码鲑鱼生长激素成熟肽的序列克隆在大肠杆菌分泌型表达载体PIN-Ⅲ-ompA内,直接位于编码大肠杆菌外膜蛋白A信号肽序列的下游,在Lpp-Lac杂合启动子控制下,经IPTG诱导,分子量约23 000的鲑鱼生长激素在大肠杆菌中获得高效表达,该产物具有天然鲑鱼生长激素的免疫活性,直接分泌到细胞周质,而信号肽被自动剪除.     

Electrotransformation of Streptococcus agalactiae with plasmid DNA

Abstract A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli - Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-μl aliquot containing about 5×10⁹ colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 μg. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2×10⁴ cfu μg⁻¹. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae .