Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis delta-endotoxin nomenclature committee.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

脑型一氧化氮合成酶的钙调蛋白结合区的表达及活性鉴定

用PCR法克隆出nNOS的CaM结合区基因(nNOS 2455～2988bp),并在大肠杆菌中进行了高效表达。经金属离子螯合亲和层析得到纯度为90％以上的重组蛋白．分子量为22kDa,CaM Oveday assay证实该蛋白具有CaM的结合活性。由于所表达的重组蛋白既具有序列特异性又具有CaM的结合活性．因此。可将它作为筛选nNOS特异性抑制肽的靶蛋白,亦可用于特异性抗体的制备。     

Open reading frames carried on UL/b' are implicated in shedding and horizontal transmission of rhesus cytomegalovirus in rhesus monkeys

Implicit with the use of animal models to test human cytomegalovirus (HCMV) vaccines is the assumption that the viral challenge of vaccinated animals reflects the anticipated virus-host interactions following exposure of vaccinated humans to HCMV. Variables of animal vaccine studies include the route of exposure to and the titer of challenge virus, as well as the genomic coding content of the challenge virus. This study was initiated to provide a better context for conducting vaccine trials with nonhuman primates by determining whether the in vivo phenotype of culture-passaged strains of rhesus cytomegalovirus (RhCMV) is comparable to that of wild-type RhCMV (RhCMV-WT), particularly in relation to the shedding of virus into bodily fluids and the potential for horizontal transmission. Results of this study demonstrate that two strains containing a full-length UL/b' region of the RhCMV genome, which encodes proteins involved in epithelial tropism and immune evasion, were persistently shed in large amounts in bodily fluids and horizontally transmitted, whereas a strain lacking a complete UL/b' region was not shed or transmitted to cagemates. Shedding patterns exhibited by strains encoding a complete UL/b' region were consistent with patterns observed in naturally infected monkeys, the majority of whom persistently shed high levels of virus in saliva for extended periods of time after seroconversion. Frequent viral shedding contributed to a high rate of infection, with RhCMV-infected monkeys transmitting virus to one na?ve animal every 7 weeks after introduction of RhCMV-WT into an uninfected cohort. These results demonstrate that the RhCMV model can be designed to rigorously reflect the challenges facing HCMV vaccine trials, particularly those related to horizontal transmission.     

On-line heat flux measurements improve the culture medium for the growth and productivity of genetically engineered CHO cells

With the increasingly competitive commercial production of target proteins by hybridoma and genetically engineered cells, there is an urgent requirement for biosensors to monitor and control on-line and in real time the growth of cultured cells. Since growth is accompanied by an enthalpy change, heat dissipation measured by calorimetry could act as an index for metabolic flow rate. Recombinant CHO cell suspensions producing interferon-γ were pumped to an on-line flow calorimeter. The results showed that an early reflection of metabolic change is size-specific heat flux obtained from dividing heat flow rate by the capacitance change of the cell suspension, using the on-line probe of a dielectric spectroscope. Comparison of heat flux with glucose and glutamine fluxes indicated that the former most accurately reflected decreased metabolic activity. Possibly this was due to accumulation of lactate and ammonia resulting from catabolic substrates being used as biosynthetic precursors. Thus, the heat flux probe is an ideal on-line biosensor for fed-batch culture. A stoichiometric growth reaction was formulated and data for material and heat fluxes incorporated into it. This showed that cell demand for glucose and glutamine was in the stoichiometric ratio of ∼3:1 rather than the ∼5:1 in the medium. It was demonstrated that the set of stoichiometric coefficients in the reaction were related through the extent of reaction (advancement) to overall metabolic activity (flux). The fact that this approach can be used for medium optimisation is the basis for an amino-acid-enriched medium which improved cell growth while decreasing catabolic fluxes. This revised version was published online in July 2006 with corrections to the Cover Date.     

X射线对仔鼠皮肤组织结构及c-Fos、KGF表达的影响

应用生物显微技术和免疫组织化学方法,研究了不同剂量X射线(0.0、3.5、6.5 Gy)辐射对发育期(1、5、10和20 d)昆明小鼠(Mus musculus)仔鼠皮肤组织结构和c-Fos、KGF表达的影响,探讨了c-Fos、KGF的生物活性作用及调控意义,并利用IPP专业图像分析软件对其表达强度进行定量分析.结果表...     

拟南芥白化突变体心口的基因定位与分析

EMS30是拟南芥经甲基磺酸乙酯（EMS）诱变得到的白化突变体。该突变体的叶绿体结构存在严重缺陷,同时伴随叶绿素缺失。遗传分析显示EMS30突变体的突变表型受隐性单基因控制。采用图位克隆的方法对EMS30突变基因进行定位的结果显示,该基因位于拟南芥第一条染色体的分子标记F21M12和F14N23之间的96kb区间内,该区间包含25个基因。通过生物信息学分析发现,该区间内有3个基因定位在叶绿体或与叶绿体发育相关。这些结果有助于该基因的克隆,为阐释叶绿体发育提供线索。     

氧调节蛋白150在人肝细胞癌中的过度表达及其对凋亡的作用

在前期研究中发现,氧调节蛋白150(ORP150)是与肝细胞癌相关的糖蛋白．进一步研究了ORP150的表达水平与肝细胞癌的相关性．免疫印迹、细胞免疫化学和定量PCR分别在蛋白质水平和mRNA水平检测了ORP150的表达．运用RNA干扰技术检测了其对凋亡和肝细胞癌侵袭性的影响．发现：无论是蛋白质水平还是mRNA水平,与正常肝细胞相比,ORP150在肝细胞癌中表达明显上调;经RNA干扰后,肝细胞癌的凋亡明显增加,但肿瘤细胞的侵袭性无改变．肝细胞癌中,ORP150表达上调,它可能抑制肿瘤细胞的凋亡而促进其生长．ORP150有可能成为肝细胞癌的治疗靶点．