mr1e, a conotoxin from Conus marmoreus with a novel disulfide pattern

Conotoxins are well known for their highly variable structures and functions. Here we report the identification of a novel conotoxin named mr1e from Conus marmoreus . mr1e is composed of 11 amino acid residues cross-linked by two disulfide bonds (CCHSSWCKHLC). The spacing of intercysteine loops in mr1e is exactly the same as that in α4/3 conotoxins. However, the native mr1e peptide co-eluted on reverse-phase HPLC with the regioselectively synthesized ribbon disulfide linkage isomer (C¹-C⁴, C²-C³) but not the globular linkage isomer (C¹-C³, C²-C⁴). Although this peptide has the same disulfide connectivity as the χ-conotoxins, their sequences do not share significant homology. Thus, mr1e could be defined as a novel conotoxin family. By intracranial injection into mice, mr1e showed an excitatory effect. The characterization of mr1e certainly enriches our understanding of conotoxins, and also opens an avenue for further structural and functional investigation.     

Chaperone Caf1M Stabilizes Hybrid Proteins Containing Sequences of F1 Antigen Subunit from Yersinia pestis

The Yersinia pestis(causative agent of plague) capsule antigen is a homopolymer of Caf1 protein. Export of the subunits is mediated by the periplasmic chaperone Caf1M. To study the mechanism of Caf1M activity, two hybrid genes including coding sequences for the Caf1 signal peptide, human granulocyte–macrophage colony-stimulating factor (GM-CSF) or interleukin-1 (IL-1) receptor antagonist, and mature Caf1 were constructed and expressed in Escherichia coli.We have shown that in the absence of Caf1M the majority of Caf1 moieties within the hybrid proteins undergo proteolysis in the periplasmic space, presumably by the DegP protease. The coexpression of a gene for chaperone Caf1M significantly increased the amount of full-size hybrid proteins in the periplasm, probably as a result of stabilization of the subunit's spatial structure within the hybrid. This effect was not observed in JCB571 cells, which lack periplasmic disulfide isomerase DsbA, essential for Caf1M activity.     

Differences in gene expression profile among SH-SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor

Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF-induced neurite-positive (NIN+) subclones that showed high responsiveness to NGF-induced neurite outgrowth and three NGF-induced neurite-negative (NIN-) subclones that abolished NGF-induced neurite outgrowth from parental SH-SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506-mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF-induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF-induced increases in mRNAs for the immediate-early genes (IEGs) c-fos and NGF inducible (NGFI) genes NGFI-A, NGFI-B and NGFI-C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN- cell line, particularly in terms of axonal vesicle-related genes and growth cone guidance-related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN- cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH-SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.     

Characterization of prion susceptibility in Neuro2a mouse neuroblastoma cell subclones

In this study, we established Neuro2a (N2a) neuroblastoma subclones and characterized their susceptibility to prion infection. The N2a cells were treated with brain homogenates from mice infected with mouse prion strain Chandler. Of 31 N2a subclones, 19 were susceptible to prion as those cells became positive for abnormal isoform of prion protein (PrP(Sc)) for up to 9 serial passages, and the remaining 12 subclones were classified as unsusceptible. The susceptible N2a subclones expressed cellular prion protein (PrP(C)) at levels similar to the parental N2a cells. In contrast, there was a variation in PrP(C) expression in unsusceptible N2a subclones. For example, subclone N2a-1 expressed PrP(C) at the same level as the parental N2a cells and prion-susceptible subclones, whereas subclone N2a-24 expressed much lower levels of PrP mRNA and PrP(C) than the parental N2a cells. There was no difference in the binding of PrP(Sc) to prion-susceptible and unsusceptible N2a subclones regardless of their PrP(C) expression level, suggesting that the binding of PrP(Sc) to cells is not a major determinant for prion susceptibility. Stable expression of PrP(C) did not confer susceptibility to prion in unsusceptible subclones. Furthermore, the existence of prion-unsusceptible N2a subclones that expressed PrP(C) at levels similar to prion-susceptible subclones, indicated that a host factor(s) other than PrP(C) and/or specific cellular microenvironments are required for the propagation of prion in N2a cells. The prion-susceptible and -unsusceptible N2a subclones established in this study should be useful for identifying the host factor(s) involved in the prion propagation.     

拟南芥中光信号系统中关键基因CRY1、CRY2和COP1启动子的表达模式分析

通过构建表达光信号系统关键基因CRY1、CRY2和COP1启动子与GUS融合基因的拟南芥转基因植株,并对转基因植株进行GUS组织化学染色的结果表明,CRY1、CRY2和COP1的表达模式不受光条件的调控,并且在各器官有广泛的表达。分别分析CRY1基因启动子在cop1突变体以及COP1基因启动子在cry1突变体遗传背景中表达模式的结果表明,CRY1和COP1在转录水平上不存在明显的相互调控关系。     

Generation of deletion subclones for sequencing by partial digestion with restriction endonucleases

A method for creating a group of deletion subclones for DNA sequencing by partial digestion of M13 bacteriophage constructions is outlined. The M13 construct is linearized at a unique site and then subjected to partial digestion with a frequent-cutting restriction endonuclease. The insert is truncated at different locations. The vector DNA is also partially digested. The products of a single partial digestion are repaired, recircularized by ligation, and used for bacterial transfection to generate subclones with a spectrum of deletions in the insert; most deletions in the vector DNA will disrupt vital viral genes and will thus disappear in the transfection. The subclones are sorted by size by gel electrophoresis of single-stranded viral DNA. This method is simpler and thus may be more reliable than established subcloning schemes.     

Molecular Phylogeny,Evolution, and Functional Divergence of the LSD1-Like Gene Family: Inference from the Rice Genome

The identification of LSD1-like genes in parasite, green algae, moss, pine, and monocot and dicot species allowed us to trace the phylogenetic history of this gene family. Computational analysis showed that the diversification of members of this family could be dated back to the early stage of plant evolution. The evolution of plant LSD1-like genes was possibly shaped by two duplication events. These proteins, which contain three copies of the LSD1 zinc finger (zf-LSD1) domain within their entire polypeptides and play crucial roles in modulating disease defense and cell death, resulted from the second duplication. A gain of zf-LSD1 domain model was reasonable for explaining the origination of three-zf-LSD1 domain-containing proteins. The zf-LSD1 domain phylogeny showed that the middle (M) and C-terminal (C) domains originated from a common ancestor; the N-terminal (N) domain might be more ancient than the former two. The divergence of the N, M, and C domains was well before the monocot-dicot split. Coevolution analysis revealed that four intramolecular domain pairs, including the N domain and the interregion between the M and the C domains (INTER2), the M and C domain, the N- and C-terminus, and the M domain and C-terminus, possibly coevolved during the evolution of three-zf-LSD1 domain-containing proteins. The three zf-LSD1 domains are evolutionary conserved. Thus, the differences at the N- and C-terminus would be crucial for functional specificity of LSD1 genes. Strong functional constraints should work on the zf-LSD1 domains, whereas reduced functional constraint was found in the INTER2 region. Functional divergence analysis showed that three-zf-LSD1 domain-containing proteins were significantly functionally divergent from those proteins containing only one zf-LSD1 domain, a result demonstrating that shifted evolutionary rates between the two clusters were significantly different from each other. [Reviewing Editor: Dr. Joshua Plotkin]     

Variation in the inheritance of expression among subclones for unselected (uidA) and selected (bar) transgenes in maize (Zea mays L.)

Variation in the inheritance of expression among subclones for an unselected (uidA) and a selected (bar) transgene was analyzed in two individual transformation events in maize. The unselectable gene (uidA) and the selectable gene (bar), on two separate plasmids, were transferred to maize (Hi-II derivative) by particle bombardment of embryogenic calli or suspension cells. A total of 188 fertile T1 plants were obtained from one transformant (transformation event BG which integrated uidA and bar). A total of 98 fertile T1 plants were obtained from a second transformant (transformation event B which integrated bar). Through self-pollination and/or cross-pollination in the greenhouse, approximately 10 000 T2 progeny were obtained from event BG, and more than 1000 T2 progeny were obtained from event B. Segregation of transgene expression was analyzed statistically in a total of 2350 T2 progeny from 40 T1 subclones of event BG and in 217 T2 progeny from six T1 subclones from event B. Variation in the inheritance of expression among subclones for the two transgenes (uidA and bar) was observed in the two transformants. A significant difference was observed between the use of the female or male as the transgenic parent in the inheritance of expression for the two transgenes in event BG. No inheritance through the pollen was observed in two of four T1 subclones analyzed in event B. Co-expression analysis of event BG showed that both transgenes were co-expressed in 67.7% of the T2 plants which expressed at least one of the two transgenes. Of the T2 expressing plants, 30.4% expressed only bar, and 1.9% expressed only uidA. Inactivation of the unselected (uidA) and the selected (bar) transgenes was observed in individual T2 plants.     

转双抗虫基因三倍体毛白杨外源基因表达及性状相关性分析

用部分改造的BtCry1Ac基因与慈菇蛋白酶抑制剂（API）基因构建的双抗虫基因表达载体,通过农杆菌介导法转化了三倍体毛白杨Populus tomentosa Carr.,获得一批转双抗虫基因株系。对转基因株系的抗虫毒蛋白表达进行了ELISA和Western Blot检测,同时用转基因株系叶片对杨扇舟蛾Clostera anachoreta Fabricius和舞毒蛾Lymantria dispar L.幼虫进行室内饲虫试验,并对各项外源基因表达指标进行了相关分析。结果表明：在检测的28个转基因株系中,对杨扇舟蛾高抗株系占总参试系号的41％,中抗系号占35.0％,低抗系号占24％,对舞毒蛾高抗株系占参试系号的70％。转基因植株可明显抑制存活幼虫的生长发育,且不同转基因株系饲养的幼虫发育存在显著差异。连续两年相关分析表明,不同转基因株系幼虫死亡率间存在极显著相关。转基因植株对舞毒蛾和杨扇舟蛾均表达出抗虫性,并存在极显著相关。ELISA检测结果表明,不同转基因株系Bt毒蛋白表达量存在差异,变化在0.0011％～0.0161％。转基因植株对害虫的杀虫效果与Bt杀虫蛋白的表达量存在显著相关,表明Bt毒蛋白在抗虫效果中占有重要地位。转基因植株对卡那霉素表现出一定的抗性,但与抗虫程度相关不明显,单纯用卡那霉素作为筛选手段并不能完全反映植株的真实抗虫效果。     

甲型H5N1流感病毒M2与HA双基因载体疫苗在小鼠体内的免疫学评价

高致病性H5N1亚型禽流感病毒 (AIV) 严重威胁到人类健康,因此研制高效、安全的禽流感疫苗具有重要意义。以我国分离的首株人H5N1亚型禽流感病毒 (A/Anhui/1/2005) 作为研究对象,PCR扩增基质蛋白2 (M2) 和血凝素 (HA) 基因全长开放阅读框片段,构建共表达H5N1亚型AIV膜蛋白基因 M2和HA的重组质粒pStar-M2/HA。此外,还通过同源重组以293细胞包装出表达M2基因的重组腺病毒Ad-M2以及表达HA基因的重组腺病毒Ad-HA。用间接免疫荧光 (IFA) 方法检测到了各载体上插入基因的表达。按初免-加强程序分别用重组质粒pStar-M2/HA和重组腺病毒Ad-HA+Ad-M2免疫BALB/c小鼠,共免疫4次,每次间隔14 d。第1、3次用DNA疫苗,第2、4次用重组腺病毒载体疫苗,每次免疫前及末次免疫后14 d采集血清用于检测体液免疫应答,末次免疫后14 d采集脾淋巴细胞用于检测细胞免疫应答。血凝抑制 (HI) 实验检测到免疫后小鼠血清中的HI活性。ELISA实验检测到免疫后小鼠血清中抗H5N1亚型流感病毒表面蛋白的IgG抗体。ELISPOT实验检测到免疫后小鼠针对M2蛋白和HA蛋白的特异性细胞免疫应答。流感病毒M2与HA双基因共免疫的研究,为研究开发新型重组流感疫苗奠定了基础。     

I型鸭肝炎病毒VP1、3D基因克隆及其在大肠杆菌中的表达

根据GenBank中的I型鸭肝炎病毒全基因序列设计了扩增I型鸭肝炎病毒VP1、3D基因的引物, 用该特异性表达引物从I型鸭肝炎病毒cDNA模板中扩增得到目的基因VP1、3D, 用相同的限制性内切酶酶切目的基因和表达载体pET32a后构建重组表达载体, 转化宿主BL21(DE3), 用不同浓度的IPTG诱导VP1、3D基因的表达, 收集菌液进行SDS-PAGE电泳, Western-blotting分析蛋白免疫原性。结果表明, VP1、3D在大肠杆菌中表达量较高, 表达产物的分子量约为48 kD、68 kD, 并能被兔抗DHV-1血清所识别。I型鸭肝炎病毒VP1、3D蛋白在大肠杆菌中表达产物具有免疫原性。     

Comprehensive analysis of pseudogene HSPB1P1 and its potential roles in hepatocellular carcinoma

The incidence and mortality rate of hepatocellular carcinoma (HCC) nowadays is still at high levels. The regulatory roles of pseudogene in cancers have been gradually recognized in recent years. However, comprehensive investigation of abnormally expressed pseudogene and related mechanisms in HCC remains lacking. GSE124535 dataset was used to identify differentially expressed pseudogenes in HCC tissues compared with normal tissues. Prognostic value of these differentially expressed pseudogenes was analyzed at GEPIA. StarBase used to analyze microRNAs (miRNAs) can bind with pseudogene, while the targets for these miRNAs were analyzed at miRTarBase. Protein–protein interaction (PPI) network was then established for miRNA targets, after that hub genes were selected. Expression correlation of pseudogene and hub genes was analyzed at StarBase. In total, 16 upregulated and 17 downregulated pseudogenes were identified. Pseudogene HSPB1P1 was identified abnormally expressed in 20 types of human cancers and could be used as an indicator for poorer overall survival of patients with HCC. Functional analyses showed that HSPB1P1 was strongly correlated with signaling pathways related to cancer progression. Further studied revealed that HSPB1P1 could direct regulate the EZH2 expression in HCC. In summary, our study indicated that HSPB1P1 was a predictor for poorer overall survival of patients with HCC and may be potential therapeutic target against HCC.     

Gene cluster containing the genes for tyrocidine synthetases 1 and 2 from Bacillus brevis: evidence for an operon.

From a genomic library of the tyrocidine producer Bacillus brevis ATCC 8185 constructed in the bacteriophage vector EMBL3, a recombinant phage which contains the structural genes coding for tyrocidine synthetases 1 and 2, TycA and TycB, was identified. The location of the tycA gene within the 16-kilobase insert of this clone, EMBL25-1, was mapped by hybridization studies by using the previously isolated tycA DNA as a probe. Restriction analyses, the construction of subclones, and the analysis of proteins encoded by the subclones located the tycB gene at the 3' end of the tycA gene and revealed that the two genes are transcribed in the same direction. Nuclease S1 protection studies and DNA sequencing studies of the intergenic region indicated that tycA and tycB are separated by a 94-base-pair noncoding region and suggested that these genes are organized as an operon.     

Both mating types in the heterothallic fungus Ophiostoma quercus contain MAT1-1 and MAT1-2 genes

In heterothallic Ascomycota, two opposite but distinct mating types control all sexual processes. Using mating crosses, mating types were assigned to ten isolates of the heterothallic fungal species Ophiostoma quercus. Primers were subsequently designed to target the MAT1-1-1, MAT1-1-3 (of the mating type 1 idiomorph), and MAT1-2-1 (of the mating type 2 idiomorph) genes in these isolates. Results showed that all isolates contained the full gene sequence for the MAT1-2-1 gene. In addition, fragments of the MAT1-1-1 and MAT1-1-3 genes were sequenced from all isolates. These results were unexpected, as each isolate from a heterothallic species would typically contain only one of the two possible MAT idiomorphs.     

Reduced susceptibility to HIV-1 infection of ethyl-methanesulfonate-treated CEM subclones correlates with a blockade in their protein kinase C signaling pathway.

We have described the isolation of chemically induced CEM subclones that express CD4 receptors and bind soluble gp120, yet show a markedly reduced susceptibility to infection with HIV-1. Two subclones were found to have an abnormal response to the protein kinase C (PKC) activator PMA. PMA treatment induced CD3 and CD25 (IL-2R) receptors on the parental line and on other ethyl-methanesulfonate-derived subclones, but not on these two mutants. Direct assays of PKC activity were conducted. Total cellular PKC enzymatic activity was found to be normal in these subclones. PMA-induced CD4 down-modulation occurred normally. In addition, activation of c-raf kinase was normal. Since HIV-1 long terminal repeat contains two functional nuclear factor kB (NF-kB) regulatory elements, we studied the ability of PMA to induce NF-kB binding activity by different assays. Chloramphenicol acetyl transferase (CAT) assays using the HIV-1 (-139)long terminal repeat-CAT construct showed no PMA induction of CAT activity in these subclones (unlike the parental line and other subclones). Okadaic acid, an inhibitor of phosphatases 1 and 2A, did not overcome the defect in these subclones. Gel retardation assays, using a 32P-probe containing the HIV-1 NF-kB probe and nuclear extracts from PMA-treated cells, showed significantly reduced induction of nuclear NF-kB binding proteins in these two subclones compared with wild type CEM and a control subclone. Deoxycholate treatment of cytoplasmic extracts from these subclones released much reduced NF-kB binding proteins from their cytoplasmic pools. Thus, reduced levels of PKC-induced nuclear NF-kB activity in two T cell subclones did not affect their normal cell growth, but correlated with a pronounced reduction in their susceptibility to HIV-1 infection.