Biphasic transitions of a hairpin hexanucleotide triplex DNA

The conformational transitions (helix-coil transitions) of three hairpin triple helices, models 5'-(A-G)(3) + 5'-(T-C)(3)-T(4)-((br)C-T)(3) [CY], 5'-(A-G)(3) + 5'-(T-(br)C)(3)-T(4)-(C-T)(3) [YC] and 5'-(A-G)(3) + 5'-(T-(br)C)(3)-T(4)-((br)C-T)(3) [YY], are characterized in this work by UV spectroscopy. Melting of these triplexes is biphasic, and the profiles are used to obtain the thermodynamic parameters. The thermodynamic properties of the hairpin triplex are T(m) = 19.45 degrees C and DeltaH(vH) = 293.12 kJ mol(-1) for CY, T(m) = 22.85 degrees C and DeltaH(vH) = 256.63 kJ mol(-1) for YC and T(m) = 28.47 degrees C and DeltaH(vH) = 234.68 kJ mol(-1) for YY at pH 4.4. Those of the duplex are T(m) = 30.50 degrees C and DeltaH(vH) = 427.09 kJ mol(-1) for CY, T(m) = 32.96 degrees C and DeltaH(vH) = 374.47 kJ mol(-1) for YC and T(m) = 33.24 degrees C and DeltaH(vH) = 329.67 kJ mol(-1) for YY at pH 4.4. The distinct transitions of triplex to duplex and duplex to single strands are analyzed using the nearest-neighbor Ising model. Electrostatic effects on each conformation are also analyzed.     

超抗原SEA与抗黑色素瘤ScFv融合基因的构建及表达

采用酶切连接和重叠PCR连接两种方法将抗黑色素瘤单链抗体基因和去除N端信号肽的金黄色葡萄球菌肠毒素A基因进行融合,并将融合基因克隆于pET28a表达载体上,转化大肠杆菌BL21(DE3)。用NiNTA系统对表达产物进行分离、纯化。MTT法检测融合蛋白对黑色素瘤细胞的体外抑制率。结果表明6HisScFvSEA融合蛋白可在E.coli BL21(DE3)中稳定表达,表达量占菌体蛋白的30%,主要以包涵体的形式存在。融合蛋白可通过激活效应细胞对表达相关抗原的黑色素瘤细胞发挥抑制作用。     

气候变暖对青藏高原牧草营养含量及其体外消化率影响模拟研究

全球气候变暖,气温上升的趋势逐步被众人接受,而青藏高原这一独特地理单元的生态系统对气候变暖十分敏感.为更好地了解气候变暖对青藏高原牧草品质的影响,利用大板山北坡3 200～3 800 m的海拔梯度,以温度为主要影响因子,用海拔高度不同造成的温差模拟全球变暖带来的升温效应,研究气候变暖对青藏高原牧草营养含量及其体外消化率的影响.针对羊茅(Festuca ovina)、早熟禾(Poa annua)、草(Koeleria cristata)、矮嵩草(Kobresia humilis)和黑褐苔草 (Carex alrofusca) 5种生长在不同海拔梯度的高原牧草中酸性洗涤纤维(ADF)、木质素(ADL)、粗蛋白(CP)、粗脂肪(EE)、无氮浸出物(NFE)、灰分等营养含量及其经绵羊瘤胃液培养后的体外消化率差异,经过1999和2000年两年的测定分析,结果表明:随着温度升高,牧草CP、EE和NFE的百分含量都呈现降低的趋势;牧草ADF和ADL百分含量与温度存在正相关关系,随着温度升高牧草ADF、ADL百分含量都呈增加的趋势;牧草体外消化率与牧草生长的环境温度存在负相关关联,随着温度升高牧草体外消化率呈降低趋势.模拟研究表明,就温度这一重要环境因素而言,未来气候变暖尤其是夜间温度的升高引起青藏高原牧草营养品质的变化,牧草CP、EE、NFE含量的降低,中性洗涤纤维(NDF)、ADL含量的增加,牧草消化率降低,从而不利于反刍动物对牧草的消化利用.     

Molecular dynamics simulations on the oligomer-formation process of the GNNQQNY peptide from yeast prion protein Sup35

Oligomeric intermediates are possible cytotoxic species in diseases associated with amyloid deposits. Understanding the early steps of fibril formation at atomic details may provide useful information for the rational therapeutic design. In this study, using the heptapeptide GNNQQNY from the yeast prion-like protein Sup35 as a model system, for which a detailed atomic structure of the fibril formed has been determined by x-ray microcrystallography, we investigated its oligomer-formation process from monomer to tetramer at the atomistic level by means of a molecular dynamics simulation with explicit water. Although the number of simulations was limited, the qualitative statistical data gave some interesting results, which indicated that the oligomer formation might start from antiparallel beta-sheet-like dimers. When a new single peptide strand was added to the preformed dimers to form trimers and then tetramers, the transition time from disorder aggregates to regular ones for the parallel alignment was found to be obviously much less than for the antiparallel one. Moreover, the parallel pattern also statistically stayed longer, providing more chances for oligomer extending, although the number of parallel stack events was almost equal to antiparallel ones. Therefore, our simulations showed that new strands might prefer to extend in a parallel arrangement to form oligomers, which agrees with the microcrystal structure of the amyloid fibril formed by this peptide. In addition, analysis of the pi-pi stacking of aromatic residues showed that this type of interaction did not play an important role in giving directionality for beta-strand alignment but played a great influence on stabilizing the structures formed in the oligomer-formation process.     

Identification of Two Species of Yeast-like Symbiotes in the Brown Planthopper, <Emphasis Type="Italic">Nilaparvata lugens</Emphasis>

To determine the species of the yeast-like symbionts (YLS) in the brown planthoppers (BPH), Nilaparvata lugens, YLS were first isolated and purified by ultracentrifugation from the fat bodies of BPH, and then 18S rDNA and internal transcribed spacer (ITS)–5.8S rDNA sequences of YLS were amplified with the different general primers for fungi. The results showed that the two different 18S and ITS–5.8S rDNA sequences of YLS were obtained. One 2291-bp DNA sequence, which contained 18S and ITS–5.8S rDNA, showed the high similarity to Cryptococcus and was named Cryp-Like symbiotes. Another 1248-bp DNA sequence, which contained a part of 18S and ITS–5.8S rDNA, showed the high similarity to Pichia guilliermondii and was named Pichia-Like symbiotes. It was further proved that Cryp- and Pichia-Like symbiotes existed in BPH through nested PCR with specific primers for two symbiotes and in situ hybridization analysis using digoxigenin-labeled probes. Our results showed that BPH harbored more than one species of eukaryotic YLS, which suggested that diversity of fungal endosymbiotes may be occurred in planthoppers, just like bacterial endosymbiotes.     

Auxin-related gene families in abiotic stress response in Sorghum bicolor

Sorghum, a C4 model plant, has been studied to develop an understanding of the molecular mechanism of resistance to stress. The auxin-response genes, auxin/indole-3-acetic acid (Aux/IAA), auxin-response factor (ARF), Gretchen Hagen3 (GH3), small auxin-up RNAs, and lateral organ boundaries (LBD), are involved in growth/development and stress/defense responses in Arabidopsis and rice, but they have not been studied in sorghum. In the present paper, the chromosome distribution, gene duplication, promoters, intron/exon, and phylogenic relationships of Aux/IAA, ARF, GH3, and LBD genes in sorghum are presented. Furthermore, real-time PCR analysis demonstrated these genes are differently expressed in leaf/root of sorghum and indicated the expression profile of these gene families under IAA, brassinosteroid (BR), salt, and drought treatments. The SbGH3 and SbLBD genes, expressed in low level under natural condition, were highly induced by salt and drought stress consistent with their products being involved in both abiotic stresses. Three genes, SbIAA1, SbGH3-13, and SbLBD32, were highly induced under all the four treatments, IAA, BR, salt, and drought. The analysis provided new evidence for role of auxin in stress response, implied there are cross talk between auxin, BR and abiotic stress signaling pathways.     

Gene cloning and heterologous expression of a serine protease from Streptomyces fradiae var.k11

The gene sfp1, which encodes a predicted serine proteinase designated SFP1, was isolated by the screening of a gene library of the feather-degrading strain Streptomyces fradiae var.k11. The open reading frame of sfp1 encodes a protein of 454 amino acids with a calculated molecular mass of 46.19 kDa. Sequence analysis reveals that SFP1 possesses a typical pre-pro-mature organization that consists of a signal sequence, an N-terminal propeptide region, and a mature proteinase domain. The pre-enzyme of SFP1 was expressed in Escherichia coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated that the mature enzyme of SFP1 was formed by autolysis of the propeptide after its expression. The purified SFP1 is active under a broad range of pH and temperature. SFP1 has pH and temperature optima of pH 8.5 and 65 degrees C for its caseinolytic activity and pH 9 and 62 degrees C for its keratinolytic activity. SFP1 was sharply inhibited by the serine proteinase inhibitor phenylmethyl sulfonyl fluoride and exhibited a good stability to solvents, detergents, and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicates that SFP1 has a considerable caseinolytic and keratinolytic activity as does proteinase K.     

Comparative proteomic analysis of virulent Korean Mycobacterium tuberculosis K-strain with other mycobacteria strain following infection of U-937 macrophage

In Korea, the Mycobacterium tuberculosis K-strain is the most prevalent clinical isolates and belongs to the Beijing family. In this study, we conducted comparative porteomics of expressed proteins of clinical isolates of the K-strain with H37Rv, H37Ra as well as the vaccine strain of Mycobacterium bovis BCG following phagocytosis by the human monocytic cell line U-937. Proteins were analyzed by 2-D PAGE and MALDITOF-MS. Two proteins, Mb1363 (probable glycogen phosphorylase GlgP) and MT2656 (Haloalkane dehalogenase LinB) were most abundant after phagocytosis of M. tuberculosis K-strain. This approach provides a method to determine specific proteins that may have critical roles in tuberculosis pathogenesis.     

Mapping of QTLs prolonging the latent period of Puccinia triticina infection in wheat

Slow rusting is considered a crucial component of durable resistance to wheat leaf rust caused by Puccinia triticina and is often expressed in the form of a prolonged latent period. Selection for a longer latent period is considered an effective approach to developing wheat cultivars with improved durable resistance to leaf rust. A recombinant inbred line (RIL) population derived from CI 13227 (long latent period) × Suwon 92 (short latent period) was phenotyped for latent period in two greenhouse experiments in separate years, and amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers were analyzed in the same population. Among the RILs, the frequency distribution for latent period was continuous, and latent period was highly correlated between years (r=0.94, P<0.0001). A quantitative trait locus (QTL) prolonging the latent period of P. triticina, designated as QLrlp.osu-2DS, explained 42.8% and 54.5% of the phenotypic and genetic variance in the two experiments, respectively. QLrlp.osu-2DS was mapped on the distal region of chromosome 2DS. Two other QTLs for latent period, QLrlp.osu-2B and QLrlp.osu-7BL, were localized on chromosome 2B and the long arm of chromosome 7B, respectively. Multiple regression analysis showed that these three QTLs collectively explained 58.0% and 73.8% of the phenotypic and genetic variance over two experiments, respectively. Fourteen RILs that carried all three alleles for long latent period at three AFLP loci flanking QLrlp.osu-2DS, QLrlp.osu-2B, and QLrlp.osu-7BL had a mean latent period of 12.5 days, whereas 13 RILs without any long-latent-period alleles at the corresponding loci had a mean latent period of 7.4 days. Three SSR markers closely linked to these QTLs have potential to be applied in marker-assisted selection for prolonged latent period in wheat.     

近岸污染指示半知菌灰黄青霉(Penicillium griseo fulvum)的分子检测

【目的】通过分子方法检测近海污染环境优势种灰黄青霉,并为由此而推断污染程度做准备。【方法】根据GenBank中青霉属不同种和相近属种的ITS序列差异和灰黄青霉特有的IAO序列,设计了污染区优势种灰黄青霉的特异性引物AS1/RS4和IAO1/IAO2,建立相应的特异探针检测体系。通过PCR和套式PCR技术,分析比较两对特异序列检测灰黄青霉的差异。【结果】建立的分子检测体系可以排除其它近似或相关菌株干扰,从环境中扩增到目的基因片段。利用引物AS1/RS4作为核酸探针,通过套式PCR菌株DNA的检测灵敏度可达到10fg/μL,当仅有10个数量级分生孢子时即可检测出,从沉积物中检测灵敏度为102个数量级孢子/0.25g。特异酶基因IAO1/IAO2检测灵敏度较前者稍低。【结论】利用特异序列作为探针检测污染环境优势种灰黄青霉的方法可行,在一定范围内,灰黄青霉的出现频率及数量对污染程度有较好的指示作用。