Probing the kinetic cooperativity of beta-sheet folding perpendicular to the strand direction

In an attempt to determine how the folding dynamics of multistranded beta-sheets vary with the strand number, we have studied the temperature-induced relaxation kinetics of a four-stranded beta-sheet, DPDPDP. Our results show that the thermally induced relaxation of DPDPDP occurs on the nanosecond time scale; however, a comparison of the current results with those obtained on a sequence-related, three-stranded beta-sheet suggests that increasing the strand number from three to four increases the folding free energy barrier by a minimum of 0.8 kcal/mol, depending on the folding mechanism. Therefore, these results together suggest that the relaxation kinetics of DPDPDP can be analyzed according to a two-state model even though its folding may actually involve parallel (but degenerate or nearly degenerate) kinetic pathways. The apparent, two-state folding time of DPDPDP is determined to be approximately 0.44 micros at the thermal melting temperature, which makes it one of the fastest folders known to date.     

Occurrence and distribution of arbuscular mycorrhizal fungal species in three types of grassland community of the Tibetan Plateau

Arbuscular mycorrhizal (AM) fungal spore communities and distribution patterns were surveyed in montane scrub grassland, alpine steppe, and alpine meadow sites at altitudes ranging from 3,500 to 5,200 m a.s.l. on the Tibetan Plateau. Thirty-two representative soil samples were collected from the root zone of the dominant and common plant species in late May 2004. Twenty-three AM fungal species representing six genera (Acaulospora, Entrophospora, Glomus, Pacispora, Paraglomus, and Scutellospora) were detected and species richness varied from 5.3 ± 0.8 to 10.5 ± 2.5 per site. Some AM fungal species were restricted to one vegetation type and Glomus mosseae, Glomus intraradices, and Scutellospora calospora were detected in all three vegetation types. Glomus species were found to be the most frequent and abundant in all three vegetation types. Acaulospora occurred mostly in the alpine steppe and alpine meadow. Scutellospora occurred mostly in montane scrub grassland. At the species level, Glomus mosseae was dominant in the montane scrub, Acaulospora laevis and Pacispora scintillans were dominant in the alpine steppe, and Acaulospora laevis, Pacispora scintillans, and Glomus claroideum dominated the alpine meadow. It was evident from the distribution pattern of AM fungi in the different vegetation types that the abundance and diversity of AM fungal species were lowest in the montane scrub grassland than the other two plant communities. Climatic conditions, especially temperatures, and intensity of land use may be the most important factors influencing the AM fungal community.     

Purification and characterization of a chitinase from Serratia proteamaculans

A chitinase gene from Serratia proteamaculans 18A1 was cloned, sequenced, and expressed in Escherichia coli M15. Recombinant enzyme (ChiA) was purified by Ni-NTA affinity column chromatography. The ChiA gene contains an open reading frame (ORF), encoding an endochitinase with a deduced molecular weight 60 kDa and predicted isoelectric point of 6.35. Comparison of ChiA with other chitinases revealed a modular structure containing an N-terminal PKD-domain, a family 18 catalytic domain and a C-terminal putative chitin-binding domain. Turn over rate (K _cat) of the enzyme was determined using colloidal chitin (49.71 ± 1.15 S^?1) and crystalline β-chitin (17.20 ± 0.83 S^?1) as substrates. The purified enzyme was active over a broad range of pH (pH 4.5–9.0) and temperature (4–70°C) with a peak activity at pH 5.5 and 55°C. However, enzyme activity was found to be stable up to 45°C for longer incubation periods. Purified enzyme was shown to inhibit fungal spore germination and hyphal growth of pathogenic fungi Fusarium oxysporum and Aspergillus niger.     

Phylogeography of the Qinghai–Tibet Plateau endemic alpine herb Pomatosace filicula (Primulaceae)

In order to trace the response of alpine plants on the Qinghai–Tibet Plateau (QTP) to the Quaternary climate oscillations, the phylogeographic history of Pomatosace filicula Maxim. was investigated in the present study. Based on sequence variations of two maternally inherited plastid markers, matK and trnH-psbA, and the biparentally inherited nuclear ribosomal internal transcribed spacer (nrITS), we estimated the population genetic structure, lineage divergence timescale, and population dynamics of P. filicula. Seven plastid haplotypes and two nrITS genotypes were identified across the range-wide sampling of 200 individuals from 24 populations. Although AMOVA revealed a high level of differentiation among populations (F_ST = 0.560), no significant phylogeographic structure was detected (N_ST = 0.503, G_ST = 0.518, P > 0.05). Molecular dating suggested that the divergences between major plastid lineages and nrITS genotypes occurred during the early and middle Pleistocene, and the middle Pleistocene, respectively. This species most likely survived at multiple unglaciated sites on the QTP during the Last Glacial Maximum, with most of these sites located above 4000 m a.s.l. The species probably experienced range expansion at its distribution fringe, but demographic tests did not suggest significant population size changes. We proposed that pronounced effective gene flow (N_em = 0.393) and short generation time may have obscured the phylogeographic and demographic patterns of this species. Our findings will shed new light on the Quaternary evolutionary history of the alpine flora of the QTP.     

The use of miRNAs as reference genes for miRNA expression normalization during <Emphasis Type="Italic">Lilium</Emphasis> somatic embryogenesis by real-time reverse transcription PCR analysis

Expression profiling of miRNAs has the ability to reveal the essence of somatic embryogenesis (SE). qRT-PCR is one of the most commonly used techniques for dynamic miRNA detection but requires optimal reference genes for data reliability. This is the first report on reference gene validation for miRNA expression normalization in Lilium (Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor). In this study, seventeen miRNAs together with two snRNAs (U4, U6), one rRNA (5S rRNA) and three protein-coding genes (FP, ACT, GAPDH) were selected as reference candidates, and their expression stability was validated by qRT-PCR among eleven developing SE cultures in two lilies. Four normalization algorithms, including geNorm, BestKeeper, NormFinder and RefFinder, were also used to evaluate the stability of the reference candidates. For Lilium pumilum DC. Fisch., lpu-miR159a was the optimal reference gene during SE, followed by lpu-miR408b, while U6 was the least stable reference candidate. For Lilium davidii var. unicolor, FP presented greater stability than did half of the miRNA candidates, but the best reference gene was lda-miR162, followed by lda-miR159a. Further analysis of the expression level of miR156 and miR529 was used to evaluate the validity of the reference genes in both lilies. In general, miRNAs are superior to common protein-coding genes and snRNAs / rRNAs as reference genes for miRNA expression normalization during Lilium SE, and the most suitable reference miRNA is different between two species in the same Lilium genus. This is a pioneer study using suitable miRNAs as reference genes in Lilium and constitutes a small but essential step for the further exploration of miRNA function in Lilium, thus offering valuable references for other plants.     

凉山山系大熊猫和黑熊适宜生境预测及重叠分析

研究同域物种的分布格局及重叠状况对物种的区域整合保护管理及区域生物多样性保护具有重要实践价值。本研究基于全国第四次大熊猫调查及长期野外调查数据, 利用MaxEnt模型预测了凉山山系两种同域分布的熊科动物——大熊猫(Ailuropoda melanoleuca)和黑熊(Ursus thibetanus)的适宜生境, 基于适宜生境预测结果, 分析了两个物种的生境需求因子、生境破碎化现状及重叠状况。结果显示: (1)大熊猫和黑熊的适宜生境分布格局相似, 主要分布在凉山山系的山脊地带, 适宜生境面积分别为1,383.84 km²和2,411.49 km²; (2)两个物种的适宜生境都较为破碎, 且存在一些隔离分布区, 相较而言, 黑熊适宜生境的连通性要优于大熊猫; (3)两个物种生态位重叠度较高(D = 0.654, I = 0.901), 适宜生境重叠面积为958.29 km², 分别占大熊猫和黑熊适宜生境总面积的69.25%和39.74%; (4)两个物种对环境因子的选择和响应表现出了相似性和差异性。相似性在于对两个物种生境分布影响最大的两个因子均为距居民点距离和海拔; 差异性在于对大熊猫生境分布影响次之的因子是植被类型和最冷季均温, 而黑熊的是年最大EVI指数和距道路距离。为了更有效地保护两个物种, 应加强对人类干扰的控制和植被的恢复, 对栖息地实行连通管理, 并建立多物种保护规划。     

Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies

A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a "cosmopolitan" tagging approach to capture the genetic diversity across approximately 2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.     

A lectin (CfLec-2) aggregating Staphylococcus haemolyticus from scallop Chlamys farreri

Lectins are a family of carbohydrate-recognition proteins which play crucial roles in innate immunity. In this study, a new lectin (CfLec-2) gene was cloned from Chlamys farreri by EST and RACE approaches. The full-length cDNA of CfLec-2 was composed of 708bp, encoding a typical long form carbohydrate-recognition domain of 130 residues. The deduced amino acid sequence showed high similarity to Brevican in Homo sapiens, C-type lectin-1 and lectin-2 in Anguilla japonica. The cDNA fragment encoding the mature peptide of CfLec-2 was recombined into plasmid pET-32a (+) and expressed in Escherichia coli Rosseta-Gami (DE3). The recombinant CfLec-2 (rCfLec-2) protein exhibited aggregative activity toward Staphylococcus haemolyticus, and the agglutination could be inhibited by d-mannose but not EDTA or d-galactose, indicating that CfLec-2 was a Ca2+ independent lectin. Moreover, rCfLec-2 could suppress the growth of E. coli TOP10F'. These results suggested that CfLec-2 was perhaps involved in the recognition and clearance of bacterial pathogens in scallop.