Role of Allosteric Switch Residue Histidine 195 in Maintaining Active-Site Asymmetry in Presynaptic Filaments of Bacteriophage T4 UvsX Recombinase

Cyanophycin, or poly-l-Asp-multi-l-Arg, is a non-ribosomally synthesized peptidic polymer that is used for nitrogen storage by cyanobacteria and other select eubacteria. Upon synthesis, it self-associates to form insoluble granules, the degradation of which is uniquely catalyzed by a carboxy-terminal-specific protease, cyanophycinase. We have determined the structure of cyanophycinase from the freshwater cyanobacterium Synechocystis sp. PCC6803 at 1.5-Å resolution, showing that the structure is dimeric, with individual protomers resembling aspartyl dipeptidase. Kinetic characterization of the enzyme demonstrates that the enzyme displays Michaelis-Menten kinetics with a k_cat of 16.5 s^− 1 and a k_cat/K_M of 7.5 × 10^− 6 M^− 1 s^− 1. Site-directed mutagenesis experiments confirm that cyanophycinase is a serine protease and that Gln101, Asp172, Gln173, Arg178, Arg180 and Arg183, which form a conserved pocket adjacent to the catalytic Ser132, are functionally critical residues. Modeling indicates that cyanophycinase binds the β-Asp-Arg dipeptide residue immediately N-terminal to the scissile bond in an extended conformation in this pocket, primarily recognizing this penultimate β-Asp-Arg residue of the polymeric chain. Because binding and catalysis depend on substrate features unique to β-linked aspartyl peptides, cyanophycinase is able to act within the cytosol without non-specific cleavage events disrupting essential cellular processes.     

Epitope Mapping of a Bactericidal Monoclonal Antibody against the Factor H Binding Protein of Neisseria meningitidis

The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria meningitidis that allows the survival of the bacterium in human plasma; it is also a major component of a universal vaccine against meningococcus B.In this study, we used nuclear magnetic resonance spectroscopy, mutagenesis, and in silico modeling to map the epitope recognized by MAb502, a bactericidal monoclonal antibody elicited by fHbp. The data show that the antibody recognizes a conformational epitope within a well-defined area of the immunodominant C-terminal domain of the protein that is formed by two loops connecting different β-strands of a β-barrel and a short α-helix brought in spatial proximity by the protein folding. The identification of the protective epitopes of fHbp is an important factor for understanding the mechanism(s) of an effective immune response and provides valuable guidelines for designing variants of the protein able to induce broadly protective immunity.     

新疆玛纳斯热气泉免培养土壤细菌多样性分析

【目的】了解新疆玛纳斯热气泉土壤免培养细菌群落组成及多样性。【方法】采用免培养法直接从土壤样品中提取总DNA,利用细菌通用引物对土壤总DNA进行16S rDNA扩增,构建细菌16SrDNA文库。使用HaeⅢ限制性内切酶对阳性克隆进行限制性片段长度多态性分析(restriction fragment length polymorphism,RFLP),挑选具有不同酶切图谱的克隆进行测序、比对并构建16SrDNA系统发育树。【结果】从土壤细菌16S rDNA文库中随机挑选了170个阳性克隆,共得到29个不同的分类单元(operational taxonomic unit,OTU)。系统发育分析归为6个门:酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)和浮霉菌门(Planctomycetes)。其中厚壁菌门(Firmicutes)为绝对优势类群,占整个细菌文库的71%。29个OTUs中有14条序列与GenBank中相关序列的相似性低于97%(序列长度约1.5kb),占序列总数的48%。【结论】热气泉土壤细菌种群多样性较低,但存在大量潜在细菌新种。     

Cloning, sequencing and prokaryotic expression of cDNAs for antifreeze protein family from beetle Tenebrio molitor

Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR. Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank. The recombinant pGEX-4T-1-tmafp-XJ430 was introduced into E. coli BL21 to induce a GST fusion protein by IPTG. SDSPAGE analysis for the fusion protein shows a band of 38 kDa. pCDNA3-tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA. The titer of the antibody was 1:2000.Western blotting analysis shows that the antiserum was specifically against the antifreeze protein. Our results laid the foundation for further studies on the properties and functions of insect antifreeze proteins. __________ Translated from Hereditas (Beijing), 2006, 28(12): 1532-1540 [译自: 遗传]     

代表活性污泥中苯酚降解菌群的ERIC-PCR产物片段的多态性

对可能代表活性污泥中两个时期的主要苯酚降解菌群的：ERIC-PCR指纹图谱中的两条2．1kb的条带(P1和P8)中的DNA片段进行克隆、转化,得到193个转化子。．HinfⅠ物理图谱分析得到35种酶切类型,将两端各进行1次测序后,同源性大于90％的克隆归为一类,共得到10种序列类型。对各类型的代表克隆进行了全长测序,5种类型为P1条带所有,3种类型为P8所有,二者共享的类型为2种,丰度最高的片段为S3,P1条带中76．3％的转化子和P8条带中66．7％转化子属该类型。对所得序列进行检索分析,它们与GenBank中已知基因序列均无显著同源性。用S3特异性引物对活性污泥样品及其它在LB、dCGY、MP和FWM培养基上的回收菌进：行扩增,除LB上的回收菌没有显示目的条带外,活性污泥和其回收菌中均检测到带有该基因组片段的菌种的存在。研究为专一性分离降酚活性污泥中的优势菌提供了分子探针。     

Increased Y-chromosome resolution of haplogroup O suggests genetic ties between the Ami aborigines of Taiwan and the Polynesian Islands of Samoa and Tonga

The Austronesian expansion has left its fingerprint throughout two thirds of the circumference of the globe reaching the island of Madagascar in East Africa to the west and Easter Island, off the coast of Chile, to the east. To date, several theories exist to explain the current genetic distribution of Austronesian populations, with the “slow boat” model being the most widely accepted, though other conjectures (i.e., the “express train” and “entangled bank” hypotheses) have also been widely discussed. In the current study, 158 Y chromosomes from the Polynesian archipelagos of Samoa and Tonga were typed using high resolution binary markers and compared to populations across Mainland East Asia, Taiwan, Island Southeast Asia, Melanesia and Polynesia in order to establish their patrilineal genetic relationships. Y-STR haplotypes on the C2 (M38), C2a (M208), O1a (M119), O3 (M122) and O3a2 (P201) backgrounds were utilized in an attempt to identify the differing sources of the current Y-chromosomal haplogroups present throughout Polynesia (of Melanesian and/or Asian descent). We find that, while haplogroups C2a, S and K3-P79 suggest a Melanesian component in 23%-42% of the Samoan and Tongan Y chromosomes, the majority of the paternal Polynesian gene pool exhibits ties to East Asia. In particular, the prominence of sub-haplogroup O3a2c* (P164), which has previously been observed at only minimal levels in Mainland East Asians (2.0-4.5%), in both Polynesians (ranging from 19% in Manua to 54% in Tonga) and Ami aborigines from Taiwan (37%) provides, for the first time, evidence for a genetic connection between the Polynesian populations and the Ami.     

AFLP-derived SCARs facilitate construction of a 1.1 Mb sequence-ready map of a region that spans the Vf locus in the apple genome

The availability of high-density anchored markers is a prerequisite for reliable construction of a deep coverage BAC contig, which leads to creation of a sequence-ready map in the target chromosomal region. Unfortunately, such markers are not available for most plant species, including woody perennial plants. Here, we report on an efficient approach to build a megabase-size sequence-ready map in the apple genome for the Vf region containing apple scab resistance gene(s) by targeting AFLP-derived SCAR markers to this specific genomic region. A total of 11 AFLP-derived SCAR markers, previously tagged to the Vf locus, along with three other Vf-linked SCAR markers have been used to screen two apple genome BAC libraries. A single BAC contig which spans the Vf region at a physical distance of approximately 1,100 kb has been constructed by assembling the recovered BAC clones, followed by closure of inter-contig gaps. The contig is 4 ×deep, and provides a minimal tiling path of 16 contiguous and overlapping BAC clones, thus generating a sequence-ready map. Within the Vf region, duplication events have occurred frequently, and the Vf locus is restricted to the ca. 290 kb region covered by a minimum of three overlapping BAC clones.     

Bst DNA聚合酶大片段在全基因组扩增中的应用

Bst DNA聚合酶大片段作为一种常用的DNA聚合酶,因其独特的特点:能引发链置换反应、高保真、耐高温等,而成为一种重要的DNA多重置换扩增酶。目的:为减少成本,设计一种高产,方便且扩增活性高的Bst DNA聚合酶大片段表达体系;探究该酶应用于胃癌石蜡包埋组织基因组DNA的扩增条件。方法:采用p TWIN1质粒作为载体克隆表达Bst DNA聚合酶大片段,应用几丁质亲和层析柱纯化该酶,使用该酶对人类基因组DNA进行不同温度下扩增,探究其最适反应温度,并据此对胃癌石蜡包埋组织基因组DNA进行扩增。结果:由此得到的Bst DNA聚合酶大片段能运用于胃癌石蜡包埋组织基因组DNA的扩增,扩增效率可达200倍,并能应用于a CGH芯片。结论:扩增得到保真性高,覆盖基因组范围大的DNA扩增产物。该应用与a CGH结合,使得对少量的癌症石蜡包埋组织DNA样本进行全基因组扩增,并进行其基因拷贝数变异研究成为可能。     

The pollination seesaw of Rhynchospora cephalotes (L.) Vahl (Cyperaceae): Influence of plant location on the role of wind and insects as pollen vectors

Ambophily (wind and insect pollination) has been reported for some genera of the typically wind-pollinated family Cyperaceae, including the genus Rhynchospora. The significance of wind and insect pollination can vary, depending on local microclimatic conditions. Rhynchospora cephalotes is an ambophilous species that can grow under different environmental conditions, either along forest edges or inside forest fragments. This study, therefore, tests the hypothesis that (a) there is greater contribution by wind than insects to fruit set of the individuals at the forest edge and (b) there is greater contribution by insects than wind to fruit set of the individuals inside the forest. Field work was carried out in a fragment of Atlantic Forest in northeastern Brazil. We quantified the number of visits by insects, wind velocity and the reproductive success (fruit set) of R. cephalotes provided by wind and insects through exclusion experiments performed at the edge of and inside the forest. We observed a greater number of visits per day by pollinating bees to individuals inside the forest (36.83 ± 7.46) than to individuals at the edge (16.66 ± 6.53). The wind speed was significantly higher at the edge (1.71 ± 0.46 m/s) than inside the forest (0.97 ± 0.18 m/s). Bees and wind are both pollen vectors of R. cephalotes, but bees were the pollen vector that contributed most to fruit set (63.3%) for individuals inside the forest, whereas wind was the primary vector for individuals at the forest edge (76.6%). This seesaw in importance of each pollen vector in the two different environments guarantees high fruit set in R. cephalotes under different microclimatic conditions.     

Measurement of Substance P Metabolites in Rat CNS

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.