Improved side-chain prediction accuracy using an ab initio potential energy function and a very large rotamer library

Accurate prediction of the placement and comformations of protein side chains given only the backbone trace has a wide range of uses in protein design, structure prediction, and functional analysis. Prediction has most often relied on discrete rotamer libraries so that rapid fitness of side-chain rotamers can be assessed against some scoring function. Scoring functions are generally based on experimental parameters from small-molecule studies or empirical parameters based on determined protein structures. Here, we describe the NCN algorithm for predicting the placement of side chains. A predominantly first-principles approach was taken to develop the potential energy function incorporating van der Waals and electrostatics based on the OPLS parameters, and a hydrogen bonding term. The only empirical knowledge used is the frequency of rotameric states from the PDB. The rotamer library includes nearly 50,000 rotamers, and is the most extensive discrete library used to date. Although the computational time tends to be longer than most other algorithms, the overall accuracy exceeds all algorithms in the literature when placing rotamers on an accurate backbone trace. Considering only the most buried residues, 80% of the total residues tested, the placement accuracy reaches 92% for chi(1), and 83% for chi(1 + 2), and an overall RMS deviation of 1 A. Additionally, we show that if information is available to restrict chi(1) to one rotamer well, then this algorithm can generate structures with an average RMS deviation of 1.0 A for all heavy side-chains atoms and a corresponding overall chi(1 + 2) accuracy of 85.0%.     

破色期番茄果实均一化cDNA沉默文库的构建和功能基因筛选模型的初步建立

番茄果实的成熟是由多基因精细调控的一个过程．利用破色期番茄果实,根据复性动力学原理在mRNA水平进行均一化操作使高丰度和低丰度的mRNA丰度接近．然后把均一化之后mRNA反转录得到cDNA,再与基因沉默载体pTRV重组,最后把构建好的载体通过电转化的方法转入到GV3101农杆菌中,从而建立起破色期番茄果实均一化cDNA沉默文库．通过番茄果实中病毒诱导基因沉默技术,对cDNA沉默文库进行初步筛选,从而确定功能基因筛选模型．在模型建立阶段,以番茄红素合成途径相关的PDS基因作为内标基因,在100个混合农杆菌样中,成功筛选到了PDS基因．     

Intergenomic single nucleotide polymorphisms as a tool for bacterial artificial chromosome contig building of homoeologous Brassica napus regions

Background

Homoeologous sequences pose a particular challenge if bacterial artificial chromosome (BAC) contigs shall be established for specific regions of an allopolyploid genome. Single nucleotide polymorphisms (SNPs) differentiating between homoeologous genomes (intergenomic SNPs) may represent a suitable screening tool for such purposes, since they do not only identify homoeologous sequences but also differentiate between them.

Results

Sequence alignments between Brassica rapa (AA) and Brassica oleracea (CC) sequences mapping to corresponding regions on chromosomes A1 and C1, respectively were used to identify single nucleotide polymorphisms between the A and C genomes. A large fraction of these polymorphisms was also present in Brassica napus (AACC), an allopolyploid species that originated from hybridisation of A and C genome species. Intergenomic SNPs mapping throughout homoeologous chromosome segments spanning approximately one Mbp each were included in Illumina’s GoldenGate® Genotyping Assay and used to screen multidimensional pools of a Brassica napus bacterial artificial chromosome library with tenfold genome coverage. Based on the results of 50 SNP assays, a BAC contig for the Brassica napus A subgenome was established that spanned the entire region of interest. The C subgenome region was represented in three BAC contigs.

Conclusions

This proof-of-concept study shows that sequence resources of diploid progenitor genomes can be used to deduce intergenomic SNPs suitable for multiplex polymerase chain reaction (PCR)-based screening of multidimensional BAC pools of a polyploid organism. Owing to their high abundance and ease of identification, intergenomic SNPs represent a versatile tool to establish BAC contigs for homoeologous regions of a polyploid genome.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-560) contains supplementary material, which is available to authorized users.     

Isolation and characterization of the yeast aspartyl-tRNA synthetase gene

A yeast genomic library in Escherichia coli, constructed by insertion of Sau3A restriction fragments into the hybrid Saccharomyces cerevisiae-E. coli plasmid pFL1, was screened by a radioimmunoassay (RIA) for colonies expressing yeast aspartyl-tRNA synthetase (AspRS). Four clones were isolated by this technique. Data obtained by Southern and restriction analysis of the inserts showed a common 3.8-kb BamHI restriction fragment which, when inserted into the plasmid pFLl, gave a positive RIA. Several controls showed that this 3.8-kb insert codes for the entire AspRS : (i) S. cerevisiae transformed by the PFL1 plasmid carrying the 3.8-kb fragment overproduces AspRS activity by a factor of ten compared to the wild-type yeast strain; and (ii) a new protein with electrophoretic behaviour similar to AspRS and immuno-reactive toward anti-AspRS appears in crude extracts of transformed yeast and E. coli.     

Structural Analysis ofArabidopsis thaliana Chromosome 5. VI. Sequence Features of the Regions of 1,367,185 bp Covered by 19 Physically Assigned P1 and TAC Clones

Nineteen Pl and TAC clones, which have been mapped on the finephysical map of the Arabidopsis thaliana chromosome 5, weresequenced according to the shotgun-based strategy, and theirstructural features were analysed. The total length of the regionssequenced in this study was 1,367,185 bp. Combining this withthe regions covered by 90 P1 and TAC clones proviously reported,the total length of chromosome 5 sequenced to date becomes 8,058,855bp. On the basis of similarity search against protein and ESTdatabases and gene modeling with computer programs, a totalof 330 potential protein-coding regions were identified, bringingan average density of the genes to approximately one gene per4.1 kb. Introns were identified in 81.0% of the potential proteingenes for which the entire gene structure was predicted, withan average number per gene of 4.2 and an average length of theintrons of 180 bp. The RNA-coding genes identified were 9 tRNAgenes corresponding to 8 amino acid species and 2 genes forU2 nuclear RNA. These sequence features are essentially identicalto those in the previously reported sequences. The sequencedata and gene information are available on the World Wide Webdatabase KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

人抗E．Coli J5噬菌体抗体制备的初步研究

以E．Coli J5株对合人Ig基因的噬菌体抗体库进行淘筛富集,免疫印迹筛选,以及ELISA检测,结果获得4株能与E．Coli J5株结合的阳性克隆,且阳性克隆结合抗原的活性可分别被E．Coli J5株、E．Coli Rc-LPS和抗E．Coli J5株核心糖脂域MAb抑制．PCR检测表明,4株阳性克隆均分别带有约660bp大小的重链和轻链基因片段．SDS－PAGE与蛋白质印迹的结果显示,经IPTG诱导的阳性克隆能表达分子量约为50000大小的蛋白,提示该4株阳性克隆能够表达具有一定抗原结合活性的人源Fab片段．     

Generation of a naïve/synthetic antibody specific to botulinum neurotoxin via motif-grafting

In this study, we describe a new approach to the production of naïve/synthetic human antibodies against the botulinum neurotoxin (BoNT). First, peptides that bind to BoNT serotype A (BoNT/A) were screened from a phage display of a combinatorial peptide library. One peptide, designated ANT 12-2 (TLPSPLALLTVH), was determined to interact with BoNT/A, as well as with other serotypes of BoNT. This peptide specifically reacted with the native form of BoNT/A, but not with its formalin-inactivated form. Next, a hybrid naïve/synthetic human Fab library was generated via the grafting of a peptide motif from ANT 12-2 into HCDR3 with randomized flanking residues. Through biopanning, the Fab clone, ANTHU-1, which harbors the HCDR3 sequence of VRIQRSPLALLSWGDV, was selected and confirmed in order to retain the same BoNT-binding characteristics as ANT 12-2.     

Development and characterization of nine polymorphic microsatellite markers in the sand goby Pomatoschistus minutus (Gobiidae)

A microsatellite‐enriched genomic library was constructed for the sand goby, Pomatoschistus minutus (Pallas 1770), and nine polymorphic DNA microsatellite markers of high quality were successfully optimized. Characterization of 96 individuals from the Vaccarès lagoon (France) showed moderate to high levels of polymorphism (two to 54 alleles). All the markers conformed to Hardy–Weinberg equilibrium and showed no evidence of null alleles, large allele dropout, stuttering and linkage disequilibrium between pairs of loci. These markers successfully amplify in three closely related species and can be employed to investigate population genetic structure and to clarify paternity in Pomatoschistus species.     

Microsatellite‐enriched genomic libraries as a source of polymorphic loci for Schistosoma mansoni

Microsatellite markers for Schistosoma mansoni were developed using four genomic microsatellite‐enriched libraries. Microsatellites were observed in 65.4% of all sequences. Primer pairs were designed and tested for 23 loci. Eighteen loci produced amplification products, out of which 11 were polymorphic and were further characterized on 100 individuals of S. mansoni. Two to 19 alleles per locus were detected. The average values of expected and observed heterozygosities among the 11 loci were 0.79 and 0.59, respectively.