A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.     

Introgression browser: high‐throughput whole‐genome SNP visualization

Breeding by introgressive hybridization is a pivotal strategy to broaden the genetic basis of crops. Usually, the desired traits are monitored in consecutive crossing generations by marker‐assisted selection, but their analyses fail in chromosome regions where crossover recombinants are rare or not viable. Here, we present the Introgression Browser (iBrowser ), a bioinformatics tool aimed at visualizing introgressions at nucleotide or SNP (Single Nucleotide Polymorphisms) accuracy. The software selects homozygous SNPs from Variant Call Format (VCF) information and filters out heterozygous SNPs, multi‐nucleotide polymorphisms (MNPs) and insertion–deletions (InDels). For data analysis iBrowser makes use of sliding windows, but if needed it can generate any desired fragmentation pattern through General Feature Format (GFF) information. In an example of tomato (Solanum lycopersicum) accessions we visualize SNP patterns and elucidate both position and boundaries of the introgressions. We also show that our tool is capable of identifying alien DNA in a panel of the closely related S. pimpinellifolium by examining phylogenetic relationships of the introgressed segments in tomato. In a third example, we demonstrate the power of the iBrowser in a panel of 597 Arabidopsis accessions, detecting the boundaries of a SNP‐free region around a polymorphic 1.17 Mbp inverted segment on the short arm of chromosome 4. The architecture and functionality of iBrowser makes the software appropriate for a broad set of analyses including SNP mining, genome structure analysis, and pedigree analysis. Its functionality, together with the capability to process large data sets and efficient visualization of sequence variation, makes iBrowser a valuable breeding tool.     

Design and synthesis of cleavable biotinylated dideoxynucleotides for DNA sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Knowledge of the anti-drug antibody (ADA) status is necessary in early research studies. Because specific assay materials are sparse and time is pressing, a generic assay format with drug tolerance for detection of ADAs in serum samples from mice exposed to immunoglobulin G (IgG) or antigen-binding fragments (Fabs) is highly desirable. This article describes a generic immune complex assay in the sandwich enzyme-linked immunosorbent assay (ELISA) format based on (i) transformation of free ADAs to immune complexes by preincubation with excess drug, (ii) the use of a murine anti-human Fab constant domain Fab as capture reagent, (iii) detection of the immune complexes by a peroxidase-labeled rabbit anti-murine Fc antibody, and (iv) ADA-positive control conjugates consisting of human Fab and murine IgG. Results of the experiments suggest that the generic immune complex assay for mouse serum samples was at least equivalent to specific ADA immune assays and even superior regarding drug tolerance. The generic immune complex assay confers versatility as it detects ADAs in complex with full-length IgG as well as with Fabs independent of the target specificity in mouse serum samples. These features help to save the sparse amounts of specific antibodies available in early research and development and speed up drug candidate selection.     

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.     

Molecular phylogenetic studies of Lachnum and its allies based on the Japanese material

Molecular phylogenetic studies were carried out based on ITS-5.8S rDNA, the D1–D2 region of the large subunit rRNA gene, RPB2, and combined data of D1–D2 and RPB2 as well as these three genes on 36 species among 7 genera for Lachnum and allied genera in the family Hyaloscyphaceae. In the combined data of all three regions, seven strongly supported clades were obtained. The same clades were also recognized in most of the trees based on each gene, and the combined data of D1–D2 and RPB2, although some of them were not strongly supported. Four clades represented Albotricha, Brunnipila, Incrucipulum, and Lachnellula, respectively, whereas Lachnum was distributed to the remaining three clades. The molecular phylogenies strongly supported a group of species with granulate hairs, and we suggest the concept of Lachnaceae should be restricted to these species. Based on the molecular phylogenetic analysis, three new combinations—Incrucipulum longispineum, I. radiatum, and Lachnellula pulverulentum from Lachnum—are proposed.     

在墨尔本召开的第18届国际植物学大会做出的有关名称发表要求的变化——电子出版物对你意味着什么？

《国际植物命名法规》的修订由每六年一次的国际植物学大会（IBC）命名分会来决定。第18届国际植物学大会在澳大利亚墨尔本举行;命名分会于2011年7月18日至22日召开,其决议获得7月30日的全体会议通过。“墨尔本法规”有几个重要的变化,将影响新名称的发表。这些变化中的两个将在“墨尔本法规”出版前的几个月,即于2012年1月1日起生效。通过以移动文档格式（Portable Document Format;pdf）在线发表的具有国际标准连续出版物号（ISSN）或国际标准图书编号（ISBN）的电子出版物,将构成有效发表。新分类群名称的合格发表所必须的拉丁文描述或特征集要将更改为拉丁文或英文描述或特征集要。此外,自2013年1月1日起,被处理为真菌的生物的新名称必须在原始资料（某一名称合格发表时与之有关的所有资料）中引证一个由一家公认的存储库（例如MycoBank）签发的标识码,才构成合格发表。本文提供了有关电子出版物的新规则的草案文本,并概述了相应的最佳做法。     

A generic rate equation for catalysed,template-directed polymerisation

Biosynthetic networks link to growth and reproduction processes through template-directed synthesis of macromolecules such as polynucleotides and polypeptides. No rate equation exists that captures this link in a way that it can effectively be incorporated into a single computational model of the overall process. This paper describes the derivation of such a generic steady-state rate equation for catalysed, template-directed polymerisation reactions with varying monomer stoichiometry and varying chain length. The derivation is based on a classical Michaelis–Menten mechanism with template binding and an arbitrary number of chain elongation steps that produce a polymer composed of an arbitrary number of monomer types. The rate equation only requires the identity of the first dimer in the polymer sequence; for the remainder only the monomer composition needs be known. Further simplification of a term in the denominator yielded an equation requiring no positional information at all, only the monomer composition of the polymer; this equation still gave an excellent estimate of the reaction rate provided that either the monomer concentrations are at least half-saturating, or the polymer is very long.     

Interspecific competition between two species of the bean weevil

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Contributions from the Entomological Laboratory, Kyoto University, No. 110.