Hemocyanin gene family evolution in spiders (Araneae), with implications for phylogenetic relationships and divergence times in the infraorder Mygalomorphae

Hemocyanins are multimeric copper-containing hemolymph proteins involved in oxygen binding and transport in all major arthropod lineages. Most arachnids have seven primary subunits (encoded by paralogous genes a–g), which combine to form a 24-mer (4 × 6) quaternary structure. Within some spider lineages, however, hemocyanin evolution has been a dynamic process with extensive paralog duplication and loss. We have obtained hemocyanin gene sequences from numerous representatives of the spider infraorders Mygalomorphae and Araneomorphae in order to infer the evolution of the hemocyanin gene family and estimate spider relationships using these conserved loci. Our hemocyanin gene tree is largely consistent with the previous hypotheses of paralog relationships based on immunological studies, but reveals some discrepancies in which paralog types have been lost or duplicated in specific spider lineages. Analyses of concatenated hemocyanin sequences resolved deep nodes in the spider phylogeny and recovered a number of clades that are supported by other molecular studies, particularly for mygalomorph taxa. The concatenated data set is also used to estimate dates of higher-level spider divergences and suggests that the diversification of extant mygalomorphs preceded that of extant araneomorphs. Spiders are diverse in behavior and respiratory morphology, and our results are beneficial for comparative analyses of spider respiration. Lastly, the conserved hemocyanin sequences allow for the inference of spider relationships and ancient divergence dates.     

Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitate Efficient in Vitro Selection and Maturation of Antibodies

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.     

Glyphosate effects on the gene expression of the apical bud in soybean (Glycine max)

Glyphosate is a broad spectrum, non-selective herbicide which has been widely used for weed control. Much work has focused on elucidating the high accumulation of glyphosate in shoot apical bud (shoot apex). However, to date little is known about the molecular mechanisms of the sensitivity of shoot apical bud to glyphosate. Global gene expression profiling of the soybean apical bud response to glyphosate treatment was performed in this study. The results revealed that the glyphosate inhibited tryptophan biosynthesis of the shikimic acid pathway in the soybean apical bud, which was the target site of glyphosate. Glyphosate inhibited the expression of most of the target herbicide site genes. The promoter sequence analysis of key target genes revealed that light responsive elements were important regulators in glyphosate induction. These results will facilitate further studies of cloning genes and molecular mechanisms of glyphosate on soybean shoot apical bud.     

Nuclear and chloroplast DNA phylogeography reveals Pleistocene divergence and subsequent secondary contact of two genetic lineages of the tropical rainforest tree species Shorea leprosula (Dipterocarpaceae) in South‐East Asia

Tropical rainforests in South‐East Asia have been affected by climatic fluctuations during past glacial eras. To examine how the accompanying changes in land areas and temperature have affected the genetic properties of rainforest trees in the region, we investigated the phylogeographic patterns of a widespread dipterocarp species, Shorea leprosula. Two types of DNA markers were used: expressed sequence tag‐based simple sequence repeats and chloroplast DNA (cpDNA) sequence variations. Both sets of markers revealed clear genetic differentiation between populations in Borneo and those in the Malay Peninsula and Sumatra (Malay/Sumatra). However, in the south‐western part of Borneo, genetic admixture of the lineages was observed in the two marker types. Coalescent simulation based on cpDNA sequence variation suggested that the two lineages arose 0.28–0.09 million years before present and that following their divergence migration from Malay/Sumatra to Borneo strongly exceeded migration in the opposite direction. We conclude that the genetic structure of S. leprosula was largely formed during the middle Pleistocene and was subsequently modified by eastward migration across the subaerially exposed Sunda Shelf.     

Bioavailable affinity label for collagen prolyl 4-hydroxylase

Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification catalyzed by the nonheme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report that 4-oxo-5,6-epoxyhexanoate, a mimic of the α-ketoglutarate co-substrate, inactivates human P4H. The inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caenorhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H. Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treatment of a variety of fibrotic diseases.     

从烟草EST微卫星标记中筛选合子胚中优势表达的基因

为获得烟草合子胚中的优势表达基因,利用CAP3程序对来自烟草公共数据库的EST序列进行组装,利用MISA程序从组装后的EST中筛选SSR位点,将多态性的SSR位点在烟草合子文库中进行扩增并对等位基因进行分析。结果表明:具有多态性的16个SSR标记中,有9个基因能从烟草的合子库中成功扩增得到。该研究为筛选烟草合子胚中优势表达基因提供新的途径。     

Development of R4 Gateway Binary Vectors (R4pGWB) Enabling High-Throughput Promoter Swapping for Plant Research

We developed a new series of Gateway binary vectors, R4pGWBs, that are plant transformation vectors designed for one-step construction of chimeric genes between any promoter and any cDNA. The structure of R4pGWBs is almost the same as the promoterless type of improved pGWBs (ImpGWBs), except that the attR1 site is replaced with attR4, which enables tripartite recombination of these vectors with promoter- and cDNA-entry clones. While ImpGWBs are suitable for promoter analysis and constitutive expression of cDNAs in higher plants, R4pGWBs have a great advantage in expressing a cDNA under the regulation of desired promoters.     

High‐throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label

Quantifying the concentration and purity of a target protein is essential for high‐throughput protein expression test and rapid screening of highly soluble proteins. However, conventional methods such as PAGE and dot blot assay generally involve multiple time‐consuming tasks requiring hours or do not allow instant quantification. Here, we demonstrate a new method based on the Photoactive yellow protein turn Off/On Label (POOL) system that can instantly quantify the concentration and purity of a target protein. The main idea of POOL is to use Photoactive Yellow Protein (PYP), or its miniaturized version, as a fusion partner of the target protein. The characteristic blue light absorption and the consequent yellow color of PYP is absent when initially expressed without its chromophore, but can be turned on by binding its chromophore, p‐coumaric acid. The appearance of yellow color upon adding a precursor of chromophore to the co‐expressed PYP can be used to check the expression amount of the target protein via visual inspection within a few seconds as well as to quantify its concentration and purity with the aid of a spectrometer within a few minutes. The concentrations measured by the POOL method, which usually takes a few minutes, show excellent agreement with those by the BCA Kit, which usually takes ～1 h. We demonstrate the applicability of POOL in E. coli, insect, and mammalian cells, and for high‐throughput protein expression screening.     

Assessing in vivo dynamics of multiple quality attributes from a therapeutic IgG4 monoclonal antibody circulating in cynomolgus monkey

Characterization of biopharmaceutical proteins and assessment and understanding of the critical quality attributes (CQAs) is a significant part of biopharmaceutical product development and is routinely performed in vitro. In contrast, systematic analysis of the quality attributes in vivo is not as widespread, although metabolism and clearance of multiple variants of therapeutic proteins administered to non-human primates and human subjects may have a different impact on safety, efficacy and exposure. The major hurdles of such studies are usually sample availability and technical capability. In this study, we used affinity purification coupled with liquid chromatography and mass spectrometric analysis of the digested protein for consistent and simultaneous detection of the full amino acid sequence of a therapeutic IgG4 monoclonal antibody, MAB1. This methodology allowed us to assess in vivo changes of all sequence-related modifications and quality attributes of MAB1 over the duration of a preclinical pharmacokinetic study in cynomolgus monkeys.