The Evolutionary History of the Chymase Locus -a Locus Encoding Several of the Major Hematopoietic Serine Proteases

Several hematopoietic cells of the immune system store large amounts of proteases in cytoplasmic granules. The absolute majority of these proteases belong to the large family of chymotrypsin-related serine proteases. The chymase locus is one of four loci encoding these granule-associated serine proteases in mammals. The chymase locus encodes only four genes in primates, (1) the gene for a mast-cell-specific chymotryptic enzyme, the chymase; (2) a T-cell-expressed asp-ase, granzyme B; (3) a neutrophil-expressed chymotryptic enzyme, cathepsin G; and (4) a T-cell-expressed chymotryptic enzyme named granzyme H. Interestingly, this locus has experienced a number of quite dramatic expansions during mammalian evolution. This is illustrated by the very large number of functional protease genes found in the chymase locus of mice (15 genes) and rats (18 genes). A separate expansion has also occurred in ruminants, where we find a new class of protease genes, the duodenases, which are expressed in the intestinal region. In contrast, the opossum has only two functional genes in this locus, the mast cell (MC) chymase and granzyme B. This low number of genes may be the result of an inversion, which may have hindered unequal crossing over, a mechanism which may have been a major factor in the expansion within the rodent lineage. The chymase locus can be traced back to early tetrapods as genes that cluster with the mammalian genes in phylogenetic trees can be found in frogs, alligators and turtles, but appear to have been lost in birds. We here present the collected data concerning the evolution of this rapidly evolving locus, and how these changes in gene numbers and specificities may have affected the immune functions in the various tetrapod species.     

In vivo mutational analysis of the mupirocin gene cluster reveals labile points in the biosynthetic pathway: the "leaky hosepipe" mechanism

A common feature of the mupirocin and other gene clusters of the AT-less polyketide synthase (PKS) family of metabolites is the introduction of carbon branches by a gene cassette that contains a beta-hydroxy-beta-methylglutaryl CoA synthase (HMC) homologue and acyl carrier protein (ACP), ketosynthase (KS) and two crotonase superfamily homologues. In vivo studies of Pseudomonas fluorescens strains in which any of these components have been mutated reveal a common phenotype in which the two major isolable metabolites are the truncated hexaketide mupirocin H and the tetraketide mupiric acid. The structure of the latter has been confirmed by stereoselective synthesis. Mupiric acid is also the major metabolite arising from inactivation of the ketoreductase (KR) domain of module 4 of the modular PKS. A number of other mutations in the tailoring region of the mupirocin gene cluster also result in production of both mupirocin H and mupiric acid. To explain this common phenotype we propose a mechanistic rationale in which both mupirocin H and mupiric acid represent the products of selective and spontaneous release from labile points in the pathway that occur at significant levels when mutations block the pathway either close to or distant from the labile points.     

UV-Induced Cell Death in Plants

Plants are photosynthetic organisms that depend on sunlight for energy. Plants respond to light through different photoreceptors and show photomorphogenic development. Apart from Photosynthetically Active Radiation (PAR; 400–700 nm), plants are exposed to UV light, which is comprised of UV-C (below 280 nm), UV-B (280–320 nm) and UV-A (320–390 nm). The atmospheric ozone layer protects UV-C radiation from reaching earth while the UVR8 protein acts as a receptor for UV-B radiation. Low levels of UV-B exposure initiate signaling through UVR8 and induce secondary metabolite genes involved in protection against UV while higher dosages are very detrimental to plants. It has also been reported that genes involved in MAPK cascade help the plant in providing tolerance against UV radiation. The important targets of UV radiation in plant cells are DNA, lipids and proteins and also vital processes such as photosynthesis. Recent studies showed that, in response to UV radiation, mitochondria and chloroplasts produce a reactive oxygen species (ROS). Arabidopsis metacaspase-8 (AtMC8) is induced in response to oxidative stress caused by ROS, which acts downstream of the radical induced cell death (AtRCD1) gene making plants vulnerable to cell death. The studies on salicylic and jasmonic acid signaling mutants revealed that SA and JA regulate the ROS level and antagonize ROS mediated cell death. Recently, molecular studies have revealed genes involved in response to UV exposure, with respect to programmed cell death (PCD).     

Characterization of an Environmental DNA‐Derived Gene Cluster that Encodes the Bisindolylmaleimide Methylarcyriarubin

Bisindolylmaleimides represent a naturally occurring class of metabolites that are of interest because of their protein kinase inhibition activity. From a metagenomic library constructed with soil DNA, we identified the four gene mar cluster, a bisindolylmaleimide gene cluster that encodes for methylarcyriarubin ( 1 ) production. Heterologous expression of the mar gene cluster in E. coli revealed that the Rieske dioxygenase MarC facilitates the oxidative decarboxylation of a chromopyrrolic acid (CPA) intermediate to yield the bisindolylmaleimide core. The characterization of the mar cluster defines a new role for CPA in the biosynthesis of structurally diverse bacterial tryptophan dimers.     

Aspergillus oryzae SAICAR合成酶基因的克隆及反义表达载体的构建

目的 构建Aspergillus oryzae新的红色表现型菌株用于天然色素生产。方法 构建Aspergillus oryzaeRIB40 cDNA文库,应用BLAST网络服务对Aspergillus oryzae RIB40 cDNA文库的EST数据进行同源性比较,从EST克隆扩增Aspergillus oryzae类SAICAR合成酶基因(sh1919f片段),反向插入pUSA真核表达载体。结果 完成反义表达载体的构建。Aspergillus oryzae类SAICAR合成酶氨基酸序列与Pichia jadinii、Saccharomyces cerevisiae和Schizosac-charomyces pombe的SAICAR合成酶氨基酸序列具有高度同源性。结论 类SAICAR合成酶基因反义表达载体的构建为其转染入Aspergillus oryzae菌株筛选红色突变菌株奠定了实验基础。     

固定化米根霉发酵生产乳酸的研究进展

根霉属中的米根霉是迄今认为生产L-乳酸较为理想的菌种.利用游离的米根霉发酵乳酸时会产生菌种利用率低、生产效率低等一系列不足,因此展开了菌体固定化发酵的研究.目前米根霉固定化发酵工程的研究主要集中于载体的选择和新型生物反应器的研制.本文对此研究进展作了综合评述与讨论,认为米根霉固定化发酵乳酸可以进一步降低生产成本,提高乳酸产率.     

米根霉发酵生产L(+)-乳酸研究进展

米根霉是生产绿色平台生物化学品L(+) 乳酸的理想菌种 ,目前集中在发酵工艺的优化、新型生物反应器的设计以及细胞固定化等方面的研究 ,通过控制米根霉菌体形态使之自聚集成为一定大小的球体进行乳酸发酵 ,操作简便、费用低。建议今后从应用代谢工程技术定向选育米根霉L(+) 乳酸高产菌 ,改进发酵设备、改良提取工艺 ,合理控制乳酸产品的构型等几个方面着手进行进一步研究 ,从而降低乳酸生产原料的成本 ,扩大L(+) 乳酸的应用领域。     

Advances and Perspectives in Tissue Culture and Genetic Engineering of Cannabis

For a long time, Cannabis sativa has been used for therapeutic and industrial purposes. Due to its increasing demand in medicine, recreation, and industry, there is a dire need to apply new biotechnological tools to introduce new genotypes with desirable traits and enhanced secondary metabolite production. Micropropagation, conservation, cell suspension culture, hairy root culture, polyploidy manipulation, and Agrobacterium-mediated gene transformation have been studied and used in cannabis. However, some obstacles such as the low rate of transgenic plant regeneration and low efficiency of secondary metabolite production in hairy root culture and cell suspension culture have restricted the application of these approaches in cannabis. In the current review, in vitro culture and genetic engineering methods in cannabis along with other promising techniques such as morphogenic genes, new computational approaches, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR/Cas9-equipped Agrobacterium-mediated genome editing, and hairy root culture, that can help improve gene transformation and plant regeneration, as well as enhance secondary metabolite production, have been highlighted and discussed.     

Pore network connectivity effects on gas separation in a microporous carbon membrane

In this paper, we present a critical path analysis (CPA) of transport through a “selective surface flow” membrane operating in a hydrogen purification application. Using experimental mixture permeability data as an input, the CPA allows us to estimate the structural parameters of the membrane. In addition, the CPA gives insight into the role of network connectivity, showing that the species selectively transported through the membrane (methane, in this case) and the species which the membrane is intended not to transport (hydrogen) pass through essentially distinct sub-networks within the pore network of the membrane.     

Molecular and Physiological Responses of Citrus sinensis Leaves to Long-Term Low pH Revealed by RNA-Seq Integrated with Targeted Metabolomics

Low pH-induced alterations in gene expression profiles and organic acids (OA) and free amino acid (FAA) abundances were investigated in sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] leaves. We identified 503 downregulated and 349 upregulated genes in low pH-treated leaves. Further analysis indicated that low pH impaired light reaction and carbon fixation in photosynthetic organisms, thereby lowering photosynthesis in leaves. Low pH reduced carbon and carbohydrate metabolisms, OA biosynthesis and ATP production in leaves. Low pH downregulated the biosynthesis of nitrogen compounds, proteins, and FAAs in leaves, which might be conducive to maintaining energy homeostasis during ATP deprivation. Low pH-treated leaves displayed some adaptive responses to phosphate starvation, including phosphate recycling, lipid remodeling, and phosphate transport, thus enhancing leaf acid-tolerance. Low pH upregulated the expression of some reactive oxygen species (ROS) and aldehyde detoxifying enzyme (peroxidase and superoxidase) genes and the concentrations of some antioxidants (L-tryptophan, L-proline, nicotinic acid, pantothenic acid, and pyroglutamic acid), but it impaired the pentose phosphate pathway and V_E and secondary metabolite biosynthesis and downregulated the expression of some ROS and aldehyde detoxifying enzyme (ascorbate peroxidase, aldo-keto reductase, and 2-alkenal reductase) genes and the concentrations of some antioxidants (pyridoxine and γ-aminobutyric acid), thus disturbing the balance between production and detoxification of ROS and aldehydes and causing oxidative damage to leaves.     

应用KMnO_4滴定法快速检测米根霉发酵液中富马酸含量

应用KMnO4滴定法快速检测米根霉发酵液中富马酸含量,通过研究富马酸滴定体系下的酸度以及发酵培养基中的葡萄糖和代谢副产物如乳酸、苹果酸、乙醇、草酰乙酸、丁二酸等对KMnO4滴定结果的影响,发现应用KMnO4滴定法测定米根霉发酵液中富马酸的含量,与HPLC检测法测定的含量误差在3%以内,平均回收率为96.86%,相对标准偏差(RSD)为0.27%.以上结论表明,KMnO4滴定法可有效应用于快速检测米根霉发酵液中富马酸的含量.     

Diversity and biosynthetic potential of culturable actinomycetes associated with marine sponges in the china seas

The diversity and secondary metabolite potential of culturable actinomycetes associated with eight different marine sponges collected from the South China Sea and the Yellow sea were investigated. A total of 327 strains were isolated and 108 representative isolates were selected for phylogenetic analysis. Ten families and 13 genera of Actinomycetales were detected, among which five genera represent first records isolated from marine sponges. Oligotrophic medium M5 (water agar) proved to be efficient for selective isolation, and "Micromonospora-Streptomyces" was proposed as the major distribution group of sponge-associated actinomycetes from the China Seas. Ten isolates are likely to represent novel species. Sponge Hymeniacidon perleve was found to contain the highest genus diversity (seven genera) of actinomycetes. Housekeeping gene phylogenetic analyses of the isolates indicated one ubiquitous Micromonospora species, one unique Streptomyces species and one unique Verrucosispora phylogroup. Of the isolates, 27.5% displayed antimicrobial activity, and 91% contained polyketide synthase and/or nonribosomal peptide synthetase genes, indicating that these isolates had a high potential to produce secondary metabolites. The isolates from sponge Axinella sp. contained the highest presence of both antimicrobial activity and NRPS genes, while those from isolation medium DNBA showed the highest presence of antimicrobial activity and PKS I genes.     

Evolution of MicroRNA Biogenesis Genes in the Sterlet (Acipenser ruthenus) and Other Polyploid Vertebrates

MicroRNAs play a crucial role in eukaryotic gene regulation. For a long time, only little was known about microRNA-based gene regulatory mechanisms in polyploid animal genomes due to difficulties of polyploid genome assembly. However, in recent years, several polyploid genomes of fish, amphibian, and even invertebrate species have been sequenced and assembled. Here we investigated several key microRNA-associated genes in the recently sequenced sterlet (Acipenser ruthenus) genome, whose lineage has undergone a whole genome duplication around 180 MYA. We show that two paralogs of drosha, dgcr8, xpo1, and xpo5 as well as most ago genes have been retained after the acipenserid-specific whole genome duplication, while ago1 and ago3 genes have lost one paralog. While most diploid vertebrates possess only a single copy of dicer1, we strikingly found four paralogs of this gene in the sterlet genome, derived from a tandem segmental duplication that occurred prior to the last whole genome duplication. ago1,3,4 and exportins1,5 look to be prone to additional segment duplications producing up to four-five paralog copies in ray-finned fishes. We demonstrate for the first time exon microsatellite amplification in the acipenserid drosha2 gene, resulting in a highly variable protein product, which may indicate sub- or neofunctionalization. Paralogous copies of most microRNA metabolism genes exhibit different expression profiles in various tissues and remain functional despite the rediploidization process. Subfunctionalization of microRNA processing gene paralogs may be beneficial for different pathways of microRNA metabolism. Genetic variability of microRNA processing genes may represent a substrate for natural selection, and, by increasing genetic plasticity, could facilitate adaptations to changing environments.     

TILLING-by-Sequencing+ to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis

Reverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their DNA sequence. Chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. The current study uses an optimized high-throughput TILLING by target capture sequencing technology, or TILLING-by-Sequencing⁺ (TbyS⁺), coupled with universal bioinformatic tools to identify population-wide mutations in soybeans. Four ethyl methanesulfonate mutagenized populations (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. The mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. To test the efficiency of TbyS⁺ in complex genomes, we used soybeans as a model with a focus on three desaturase gene families, GmSACPD, GmFAD2, and GmFAD3, that are involved in the soybean fatty acid biosynthesis pathway. We successfully isolated mutants from all the six gene family members. Unsurprisingly, most of the characterized mutants showed significant changes either in their stearic, oleic, or linolenic acids. By using TbyS⁺, we discovered novel sources of soybean oil traits, including high saturated and monosaturated fatty acids in addition to low polyunsaturated fatty acid contents. This technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. The obtained soybean mutants from this study can be used in subsequent soybean breeding programs for improved oil composition traits.     

Gene expression profiling in streptozotocin-induced diabetic rat liver in response to fungal polysaccharide treatment

We established the gene expression profiling in streptozotocin (STZ)-induced diabetic rat liver in response to hypoglycemic fungal polysaccharides (EPS), using oligonucleotide microarray analysis. Differentially regulated genes showing higher fold change than 2 were identified and categorized through hierarchical clustering analysis. Among the 835 genes analyzed, 244 were up-regulated, while 321 were down-regulated after diabetes induction. Interestingly, many gene expressions altered after STZ-treatment mimicked a normal rat liver by EPS therapy. Most of these genes included genes involving cell structure and motility, immunity and defense, lipid, fatty acid and steroid metabolism, protein metabolism and modification, and signal transduction. More importantly, we found a total of 36 genes that showed significant fold changes in their expression that have not previously been examined in the context of diabetes. To validate the microarray results, we further confirmed the gene expression patterns by RT-PCR using four genes of interest (carboxylesterase 2, stearoyl-coenzyme A desaturase 1, insulin-like growth factor 1, and insulin-like growth factor binding protein 2). Taken together, EPS acted as a potent regulator of gene expression for a wide variety of genes in diabetic rat liver.