In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus

An in vivo-directed evolutionary strategy was used to obtain a thermostabilized Escherichia coli hygromycin B phosphotransferase, using a host-vector system of Thermus thermophilus. Introduction of the mutant gene containing two amino acid substitutions, S52T and W238C, which was previously reported by Cannio et al. [J. Bacteriol., 180, 3237-3240 (1998)], did not confer hygromycin resistance on T. thermophilus cells at 55 degrees C; however, five spontaneously-generated independent mutants were obtained by selection of the transformants at this temperature. Each mutant gene contained one amino acid substitution of either A118V or T246A. Further selection with increasing temperature, at 58 degrees C and then 61 degrees C, led to acquisition of three more substitutions: D20G, S225P and Q226L. These mutations cumulatively influenced the maximum growth temperature of the T. thermophilus transformants in the presence of hygromycin; T. thermophilus carrying a mutant gene containing all the five substitutions was able to grow at up to 67 degrees C. This mutant gene, hph5, proved useful as a selection marker in the T. thermophilus host-vector system, either on the plasmid or by genome integration, at temperatures up to 65 degrees C.     

Plasmids with the Cre-recombinase and the dominant nat marker, suitable for use in prototrophic strains of Saccharomyces cerevisiae and Kluyveromyces lactis

Two plasmids are described which can be used to remove the "loxP-markerMX-loxP" cassettes in strains lacking the ura3 mutation. Both contain the Cre-recombinase under control of the GAL1 promoter and the natMX cassette with the dominant marker nat, which gives yeasts resistance to the antibiotic ClonNat. pNatCre contains ARSH and CEN6 for maintenance in Saccharomyces cerevisiae. pKlNatCre has a Kluyveromyces lactis replication origin and centromere in addition.     

孤南洼陷低熟油成因分析

济阳坳陷孤南洼陷低熟油的地质储量可达数千万吨，类似的由沙一段和沙四段烃源岩形成的低熟油在渤海湾盆地普遍可见。以孤南洼陷为例，讨论了低熟油勘探中的有关问题。明确提出判别低熟原油的指标是其Ｃ２９甾烷的异构化系数（其值０．３０～０．３５可作为上限值），ＯＥＰ及Ｐｒ／ｎＣ１７和Ｐｒ／ｎＣ１８可作为辅助指标。低熟原油可以是中等密度的高蜡油，其组分特征是高饱芳比和高非沥比。孤南洼陷生成低熟油的沙一段烃源岩为富含藻类有机质的油页岩，形成于半咸水—咸水、强还原的沉积环境。孤南洼陷低熟油形成阶段的Ｒｏ值上限为０．４０％～０．４５％，相应的深度为２３００～２５００ｍ；Ｒｏ值下限为０．６０％～０．６５％，相应的深度为３０００～３２００ｍ。咸水环境由富含藻类的有机质早期生烃，可能是最主要的低熟油形成机制。图２表４参８（邓春萍摘）     

莺琼盆地气源研究再认识

采用天然气重烃富集及色质分析技术，从莺琼盆地ＹＡ１３－１气田和ＬＤ２２－１气田的天然气中，检测到甾萜、芳烃、金刚烷等生物标记化合物，并结合其它有机地球化学资料，应用新的天然气有机地球研究方法，进行了气源综合对比研究，认为ＹＡ１３－１气田和ＬＤ２２－１气田的气源岩同为埋深大于５ｋｍ的高成熟海相气源岩，对比结果还表明下第三系海相泥岩可能是莺琼盆地的重要气源岩之一。     

单速率三色标记在区分业务模型中的应用

传统的IP技术已不能满足当前网络的发展，为了提供更好的服务质量，IETF制定了区分业务体系结构，本文在简要介绍区分业务模型的体系结构的基础上，重点讨论了单速率三色标记的原理及其在区分业务模型中如何实现流量调节。     

A micromethod for the differentiation of fresh from frozen fish muscle

A sensitive and rapid routine method was developed which allows a general, unambiguous and reliable differentiation between fresh and frozen-thawed fish muscle. The suggested procedure consists of (i) mild extraction of muscle in isotonic and buffered medium, (ii) rapid separation of the marker isozymes—those of the mitochondrial aspartate aminotransferase—by cellulose acetate electrophoresis, (iii) specific staining at neutral pH for enzyme activity by sandwich with an agarose-substrate-chromogen pad, and (iv) a clearing and drying process to make a permanent record.     

335铀钼矿床地质特征及找矿方向

在前人铀矿详查工作的基础上,系统分析总结了335铀钼矿床地质特征,归纳了矿化富集因素及找矿标志。2009年在矿区通过新一轮普查找矿工作,圈出了物化探综合异常,经钻探工程验证发现了铀钼矿体,取得了较好的找矿成果。最后,在综合分析成矿地质条件和物化探找矿成果的基础上,指出了矿区找矿方向。     

De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus densiflora)

Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora.     

New cassettes for single‐step drug resistance and prototrophic marker switching in fission yeast

Construction of multiply mutated strains for genetic interaction analysis and of strains carrying different epitope tags at multiple open reading frames for testing protein localization, abundance and protein–protein interactions is hampered by the availability of a sufficient number of different selectable markers. Moreover, strains with single gene deletions or tags often already exist in strain collections; for historical reasons these will mostly carry the ura4⁺ gene or the G418‐resistance kanMX as marker. Because it is rather cumbersome to produce multiply deleted or tagged strains using the same marker, or to completely reconstruct a particular strain with a different marker, single‐step exchange protocols of markers are a time‐saving alternative. In recent years, dominant drug resistance markers (DDRMs) against clonNAT, hygromycin B and bleomycin have been adapted and successfully used in Schizosaccharomyces pombe. The corresponding DDRM cassettes, natMX, hphMX and bleMX, carry the TEF promotor and terminator sequences from Ashbya gossypii as kanMX; this provides flanking homologies to enable single‐step marker swapping by homologous gene targeting. To expand this very useful toolset for single‐step marker exchange, I constructed MX cassettes containing the nutritional markers arg3⁺, his3⁺, leu1⁺ and ura4⁺. Furthermore, a set of constructs was created to enable single‐step exchange of ura4⁺ to kanMX6, natMX4 and hphMX4. The functionality of the cassettes is demonstrated by successful single‐step marker swapping at several loci. These constructs allow straightforward and rapid remarking of existing ura4⁺‐ and MX‐deleted and ‐tagged strains. Copyright © 2015 John Wiley & Sons, Ltd.     

基于特征肽测定阿胶食品中的5种杂皮源成分

建立了以特征肽为指标的超高效液相色谱串联质谱测定阿胶食品中马皮等5种杂皮源成分的检测方法。考察了样品提取方式、样品酶解条件,比较了不同色谱条件对各杂皮源成分的分离效果,优化了流动相洗脱体系。样品经处理后,于37 ℃下经胰蛋白酶酶解,产生马源寡肽A等杂皮源特征肽段,以0.1%甲酸水溶液和0.1%甲酸乙腈溶液作为流动相进行梯度洗脱,流速0.3 mL/min,柱温40 ℃,进样量2 µL,在电喷雾正离子（ESI+）模式下进行多反应监测（MRM）,基质匹配标曲定量。马源寡肽A、鹿皮特征肽在5~500 ng/mL、牛皮特征肽、猪皮特征肽、骆驼皮特征肽在10~500 ng/mL浓度范围内线性关系良好,相关系数r均大于0.999,方法定量限为0.082~1.1 mg/kg,加标回收率为86.8%~103.5%。29批次阿胶糕、阿胶饮品样品检出了马、牛皮源成分。该方法专属性强、灵敏度高、操作简单、定量准确,适用于阿胶糕等食品中杂皮源成分的检测。