PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS

Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.

Continuous-flow polymerase chain reaction microfluidics by using spiral capillary channel embedded on copper

This paper presents a novel method for performing polymerase chain reaction (PCR) amplification by using spiral channel fabricated on copper where a transparent polytetrafluoroethylene ( PTFE) capillary tube was embedded. The channel with 25 PCR cycles was gradually developed in a spiral manner from inner to outer. The durations of PCR mixture at the denaturation, annealing and extension zones were gradually lengthened at a given flow rate, which may benefit continuous-flow PCR amplification as the synthesis ability of the Taq polymerase enzyme usually weakens with PCR time. Successful continuous-flow amplification of DNA fragments has been demonstrated. The PCR products of 249, 500 and 982 bp fragments could be obviously observed when the flow rates of PCR mixture were 7.5, 7.5 and 3.0 mm s(-1), respectively, and the required amplification times were about 25, 25, and 62 min, respectively. Besides, the successful segmented-flow PCR of three samples ( 249, 500 and 982 bp) has also been reported, which demonstrates the present continuous-flow PCR microfluidics can be developed for high-throughput genetic analysis.

新外显子的起源与功能及短同源序列介导转录滑动产生嵌合RNA 的研究

新外显子的起源是一种重要的增加转录组和蛋白质组多样性的分子机制。 对于新外显子及其父本基因的进化和功能特征方面还有很多重要的问题有待于 解决。本研究首先在全基因组水平上鉴定在人和小鼠中产生的新外显子，随后 对这些外显子及其父本基因作进化和功能上的分析。我们发现新外显子倾向于 位于基因的UTR 区域，尤其是5’ UTR 区域，这表明可能有些新外显子的出现 与基因的表达调控相关。我们还发现，产生新外显子的基因具有较高的组织表 达特异性，其基因功能倾向于细胞调控和与外界环境相互作用。通过对外群中 直系同源基因的分析，我们的结果表明进化速率较高的基因更容易获得新的外 显子，纠正了先前认为的获得新外显子会加速基因进化速率的看法。 我们对哺乳类CDYL 基因家族中产生的新外显子进行了具体的进化分析和 功能研究。我们的结果表明CDYL 基因在哺乳类分化前在原先的基因上游区域 获得了一个新的启动子和三个新的外显子。随后在哺乳动物各个支系的分化中， CDYL 基因在小鼠，狗和人中分别独立的进化出一个新的外显子。同源比对的 结果表明，这些新外显子是通过内含子序列的外显子化这一分子机制产生。近 缘物种间的进化速率的计算结果表明这些新产生的外显子具有快速进化的模 式，并且其快速进化可能是由正选择所驱动。在人中，多种突变包括新外显子 的获得，启动子的改变，选择性剪切的发生使得人的CDYL 基因获得了一种新 的编码更长蛋白质的剪切体。在人Hela 细胞系中的实验表明，新产生的蛋白质 与原有的蛋白质相比都具有显著的转录抑制活性，但新的蛋白质的转录抑制活 性较弱，且两者之间存在相互干扰的关系。这一结果表明通过新外显子的获得 产生的新的蛋白质可以丰富原有的基因表达调控体系，使得生物体的调控网络 更加精确。 嵌合RNA 通常认为是由来源于不同的pre-mRNA 的外显子通过反式剪切连 接在一起形成的。这一现象在包括多种动物和植物中被广泛的报道。我们的研 究首先通过大规模表达序列（ESTs）的搜索，在酵母，果蝇，小鼠和人中鉴定 到了大量的嵌合RNA。这一结果表明形成嵌合RNA 在真核生物中是一种普遍 的生物学过程，是一种重要的增加转录组和蛋白质组的多样性的分子机制。对 嵌合RNA 的序列分析表明，仅有<20%的嵌合RNA 在接合处可以找到典型的剪切位点 GU-AG，可以用经典的反式剪切模型来解释其产生机制。然而有意思的 是，我们在大约一半的嵌合RNA 的供体基因之间找到了短的同源序列，这一发 现使我们提出了一种新的分子机制来解释这些嵌合RNA 的形成，我们称之为 “转录滑动”模型。在酵母我们，我们用实验的方法验证了短同源序列对形成嵌 合RNA 的必要性，有力地支持了我们这一模型。

干扰RNA(RNAi)作用机理及其在植物细胞工程中的应用进展

随着RNAi调控目的基因表达机理研究的深入,RNAi技术也发展为一种强有力的实验工具,用来控制目的基因的表达以获取预期的生物表型。目前在植物中至少发现存在三种不同的RNAi途径,这些途径中基因沉默信号可以放大、传递和自我调控。为了建立高效、经济的RNAi技术体系,必须解决以下几个问题:即RNAi的有效传递,稳定性的提高,非目标效应出现的减小以及目标RNA敏感位点的确定等。综述了RNAi作用机制及其在植物细胞工程中的最新应用进展,并详细探讨了其技术体系。

果树组织中总RNA提取的新方法

本研究设计了一种新的RNA提取方法 ,解决了RNA提取时容易被降解和污染这一关键问题。通过加入Rnase抑制剂 ,消除了同外源RNase对RNA的降解 ,结合DNA难呈低盐溶液(140mmol·L -1NaCl)的原理 ,去除了DNA对RNA提取液的污染 ;先后使用酚和氯仿 ,有效地去除了蛋白质和酚类物的污染 ,利用抗氧化剂PVP和巯基乙醇 ,消除了内源酚类物质氧化变色对病毒RNA逆转录的影响。采用上述方法可以在4～5h内得到纯度高、含量大、完整性好的果树总RNA ,并获得了逆转录活性较强的病毒RNA ,同时使提取RNA的成本降低。这些方法对苹果、葡萄、桃、樱桃等果树总RNA的提取均适用。

Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Coding for 16S rRNA

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Soil microbial community analysis using denaturing gradient gel electrophoresis

The most biological diversity on this planet is probably harbored in soils. Understanding the diversity and function of the microbiological component of soil poses great challenges that are being overcome by the application of molecular biological approaches. This review covers one of many approaches being used: separation of polymerase chain reaction (PCR) amplicons using denaturing gradient gel electrophoresis (DGGE). Extraction of nucleic acids directly from soils allows the examination of a community without the limitation posed by cultivation. Polymerase chain reaction provides a means to increase the numbers of a target for its detection on gels. Using the rRNA genes as a target for PCR provides phylogenetic information on populations comprising communities. Fingerprints produced by this method have allowed spatial and temporal comparisons of soil communities within and between locations or among treatments. Numerous samples can be compared because of the rapid high throughput nature of this method. Scientists now have the means to begin addressing complex ecological questions about the spatial, temporal, and nutritional interactions faced by microbes in the soil environment.

Soil microbial community analysis using terminal restriction fragment length polymorphisms

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR-fingerprinting methods. Sizing of terminal restriction fragments (T-RFs) can now be done using capillary sequencing technology allowing samples contained in 96- or 384-well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T-RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T-RFLP data. Due to biases inherent in the method, linking the size of T-RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T-RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T-RFLP method is a user-friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.

Introduction to Molecular Analysis of Ectomycorrhizal Communities

A number of methods are available for those researchers considering the addition of molecular analyses of ectomycorrhizal (EcM) fungi to their research projects and weighing the various approaches they might take. Analyzing natural EcM fungal communities has traditionally been a highly skilled, time-consuming process relying heavily on exacting morphological characterization of EcM root tips. Increasingly powerful molecular methods for analyzing EcM communities make this area of research available to a much wider range of researchers. Ecologists can gain from the body of work characterizing EcM while avoiding the requirement for exceptional expertise by carefully combining elements of traditional methods with the more recent molecular approaches. A cursory morphological analysis can yield a traditional quantification of EcM fungi based on tip numbers, a unit with functional and historical significance. Ectomycorrhizal root DNA extracts may then be analyzed with molecular methods widely used for characterizing microbiota. These range from methods applicable only to the simple mixes resulting from careful morphotyping, to community-oriented methods that identify many types in mixed samples as well as provide an estimate of their relative abundances. Extramatrical hyphae in bulk soil can also be more effectively studied, extending characterization of EcM fungal communities beyond the rhizoplane. The trend toward techniques permitting larger sample sets without prohibitive labor and time requirements will also permit us to more frequently address the issues of spatial and temporal variability and better characterize the roles of EcM fungi at multiple scales.

土壤中Frankia菌RNA提取

土壤中Ｆｒａｎｋｉａ菌ＲＮＡ提取王育英，林建群，郭秀荣，李彤，张忠泽（中国科学院沈阳应用生态研究所，１１００１５）Ｆｒａｎｋｉａ非亚科共生固氮是一类重要的固氮资源。对Ｆｒａｎｋｉａ共生生物学、生态学等特性曾进行过广泛的研究。８ｔ０年代末，Ｆｒａｎｋｉ...

Targeted RNA interference of cyclin A(2) mediated by functionalized single-walled carbon nanotubes induces proliferation arrest and apoptosis in chronic myelogenous leukemia K562 cells

Cyclin A(2) plays critical role in DNA replication, transcription, and cell cycle regulation. Its overexpression has been detected and related to many types of cancers including leukemia, suggesting that suppression of cyclin A(2) would be an attractive strategy to prevent tumor progression. Herein, we apply functionalized single wall carbon nanotubes (f-SWNTs) to carry small interfering RNA (siRNA) into K562 cells and determine whether inhibition of cyclin A(2) would be a potential therapeutic target for chronic myelogenous leukemia.

Changes in RNA, DNA, protein contents and growth of turbot Scophthalmus maximus larvae and juveniles

The growth potential of turbot Scophthalmus maximus larvae and juveniles was studied using nucleic acid-based indices and protein variables. The experiment was carried out from 4 to 60 days post hatching (dph). A significant increase in instantaneous growth rate during metamorphosis and retarded growth rate during post-metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein all showed developmental stage-specific traits. The RNA:DNA ratio decreased up to 12 dph, then increased rapidly till 19 dph and fluctuated until 35 dph followed by a decline to the end. The RNA:DNA ratio was positively correlated with growth rate of juveniles during the post-metamorphic phase, whereas this ratio was not a sensitive indicator of growth during the pre-metamorphic phase and metamorphosis. The protein:DNA ratio showed a similar tendency to the RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of S. maximus performed mainly by hyperplasia from 4 to 12 dph and hypertrophy until 21 dph during the pre-metamorphic larval phase. Growth was dominantly hypertrophical from the early- to mid-metamorphosing phase and hyperplastic thereafter. The results show that the DNA content and protein:DNA ratio can evaluate growth rates of larval and juvenile S. maximus on a cellular level.

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.