Total Synthesis and Expressions in E. coli of Three Redesigned Genes for the Mature Small Subunit (rbcS) of Rubisco Fron Tobacco, Wheat and Rice

Three structural genes, which code for the mature small subunits (rbcS) of ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco, wheat and rice, respectively,have been redesignedwith the aid of a computer,totally synthesized by combining chemical method and enzymatic ligation,and expressed in E. coli with high yields.     

Bidirectional Regulation of mRNA Translation in Mammalian Cells by Using PUF Domains

The regulation of gene expression is crucial in diverse areas of biological science, engineering, and medicine. A genetically encoded system based on the RNA binding domain of the Pumilio and FBF (PUF) proteins was developed for the bidirectional regulation (i.e., either upregulation or downregulation) of the translation of a target mRNA. PUF domains serve as designable scaffolds for the recognition of specific RNA elements and the specificity can be easily altered to target any 8‐nucleotide RNA sequence. The expression of a reporter could be varied by over 17‐fold when using PUF‐based activators and repressors. The specificity of the method was established by using wild‐type and mutant PUF domains. Furthermore, this method could be used to activate the translation of target mRNA downstream of PUF binding sites in a light‐dependent manner. Such specific bidirectional control of mRNA translation could be particularly useful in the fields of synthetic biology, developmental biology, and metabolic engineering.     

Chemical synthesis of a structural gene coding for trichosanthin

A structural gene (750 bp), which codes for a type I ribosome inactivating protein, trichosanthin, has been designed according to the codon usage of highly expressed gene in E. coli and chemically synthesized. In the synthesized gene, twenty-seven unique restriction sites were evenly dispersed with an average distance between two adjacent sites less than 50 bp to facilitate a systematic investigation on structure-functional relationship of this protein by site-directed muta-genesis. To synthesize it, the whole gene was divided into three large fragments (EP, PN and NH) which were assembled from several chemical synthetic oligonucleotides by enzymatic method. The assembly of both the fragment EP from six oligonucleotides (A-F) and the fragment PN from four oligomers (G-J) was catalyzed by T4 DNA ligase in using the single stranded DNA method [Chen, H.-B. et al, Nucl. Acids Res., 18, 871(1990)]. And fragment NH was formed from three duplexes K, L and M by the classical double stranded DNA method. Finally,     

Noise-induced coherent switch

Taking the famous genetic toggle switch as an example,we numerically investigated the effect of noise on bistability.We found that extrinsic noise resulting from stochastic fluctuations in synthesis and degradation rates and from the environmental fluctuation in gene regulatory processes can induce coherent switch,and that there is an optimal noise intensity such that the noise not only can induce this switch,but also can amplify a weak input signal.In addition,we found that the intrinsic noise introduced through the Poisson τ-leap algorithm cannot induce such a switch.     

pH敏感基因载体的研究现状

基因治疗正成为遗传病、癌症等疾病的有效治疗方法. 基因治疗实现的最大挑战是开发安全有效的基因运载载体以将目的基因从血液运送到细胞质或细胞核. 目前, 常用的基因治疗载体有病毒载体和非病毒载体. 非病毒载体由于安全性好, 易于合成, 易于修饰而得到了更为广泛的研究. 其中pH敏感载体作为功能性非病毒载体, 不仅安全性高, 而且有更好的体内基因转染效率, 为非病毒性载体在临床上的应用开辟了广阔的前景. 本文主要从pH敏感脂质和pH敏感聚合物两方面对pH敏感基因载体进行简要综述, 介绍了该两类载体的构建方法及其对基因的运载机制.     

Phosphoester modified poly(ethylenimine) as efficient and low cytotoxic genevectors

Cyclic phosphoester monomer ethyl ethylene phosphate (EEP) modified poly(ethylenimine) (PEI),denoted as PEI-EEP,was developed for gene delivery.Three PEI-EEP polymers were synthesized and their structures were characterized by 1H and 31P NMR methods.All the PEI-EEP polymers could condense DNA efficiently at N/P ratios higher than 0.5/1.The physiochemical characteristics of PEI-EEP/DNA complexes were analyzed by particle size and zeta potential measurements.The particle sizes of complexes were around 160–250...     

aprE基因表达的分子生物学和微量热法分析

利用分子生物学方法(SDS-PAGE和酶活检测法)未检测到所克隆的aprE基因在大肠杆菌中的表达产物(碱性蛋白酶), 而微量热法检测结果发现: 重组菌株的生长代谢产热曲线之间存在明显的差异. 根据这些差异, 分析了该基因的上、下游调控序列对该基因在大肠杆菌中表达的重要作用, 从而进一步对该基因进行了亚克隆, 得到了生物学方法可检测到的表达产物. 由此推测, 微量热技术有可能为检测外源基因表达及其调控, 以及为指导进一步基因工程操作提供一种新的快速灵敏的技术和方法.     

Syntheses of polyamidoamine dendrimers starting from a hexadimensional core and application in gene transfer

Gene therapy refers to the concept and practice of applying gene to treat diseases. It may be defined as a method for inserting a functioning gene into the cells of a patient to correct an inborn error of metabolism (i.e. genetic abnormality or birth defect) or to provide a new function in a cell. There are numerous diseases that may be treated by gene therapy including genetic defects, com-mon illnesses such as cancer, AIDS and chronic diseases such as diabetes[1]. A gene medicine sys-tem c…     

TRANSFER OF FOREIGN GENES BY ELECTROPORATION AND THEIR EXPRESSION IN MAMMALIAN CELLS

Expression of foreign genes transferred into mammalian cells by electroporation has been studied. The pX1TK gene, pSV2Neo gene and pUCEJ oncogene have been introduced into MLTK-cells and NIH/3T3 cells, respectively. Stable transformation transient expression of TK gene by MLTK-cells as well as stable and malignant transformation of NIH/3T3 cells have been obtained. Transient expression frequency is about 80% and stable transformation frequency is about 10~(-4). Integration of foreign genes into the cellular genome was verified with molecular hybridization. Tumor development was observed after inoculation of transformed celts into nude mice.     

超声介导微泡基因传递体系的研究

超声波能够在不损害人体正常组织的条件下,有效到达人体组织深处。超声造影剂（微泡）,能够显著提高血液和组织的背向散射强度,广泛应用于心血管疾病的检测中。超声微泡介导基因转染技术是指通过微泡的空化作用,实现超声波能量在靶向组织处的富集和释放,进而促进此处组织细胞对基因药物的内化,达到高效转染的目的。由于具有无创、高效安全的特点,超声微泡介导在基因治疗中具有很大的优势和潜力。利用微泡的超声成像功能实现对病变组织诊断成像,利用靶向组织处的超声实现时间空间可控的目的基因的释放,利用微泡在超声作用下的“声孔效应”,实现基因对多种生物膜的跨膜传递,达到基因药物的可视化传递与高效可控释放的目的。本文主要从超声造影剂的种类、超声微泡介导基因转染的机理、微泡基因传递体系的构建等方面,详细综述了这一领域最新的研究进展,并对超声微泡介导基因转染的发展进行了展望。     

STUDIES ON HETEROLOGOUS EXPRESSION OF Rhizobium meliloti nifA GENE AND OXYGEN SENSITIVITY OF ITS PRODUCT

When the R. meliloti (Rm) nifA'-lacZ fusion-carrying plasmids were introduced intothe strains of Agrobacterium tumefaciens, R. trifolii and R. astragalus, β-galactosidaseactivity was demonstrated. However, activity was not induced by microaerobiosis. Further-more, R. meliloti nifA'-lacZ fusion was also not expressed in the nodule bacteroids ofR. trifolii and R. astragalus. We speculate that some factor(s) important for the inductionof Rm nifA presumed to be the fixLJ regulatory system would not be operative in thesebacteria. Experiments using R. meliloti nifH'-lacZ/ K. Pneumoniae nifH'-lacZ fusion and theconstitutive Rm nifA system to test the nifA-dependent expression of nifH'-lacZ underaerobic and microaerobic conditions in E. coli were performed. The inhibition of the RmnifA activation of nifH'-lacZ expression in the bacteria grown in the aerobic conditionwas shown. Assays on the Rm nifA-m RNA produced by the constitutive Rm nifA in E.coli under aerobic and microaerobic conditions with the cloned ni     

基于纳米材料的基因载体

基因疗法是治疗基因变异引起的先天性遗传疾病和后天获得性疾病以及癌症的新型有效方法。外源基因在细胞中安全、高效、稳定的表达是基因治疗成功的关键,这与基因治疗所使用的载体系统息息相关。基因载体主要分为病毒载体和非病毒载体两大类:病毒载体的转染效率较高,但副作用较大;非病毒载体作为一种新型的基因传递系统,可以弥补病毒载体的缺陷,尽管其转染效率稍逊于病毒载体,但在基因治疗领域具有不可替代的作用。随着纳米技术的出现和蓬勃发展,基于纳米材料的基因载体研究受到越来越多的关注。纳米基因载体具有如下潜在的优势:它制备相对简单,易于对其进行多功能的修饰;具有良好的生物相容性,一般不会引起强烈的机体免疫反应;粒径普遍很小,容易穿过人体的组织间隙而被细胞吸收,基因转运效率较高;可以较有效保护其所携带外源基因,利于基因更高效地表达。本文主要对基于金属、无机非金属、阳离子聚合物和脂质体纳米材料作为基因载体的研究进展进行综述和展望。     

CLONING, EXPRESSION AND CHARACTERIZATION OF Pro3 GENE OF YEAST Saccharomyces cerevisiae

The proline biosynthetic pathway and Pro genes in Saccharomyces cerevisiae have just begun to be studied recently. In our laboratory, Pro2 gene of S. cerevisiae had been cloned in yeast. As described in this paper, yeast Pro3 gene was also cloned, which can complement yeast Pro3 mutants, and be expressed efficiently in E. coli. The high activities of this gene product, L-pyrroline-5-carboxylate (P5C) reductase, can be detected in both organisms. The activity of the Pro3 gene product in multiple copy plasmids is not higher than that of single copy genes in chromosomes in both yeast and E. coll. The preliminary characterization of the gene is also reported.     

Polymeric Gene Carriers Bearing Pendant β‐Cyclodextrin: The Relevance of Glycoside Permethylation on the “In Vitro” Cell Response

The incorporation of cyclodextrins (CDs) to nonviral cationic polymer vectors is very attractive due to recent studies that report a clear improvement of their cytocompatibility and transfection efficiency. However, a systematic study on the influence of the CD derivatization is still lacking. In this work, the relevance of β‐CD permethylation has been addressed by preparing and evaluating two series of copolymers of the cationic N‐ethyl pyrrolidine methacrylamide (EPA) and styrenic units bearing pendant hydroxylated and permethylated β‐CDs (HCDSt and MeCDSt, respectively). For both cell lines, CDs permethylation shows a strong influence on plasmid DNA complexation, “in vitro” cytocompatibility and transfection efficiency of the resulting copolymers over two murine cell lines. While the incorporation of the hydroxylated CD moiety increased the cytotoxicity of the copolymers in comparison with their homopolycationic counterpart, the permethylated copolymers have shown full cytocompatibility as well as superior transfection efficiency than the controls. This behavior has been related to the different chemical nature of both units and tentatively to a different distribution of units along the polymeric chains. Cellular internalization analysis with fluorescent copo­lymers supports this behavior.

     

重组质粒对大肠杆菌HB101生长影响的微量热研究

用微量热方法研究了嗜麦芽假单胞菌AT18, 受体菌大肠杆菌HB101, mel基因工程菌——大肠杆菌HB101/pWSY8和携带克隆载体pUC18质粒的大肠杆菌HB101等的生长代谢过程. 实验结果从热化学和热动力学上阐明了细菌的生长速率常数与其所含质粒的大小呈负相关. 探讨了低温处理对含不同质粒大肠杆菌生长的影响, 发现低温处理对工程菌生长影响最大.     

Adaptive evolution of rotavirus VP7 and NSP4 genes in different species

Rotavirus is recognized as the major enteric pathogen associated with high burden of worldwide epidemic diarrhea disease in human and animals. Its outer capsid structural protein VP7 elicits the production of distinct neutralizing antibodies in the host and also determines the serotype of the virus strain. As the diarrhea-related protein of rotavirus, NSP4 is becoming an attractive candidate for vaccine development. It is not clear whether rotavirus VP7 or NSP4 evolved in the same way or not, and how the rotavirus VP7 and NSP4 evolved in specific species. Using the different models, we analyzed Datasets A composed of 12 coding sequences representing 12 species and Dataset B composed of nine coding sequences representing nine species. Computational results indicate that rotavirus experienced strong purifying selection in VP7 and NSP4 across species, and there exist some positive selective sites in specific species by Branch-site model A (119S in Bovine lineage and 199T in Canine lineage for Datasets A, 69Y and 70H in Murine lineage for Datasets B). Since these sites are located in different functional sequence segments, it may be concluded that these sites are crucial to related virus function. Therefore, the results of this study would provide potential values to vaccine research and development.     

Biocompatible Infinite‐Coordination‐Polymer Nanoparticle–Nucleic‐Acid Conjugates for Antisense Gene Regulation

Herein, we report the synthesis of DNA‐functionalized infinite‐coordination‐polymer (ICP) nanoparticles as biocompatible gene‐regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3‐hydroxy‐4‐pyridinone (HOPO) ligand bearing a pendant azide. Addition of Fe^III to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the Fe^III–HOPO ICP particles by copper‐free click chemistry afforded colloidally stable nucleic‐acid nanoconstructs. The DNA–ICP particles, when cross‐linked through sequence‐specific hybridization, exhibited narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA–ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate that these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.     

A STRATEGY FOR ISOLATING DIFFERENTIATIONINDUCING COMPLEMENTARY DNAs FROM HUMAN ESOPHAGEAL CANCER CELL LINE TREATED WITH RETINOIC ACID

Treatment of the human esophageal cancer cell line EC8712 with retinoic acid (RA) stopped the cell growth significantly and gave rise to terminal differentiation of the cells characterized by increased expression of involucrin gene. Two cDNA libraries were constructed from the parental and RA-treated cells respectively. Repeated subtractive hybridization of single-stranded plasmid DNA prepared from pooled colonies of cDNA library of the parental cells with cDNA probe generated from the RA-treated cells exhausted sequences common to both libraries of the cell. The unhybridized cDNA probe represented, therefore, the genes activated after RA-treatment. By using these enriched cDNAs as probe to screen the cDNA library constructed from the RA-treated cells thirty-nine positive colonies were obtained, of which two were specifically due to RA-induction. One of these two cDNA clones, designated as pRA538, has undergone further analysis and shown differentiation-inducing effect on parental cancer cells. A novel     

Cut and Paste for Cancer Treatment: A DNA Nanodevice that Cuts Out an RNA Marker Sequence to Activate a Therapeutic Function

DNA nanotechnology uses oligonucleotide strands to assemble molecular structures capable of performing useful operations. Here, we assembled a multifunctional prototype DNA nanodevice, DOCTR, that recognizes a single nucleotide mutation in a cancer marker RNA. The nanodevice then cuts out a signature sequence and uses it as an activator for a “therapeutic” function, namely, the cleavage of another RNA sequence. The proposed design is a prototype for a gene therapy DNA machine that cleaves a housekeeping gene only in the presence of a cancer-causing point mutation and suppresses cancer cells exclusively with minimal side effects to normal cells.