用于转基因调控研究的人类ApoAI-AIV全基因簇克隆

目的 克隆人类ApoAI -AIV全基因簇用于转基因调控研究ApoAI的刺激表达药物。 方法 使用平均克隆长度为 38kb的人Cosmid基因组文库 ,通过制备逐级降低独立克隆数的文库亚库 ,以PCR对亚库中的ApoAI-AIV全基因簇首尾特征序列扩增作为亚库筛选性指标 ,筛选出独立克隆数约为 1 0数量级的文库亚库 ,进行平板单克隆菌落筛选。结果 得一个阳性克隆 ,其首尾特征序列经测序与公布序列相同。

提高转基因在质体中特异有效表达的新策略

质体基因工程已成为植物遗传改良的新途径,尤其在利用植物作为反应器合成积累药用蛋白等重要有机化合物方面有独特的应用价值。然而,质体仅具有半自主性转录和翻译机制,其内源基因的转录要依赖于核基因编码的转录因子。外源基因在质体中的表达调控受多种因素影响。新近报道了一些调控质体中转基因表达的技术策略,例如,杂种转录因子(hybrid transcription factor)介导的体系,基于噬菌体T7转录系统和基于细菌lac阻遏因子的体系等。这些技术策略的应用及改进将提高转基因在质体中特异有效表达,实现外源蛋白在质体中高水平积累。

防止转基因作物释放引发“超级杂草”产生的若干对策

转基因作物释放可能导致潜在的生态风险性 ,其中的一个重要方面是通过花粉的传播将某些转基因 (主要是抗除草剂基因 )漂入野生近缘种或近缘杂草而引发难以控制的“超级杂草”的产生。本文讨论了防止“超级杂草”产生的若干对策 ,包括物理隔离、转基因遗传调控、雄性不育性与无融合生殖机制的利用、将转基因定位于同当地杂草不亲和的基因组和叶绿体或线粒体基因组等。

动物转基因技术研究新进展及其在牛育种上的应用

动物转基因技术已应用于生命科学的各个领域,尤其在研究基因功能、转基因动物育种(抗病育种和提高生产性能)、治疗人类疾病(生产药用蛋白、干细胞治疗和种器官移植)等方面得到了广泛的应用。本文从转基因技术的历史和发展状况入手,简要介绍了几种传统的转基因技术方法,重点综述了近几年为提高转基因效率和转基因精确调控的一些新技术。另外,本文就转基因技术在牛育种方面的研究现状,以及对转基因技术在牛新品种培育和生物制药等领域的应用和前景进行了综述。

植物类胡萝卜素生物合成及其调控与遗传操作

类胡萝卜素是生物体内通过类异戊二烯途径合成的、自然界广泛存在的一大类天然色素物质的总称。近年来,类胡萝卜素生物合成基因的分离和功能鉴定与有关类胡萝卜素生物合成调控机制研究的新进展,使通过遗传操作调控植物体内类胡萝卜素生物合成途径成为可能。本文主要综述了近年来类胡萝卜素生物合成及其调控研究的进展,并介绍了应用转基因技术改变植物体内类胡萝卜素成分与含量的成功事例。

植物挥发性物质代谢的遗传修饰

植物在生命周期里能合成释放多种多样的挥发性化合物,这些物质在植物生长发育和代谢调控,植物抵御病虫侵害,以及植物与环境信息交流中行使重要功能。在介绍植物挥发性化合物的生理功能、合成途径调控和商业应用的基础上,重点论述应用基因工程调控植物挥发性物质合成的技术策略和研究进展,并讨论了植物挥发性物质遗传修饰存在的问题和发展前景。

Transcriptional Regulation of 10 Mitochondrial Genes in Different Tissues of NCa CMS System in Brassica napus L. and Their Relationship with Sterility

Northern blot analysis was conducted with mitochondrial RNA from seedling leaves, floral buds, and developing seeds of NCa CMS, maintainer line and fertile F1 using ten mitochondrial genes as probes. The results revealed that 9 out of the 10 mitochondrial genes, except for atp6, showed no difference in different tissues of the corresponding materials of NCα CMS system and that they might be constitutively expressed genes. Eight genes, such as orf139, orf222, atpl, cox1, cox2, cob, rm5S, and rm26S, showed no difference among the three tissues of all the materials detected. So the expression of these eight genes was not regulated by nuclear genes and was not tissue-specific. The transcripts of atp9 were identical among different tissues, but diverse among different materials, indicating that transcription of atp9 was neither controlled by nuclear gene nor tissue-specific. Gene atp6 displayed similar transcripts with the same size among different tissues of all the materials but differed in abundance among tissues of corresponding materials and its expression might be tissue-specific under regulation of nuclear gene. Moreover, three transcripts of orf222 were detected in the floral buds of NCa cms and fertile F1, but no transcript was detected in floral buds of the maintainer line.The transcription of orf139 was similar to that of orf222 but only two transcripts of 0.8 kb and 0.6 kb were produced. The atp9 probe detected a single transcript of 0.6 kb in NCa cms and in maintainer line and an additional transcript of 1.2 kb in fertile F1. The relationship of expression of orf222, orf139, and atp9 with NCa sterility was discussed.

Cloning of cDNA Encoding CCoAOMT from Populus tomentosa and Down-regulation of Lignin Content in Transgenic Plant Expressing Antisense Gene

cDNA encoding caffeoyl CoA O-methyltransferase (CCoAOMT) from Chinese white poplar ( Populus tomentosa Carr.) was cloned by RT-PCR and sequenced. Northern analysis displayed that the CCoAOMT was expressed specifically in the developing secondary xylem and its expression was coincident with lignification. The antisense CCoAOMT cDNA was transformed into P. tremula x P. alba mediated by Agrobacterium tumefaciens ( Smith et Townsend) Conn. Transgenic plants were identified with PCR, PCR-Southern and Southern analysis. Lignin content in 5- to 6-month-old transgenic plants was measured. One of the transgenic lines had significant reduction of 17.9% in Klason lignin content as compared with that of untransformed poplar. The results demonstrate that antisense repression of CCoAOMT is an efficient way to reduce lignin content for improving pulping property in engineered trees.

影响烤烟绿原酸含量因素的研究进展

绿原酸是烟叶中最主要的酚类物质,与烤烟的色泽、香气及质量密切相关。该文以绿原酸的遗传研究现状作为出发点,围绕生态、栽培和调制3个方面对影响烟草绿原酸含量的因素进行了分析,为有目的地改善烟叶香气品质、外观质量和服务生产实际提供理论依据。

Role of H3K27 methylation in the regulation of IncRNA expression

Once thought to be transcriptional noise, large non-coding RNAs （IncRNAs） have recently been demonstrated to be functional molecules. The cell-type-specific expression patterns of lncRNAs suggest that their transcription may be regulated epigenetically. Using a custom-designed microarray, here we examine the expression profile of IncRNAs in embryonic stem （ES） cells, lineage-restricted neuronal progenitor cells, and terminally differentiated fibroblasts. In addition, we also analyze the relationship between their expression and their promoter H3K4 and H3K27 methyla- tion patterns. We find that numerous lncRNAs in these cell types undergo changes in the levels of expression and promoter H3K4me3 and H3K27me3. Interestingly, lncRNAs that are expressed at lower levels in ES cells exhibit higher levels of H3K27me3 at their promoters. Consistent with this result, knockdown of the H3K27me3 methyltransferase Ezh2 results in derepression of these IncRNAs in ES cells. Thus, our results establish a role for Ezh2-mediated H3K27 methylation in lncRNA silencing in ES cells and reveal that lncRNAs are subject to epigenetic regulation in a similar manner to that of the protein-coding genes.

The ATF/CREB site is the key element for transcription of the human RNA methyltransferase like1(RNMTL1) gene, a newly discovered 17p13.3 gene

The human RNA methyltransferase like 1 gene (RNMTL1) is one of thirteen newly discovered genes within a 116 Kb segment of the chromosome 17pl3.3 that suffers from a high frequent loss of heterozygosity in human hepatocellular carcinoma in China[1-5]. To understand the molecular mechanisms underlying transcription control of the RNMTL1 gene in human cancers, we decline using of the conventional approach where the cis-elements bound by the known transcription factors are primary targets, and carried out the systematic analyses to dissect the promoter structure and identify/characterize the key cis-elements that are responsible for its strong expression in cell. The molecular approaches applied included 1, the primer extension for mapping of the transcription starts; 2, the transient transfection/reporter assays on a large number of deletion and site-specific mutants of the promoter segment for defining the minimal promoter and the crucial elements within; and 3, the electrophoresis mobility shift assay with specific antibodies for reconfirming the nature of the transcription factors and their cognate cis-elements. We have shown that the interaction of an ATF/CREB element (-38 to -31) and its cognate transcription factors play a predominant role in the promoter activity of the RNMTL1 gene. The secondary DNA structures of the ATF/CREB element play a more vital role in the protein-DNA interaction. Finally, we reported a novel mechanism underlying the YY1 mediated transcription repression, namely, the ATF/CREB dependent transcription-repression by YY1 is executed in absence of its own sequence-specific binding.

Research progress on the relationship between BRCA1 and hereditary breast cancer

Breast cancer gene 1(BRCA1) gene was the first breast cancel susceptibility gene discovered in familial breast cancer.It has been revealed that BRCA1 can be combined with an array of important protein involved in cell cycle regulation,DNA repair,gene transcription control and apoptosis regulation.It plays a down-regulation effect on tumor growth and an important role in maintaining genomic stability.New research suggests that it also associate with the breast cancer stem cells and microRNA.Its mutations,promoter methylation and ectopic expression may one of the main reasons for the generation and development of hereditary breast cancer.

Structural insights into selective histone H3 recognition by the human Polybromo bromodomain 2

The Polybromo （PB） protein functions as a key component of the human PBAF chromatin remodeling complex in regulation of gene transcription. PB is made up of modular domains including six bromodomains that are known as acetyl-lysine binding domains. However, histone-binding specificity of the bromodomains of PB has remained elusive. In this study, we report biochemical characterization of all six PB bromodomains＇ binding to a suite of lysine-acetylated peptides derived from known acetylation sites on human core histones. We demonstrate that bromodomain 2 of PB preferentially recognizes acetylated lysine 14 of histone H3 （H3K14ac）, a post-translational mark known for gene transcriptional activation. We further describe the molecular basis of the selective H3K14ac recognition of bromodomain 2 by solving the protein structures in both the free and bound forms using X-ray crystallography and NMR, respectively.

BRCA1 affects global DNA methylation through regulation of DNMT1

Global DNA hypomethylation at CpG islands coupled with local hypermethylation is a hallmark for breast cancer, yet the mechanism underlying this change remains elusive. In this study, we showed that DNMT1, which encodes a methylation maintenance enzyme, is a transcriptional target of BRCA1. BRCA1 binds to the promoter of the DNMT1 gene through a potential OCT1 site and the binding is required for maintaining a transcriptional active configuration of the promoter in both mouse and human cells. We further demonstrated that impaired function of BRCA1 leads to global DNA hypomethylation, loss of genomic imprinting, and an open chromatin configuration in several types of tissues examined in a BRCA1 mutant mouse model at premaligant stages. BRCA1 deficiency is also associated with significantly increased expression levels of several protooncogenes, including c-Fos, Ha-Ras, and c-Myc, with a higher expression in tumors, while premalignant mammary epithelial cells displayed an intermediate state between tumors and controls. In human clinical samples, reduced expression of BRCA1 correlates with decreased levels of DNMT1, and reduced methylation of CpG islands. Thus, BRCA1 prevents global DNA hypomethylation through positively regulating DNMT1 expression, and this provides one of mechanisms for BRCAl-associated breast cancer formation.

The promoter analysis of the human C17orf25 gene, a novel chromosome 17pl3.3 gene

The human C17orf25 gene (Accession No. AF177342) is one of thirteen genes cloned from a region displaying a high score of loss of heterozygosity within chromosome 17pl3.3 in human hepatocellular carcinoma in China[l]. To unveil the underlying mechanisms for the transcription regulation of this gene and understand its implication to the hepatocellular carcinogenesis, we looked into the relevant aspects by both bioinformatic and experimental executions. We found: 1, The abundant expression of the C17orf25 gene was evident in all the cell lines and tissue samples tested, showing little hepatoma-selectivity; 2, Its transcription starts at a single site, locating at -60 from the translation initiation codon; 3, A 58 bp fragment containing the transcription start, extending from -112 to -55, represents the minimal promoter; 4, The consensus sequence within this fragment recognized by SP1 contributes predominantly to the activity of the minimal promoter; 5, The bioinformatic analysis suggests that the C17orf25 gene may encode a protein in the family of the glyoxalase. Our data has provided some deep insight into both function and regulation of the C1 7orf25 gene in the context of the normal liver and hepatocellular carcinoma.

NECK LEAF 1, a GATA type transcription factor, modulates organogenesis by regulating the expression of multiple regulatory genes during reproductive development in rice

In the monocot rice species Oryza sativa L., one of the most striking morphological processes during reproductive development is the concurrence of panicle development with the sequential elongation of upper internodes （UPIs）. To elucidate the underlying molecular mechanisms, we cloned the rice gene NECK LEAF 1 （NL1）, which when mutated results in delays in flowering time, smaller panicles with overgrown bracts and abnormal UPI elongation patterns. The NL1 gene encodes a GATA-type transcription factor with a single zinc finger domain, and its transcripts are de- tected predominantly in the bract primordia, which normally degenerate in the wild-type plants. Overexpression of NL1 in transgenic plants often gives rise to severe growth retardation, less vegetative phytomers and smaller leaves, suggesting that NL1 plays an important role in organ differentiation. A novel mutant allele of PLASTOCHRON1 （PLAD, a gene known to play a key role in regulating leaf initiation, was identified in this study. Genetic analysis demonstrated an interaction between nil and plal, with NL1 acting upstream of PLA1. The expression level and spatial pattern of PLA1 were found to be altered in the nil mutant. Furthermore, the expression of two regulators of flowering, Hd3a and OsMADS1, was also affected in the nil mutant. On the basis of these findings, we propose that NL1 is an intrinsic factor that modulates and coordinates organogenesis through regulating the expression of PLA1 and other regulatory genes during reproductive development in rice.

Pluronic L-81 ameliorates diabetic symptoms in db/db mice through transcriptional regulation of microsomal triglyceride transfer protein

AIM: To test whether oral L-81 treatment could im-prove the condition of mice with diabetes and to investigate how L-81 regulates microsomal triglyceride transfer protein (MTP) activity in the liver. METHODS: Genetically diabetic (db/db) mice were fed on chow supplemented with or without L-81 for 4 wk. The body weight, plasma glucose level, plasma lipid profile, and adipocyte volume of the db/db mice were assessed after treatment. Toxicity of L-81 was also evaluated. To understand the molecular mecha-nism, HepG2 cells were treated with L-81 and the effects on apolipoprotein B (apoB) secretion and mRNA level of the MTP gene were assessed.RESULTS: Treatment of db/db mice with L-81 sig-nificantly reduced and nearly normalized their body weight, hyperphagia and polydipsia. L-81 also markedly decreased the fasting plasma glucose level, improved glucose tolerance, and attenuated the elevated levels of plasma cholesterol and triglyceride. At the effective dosage, little toxicity was observed. Treatment of HepG2 cells with L-81 not only inhibited apoB secretion, but also signif icantly decreased the mRNA level of the MTP gene. Similar to the action of insulin, L-81 exerted its effect on the MTP promoter. CONCLUSION: L-81 represents a promising candidate in the development of a selective insulin-mimetic mol-ecule and an anti-diabetic agent.

Molecular Modification of a HSV-1 Protein and Its Associated Gene Transcriptional Regulation

The molecular modifications of Herpes Simplex Virus Type I （HSV-1） proteins represented by acetylation and phosphorylation are essential to its biological functions. The cellular chromatin-remodeling/ assembly is involved in HSV-1 associated gene transcriptional regulation in human cells harboring HSV-1 lytic or latent infections. Further investigation on these biological events would provide a better understanding of the mechanisms of HSV-1 viral gene transcriptional regulation

Analysis of the 5' flanking sequence of the human norepinephrine transporter gene

The human norepinephrine transporter(NET) gene was cloned and structurally analyzed. The far 5’ fragment containing exon 1 (a non-coding exon) and exon 2 was sequenced. The transcription start site of the gene in human brain stem tissue was determined by primer extension analysis. It was found that the gene could be transcribed from multiple starting points. The 5’ flanking sequence contains a proximal G-C rich region, one possible GSG elemeflt and several SP1 sites. However it does not contain TATA box and CAAT box motifS. Gel shift analysis with nuclear extracts from different tissues of mouse shows that the G-C rich region may be involved in tissue specific expression of the gene.

Novel Tuning Buttons in Tomato Fruit Ripening:miRNAs and siRNAs

Tomato has emerged as an emblematic model plants for fleshy fruit research and tomato fruit ripening is a complex and highly coordinated developmental process.The many physiology and biochemical processes associated with tomato fruit ripening require changes in the expression of hundreds to thousands of genes.Gene expression is regulated by transcriptional and post-transcriptional pathways,one of the recently discovered mechanisms in plants was small RNAs mediated gene silencing at post-transcriptional(PTGS) level.Intriguingly,several mi RNAs and endogenous si RNAs were revealed to be involved in the fruit ripening process which opened a new avenue in the field of fleshy fruit biology.This review compiled the most recent advances made in deciphering the regulation functions of mi RNAs and si RNAs in tomato fruit ripening.It also emphasized the new perspectives now possible in the small RNAs regulation research in tomato fruit ripening and senescence.