高等植物启动子的研究进展

从高等植物启动子的基本结构、启动子克隆方法入手,着重介绍了组成型、组织特异性及诱导型启动子的研究进展及其在植物基因工程方面的应用情况,提出了植物启动子研究中存在的问题与展望。     

Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNa polymerase III

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to -amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.     

两个人组蛋白H1基因的亚克隆和它们启动子核苷酸顺序的比较

从人基因库中分离得到的两个含有不同变种组蛋白H_1基因的λ克隆(λHh8和λHh9),分别把它们含有该基因的片段(8c和9a)插入载体puc8质粒中,得到了亚克隆pHh8c和pHh9a。对这两个人组蛋白H_1基因的启动子(Promoter)和部份编码蛋白质的区域作了核苷酸顺序的分析和比较,确定了它们的启动要素和同系顺序。     

人类RELM-beta基因启动子的结构与功能分析

为克隆人类抵抗素样分子β（resistin-like molecule beta, RELMβ）基因的上游启动子序列,并观察其不同截短片段的启动子活性,以人类基因组DNA为模板,通过PCR扩增方法获得-871～+50 bp、-729～+50 bp、-471～+50 bp、-438～+50 bp、-371～+50 bp大小的RELMβ启动子片段,将其定向克隆入pGL3-Basic载体,构建荧光素酶报告基因载体,并制备转录因子CDX-2结合位点的突变或缺失体.在阳离子脂质体的介导下,报告基因载体分别瞬时转染人胚肾293细胞、结肠癌HCT116和SW480细胞、宫颈癌HeLa细胞.结果发现,各RELMβ启动子片段在293、HCT116、SW480细胞中均有活性,但在HeLa细胞中活性缺失;-471～-438 bp区存在RELMβ启动子的核心调控元件.针对该区域CDX-2转录因子结合位点进行突变,能导致RELMβ启动子活性显著降低;凝胶电泳迁移率实验表明,该区段能结合CDX-2.结果提示,成功克隆了具有活性的RELMβ启动子序列,CDX-2为其重要的转录因子,为研究RELMβ基因的转录调控机制奠定了实验基础.     

Z-DNA的形成及其与基因转录的关系

Z-DNA是一种非常独特的DNA二级结构.与B-DNA相比,Z-DNA最显著的结构特征是左手螺旋和磷酸-核糖骨架呈“zigzag”状. 虽然目前对Z-DNA功能的了解还不确切,但毫无疑问,Z-DNA与基因的转录和调控密切相关. 一方面,在体内Z-DNA在基因转录过程中产生;另一方面,分布于启动子等不同区域的Z-DNA又可以反过来调控基因的转录, 即Z-DNA能够增强一些基因转录,也能抑制某些基因的表达,但其调控机制还不清楚.这种调控似乎与Z-DNA在启动子中的位置、基因和细胞类型有关.研究Z-DNA的形成及其与基因转录的关系对理解基因转录调控理论具有十分重要的意义.     

猪CuZnSOD基因启动子的克隆鉴定及分析

猪铜锌超氧化物歧化酶(CuZnSOD)是一种重要的抗氧化酶,其功能已被广泛研究,但CuZnSOD基因的转录调控尚不明确。为了研究猪CuZnSOD基因的核心启动子区域,并对其转录调控机制进行探讨,运用PCR方法从猪基因组克隆CuZnSOD基因5′上游调控区853 bp的片段,然后通过巢式PCR方法获得5′末端逐渐缺失的启动子系列片段,并将这些片段定向插入到荧光素酶报告基因表达载体(pGL3-Basic)中。瞬时转染小鼠胚胎细胞(NIH/3T3),利用双荧光素酶报告基因检测不同长度启动子活性。检测结果显示,在CuZnSOD基因5′上游调控区-87 bp和-266 bp处分别存在2个潜在转录起始位点,-383 bp~+67 bp启动区活性最强,进一步缺失分析发现-75 bp~-32 bp区域内含有猪CuZnSOD基因转录所必需的基础启动子序列,其中存在多个潜在的转录因子结合位点,研究结果提示这些转录因子结合位点可能是参与CuZnSOD基因转录的重要调控序列。     

Effect of lithium on the melatonin production in the pineal gland of viscacha

Melatonin is synthesized primarily in the pineal gland. Lithium affects the circadian rhythms that may explain its therapeutic effectiveness in the treatment of bipolar disorder. The objective of this study was to investigate the effect of lithium on the biochemical parameters involved in melatonin synthesis in the pineal gland of viscacha. Viscachas were daily intraperitoneally injected with lithium chloride or saline solution for one month. Pineal mRNAs encoding β₁-adrenoceptor and arylalkylamine-N-acetyltransferase enzyme (AA-NAT) were studied by in situ hybridization. Pineal melatonin concentrations were determined by radioimmunoassay, and AA-NAT and hydroxyindol-O-methyltransferase (HIOMT) activities were investigated by radiometric assays. The only parameters that decreased significantly were the expression of AA-NAT mRNA and pineal melatonin levels. Our data suggest that lithium treatment may decrease melatonin synthesis in the viscacha pineal gland by a complex mechanism that involves currently unknown events that are beyond a decrease in the expression of AA-NAT enzyme.     

Structural and functional analyses of Arabidopsis thaliana chlorophyll a/b-binding protein (cab) promoters

Arabidopsis thaliana carries three functional copies of the chlorophyll a/b-binding protein (cab) gene which code for an identical mature protein. DNA sequence comparison of all three cab promoters indicated that cab2 and cab3 are more closely related compared to cab1. Although the highest degree of homology was found between the TATA box and -256 of cab3 promoter, suggesting that this region plays a major role in promoter function, this promoter regions are only 47% homologous. To study whether these promoters are regulated by identical cis-acting regulatory elements, the promoters were mutated by progressive deletions and the effects on the promoter activity were measured in either transformed plants or cultured cells. It was found that the minimum sequence necessary for the light-dependent tissue-specific promoter activity of the cab3 is the 89 bp DNA fragment (between -74 and -164) at the region of the TATA and the CCAAT boxes. However, an additional 45 bp DNA fragment (between -164 and -209) upstream of the CCAAT box was necessary for the full promoter activity in the leaves. The regulatory element in the 45 bp region appears to be a positive regulator or enhancer which is specific to photosynthetic cells, since the region did not enhance the promoter activity in cultured cells. This region contains an octamer, TGCCACGT (cab2) or TGCCACAT (cab3), which is similar to the previously identified element, TGACACGT from Arabidopsis cab1 promoter. The upstream regions of the cab promoters appear to contain additional elements which are functionally distinct in each promoter since the upstream region of cab1 activated a non-functional nos promoter whereas that of cab3 did not.     

人端粒酶催化亚基基因启动子调控的肿瘤细胞特异表达载体

为获得端粒酶阳性肿瘤细胞特异表达载体用于癌症的基因治疗 ,克隆并构建了人端粒酶催化亚基 (hTERT)基因启动子调控的萤光素酶报告载体 .用脂质体转染法将其分别转染肿瘤细胞和正常细胞 ,检测其在肿瘤细胞和正常细胞中的转录活性 .hTERT启动子在所检测的 4种端粒酶阳性的肿瘤细胞中具有明显的转录活性 ,平均为阳性对照的 4 4 3% ;而在端粒酶阴性的正常人胚肺成纤维细胞中则无明显的转录活性 .提示hTRET启动子的转录活性在端粒酶阳性的肿瘤细胞中明显上调 ,由hTERT启动子构建的载体可能是一种新颖和有前景的肿瘤细胞特异性表达的基因治疗载体     

Studies on the Methylation Status of CpG Sequences Located in Promoters of Selected Tumour Suppressor Genes in Breast Cancer Cells

In the tested samples of sporadic breast cancer (100 cases), hypermethylation of CpG sequences located in ERα promoter was observed in 62 cases. It correlated with: (i) deficiency of ERα protein in 45%, (ii) hypermethylation of BRCA1 promoter in 95%, and (iii) nonmethylated E-cadherin promoter in 90%. Fifty-eight percent of the patients with nonmethylated E-cadherin promoter (56 cases) did not show metastasis to lymphatic nodes. The analysis of the methylation level of the promoter of ERα, BRCA1, and E-cadherin, frequently connected with their activity, shows that it can be an important parameter in the diagnosis and therapeutic strategies in sporadic breast cancer.