人Ha-ras基因中蛋白质特异结合DNA位点的鉴定

利用迁移率改变法和DNaseⅠ足纹法观察了Ha-ras癌基因5’端上游序列与特异结合蛋白的相互作用。用限制性内切酶XmaⅠ消化6.6kb Ha-ras基因得到约10个片段,进行3’-末端标记,与T24细胞核提取液反应,经低离子强度聚丙烯酰胺凝胶电泳,发现与蛋白质特异结合的416bp片段,位于Ha-ras基因5’-端上游1230—1646区域内,靠近转录起始点1660bp处(CAP位置)。另一个与蛋白质特异结合的389bp片段,位于转录起始点上游162—551处。     

AFP基因细胞专一性的表达依赖于某些核蛋白

Southern blotting分析没有发现成年大鼠肝、胚肝及肝癌细胞AFP基因5′端及上游有任何不同。以AFP基因转录起始点到5′端上游255 bp DNA片段为探针进行Southwestern blotting分析,发现表达AFP基因的细胞核蛋白中存在与其结合的核蛋白,这些在成年大鼠肝、肺、脾、心和肾细胞核蛋白中不存在。含有结合蛋白的肝癌核蛋白部分能使作为RNA聚合酶Ⅱ来源的成年大鼠肝细胞核蛋白部分具备较高的体外转录活性,表明基因细胞专一的表达确与某些结合蛋白有关。     

癌基因Ha-ras在转化和未转化细胞中表达和调控的差异

以前的工作曾用人胃癌基因Ha-ras转化了大鼠全胚细胞系Ratl细胞,得到转化细胞Rat3-3。克隆了Ha-ras癌基因6.6kb及其上游区2.5kb DNA片段,并发现2.5kb有Alu重复顺序,说明这个片段是来源于人胃癌细胞,虽族观察到p21蛋白编码12位点突变,我们又发现转化的Rat3-3细胞的Ha-ras mRNA水平比未转化的Rat1高大约五倍;通过DNase I超敏感实验证明只有转化细胞核中的Ha-ras基因对DNaseI敏感,1μg/mL的DNaseI就有明显的降解,而未转化细胞Rat1细胞核的Ha-ras基因在15μg/mL的DNaseI中也未发现有任何降解;另外还发现转化细胞核有一种能为Ha-ras基因上游区2.5kb特异结合的核蛋白,分子量大约35kD,此核蛋白不能与6.6kb Ha-ras基因本身结合,在未转化细胞中未发现此蛋白。从这些结果推测,癌基因Ha-ras的活化,除了点突变外,还可能存在另一条活化途径,即它的上游区可能有类似增强子的调控区。     

肝癌细胞AFP基因高水平的转录是受核蛋白成分的促进

利用大鼠甲胎蛋白(AFP)基因片段作为模板,分析大鼠肝癌细胞核蛋白成分对体外转录活性的影响,发现大鼠肝癌含有促进AFP基因体外转录的核蛋白。作为对照．没有发现任何成年大鼠肝核蛋白可以促进AFP基因的体外转录。为了确定促进AFP基因体外转录的核蛋白作用部位,对AFP基因模板5＇端上游序列进行了不同程度的删除,进一步分析核蛋白对删掉5’端上游序列后的模板体外转录的影响,结果表明,AFP基因转录的起始点到255bp这段DNA序列是大鼠肝癌核蛋白促进AFP基因转录必不可少的。以SV40DNA经Pst I酶酶切所得的DNA片段(1216bp和4027bp)代替AFP基因片段作为模板,不存在核蛋白促进体外转录的现象。用AFP基因转录的起始点到 255bp这段的DNA为探针,进行Southwestm印迹分析,结果发现了8种与探针结合的核蛋白。     

AFP基因细胞专一性的表达依赖于某些核蛋白

Ｓｏｕｔｈｅｒｎ ｂｌｏｔｔｉｎｇ分析没有发现成年大鼠肝、胚肝及肝癌细胞ＡＦＰ基因５＇端及上游有任何不同。以ＡＦＰ基因转录起始点到５＇端上游２５５ｂｐＤＮＡ片段为探针进行Ｓｏｕｔｈｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ分析,发现表达ＡＦＰ基因的细胞核蛋白中存在与其结合的核蛋白,这些在成年大鼠肝、肺、脾、心和肾细胞核蛋白中不存在。含有结合蛋白的肝癌核蛋白部分能使作为ＲＮＡ聚合酶Ⅱ来源的成年大鼠肝细胞核蛋白部     

外源RNA对小鼠白蛋白基因表达及DNaseⅠ敏感性的影响

兔肝RNA诱导培养的小鼠成纤维细胞 ,大鼠肝RNA注射入小鼠前列腺 ,用免疫组织化学染色检测外源RNA对小鼠白蛋白基因表达的影响 ;不同RNA诱导培养的小鼠成纤维细胞 ,提取细胞核 ,DNaseⅠ消化 ,PCR法扩增小鼠白蛋白基因 ,检测白蛋白基因消化情况。发现外源RNA可促进小鼠白蛋白基因表达并增加该基因DNaseⅠ敏感性     

凡纳滨对虾DNaseⅠ基因的克隆及原核表达

利用RT-PCR和RACE技术,从凡纳滨对虾(Litopenaeus vannamei)肝胰腺中克隆了DNaseⅠ基因的全长cDNA序列。该序列全长1614bp,包含1209bp的开放阅读框,编码一个含403个氨基酸的蛋白;5′非翻译区为116bp,3′非翻译区为289bp。实时定量PCR分析结果表明,DNaseⅠ基因在肝胰腺的表达量是其他器官表达量的16～162倍,表明凡纳滨对虾DNaseⅠ基因属于胰腺型表达。本研究还利用酶切重组构建原核表达载体,并在大肠杆菌(Escherichia coli)中成功表达出了有活性的重组DNaseⅠ蛋白。     

C-myc基因表达调控的研究

本文利用cDNA探针,以Southern杂交在C-myc 5′上游区域发现了一段活跃转录的序列(文中称“旁侧基因”)。对该基因克隆及进一步分析表明,能与cDNA杂交的序列存在于距C-myc 5′端约2.4kb的Sma Ⅰ片段中。Northern杂交显示,该旁侧基因在不同组织均有表达,产物为5.8kb,推断该基因为15～3.5kb。以旁侧基因部分序列为探针,在不同种属的基因组中检测出单拷贝序列,提示该基因在进化上的保守性。RNase Mapping分析发现此旁侧基因与C-myc转录方向相同,转录终止在C-myc5’上游约2.4～3.4kb区域。根据基因领域效应及本文结果,我们推测,在正常细胞中,由于旁侧基因的领域效应,使C-myc保持相对静止状态,而在肿瘤细胞中,染色体转位或原病毒插入,破坏了旁侧基因的领域效应,使C-myc表达增高。     

羟基脲诱导前后HEL细胞内GATA转录因子与人β—类珠蛋白基因调控元件的结合模式分析

HEL细胞是一株人红白血病细胞株,其中成年型β-珠蛋白基因不能表达。我们以往的试验证明,羟基脲诱导以后,能使HEL细胞内成年型β-珠蛋白基因表达。本文以此为模型,探索了β-珠蛋白基因在HEL细胞内诱导表达的分子机制。结果表明,羟基脲诱导之后,与人β-珠蛋白基因5’远侧端DNaseⅠ超敏感点2核心DNA序列以及近侧端启动子DNA序列相结合的GATA-1蛋白因子量明显增多,而GATA-2蛋白因子量明显减少。结果显示,GATA蛋白家族各成员在HEL细胞分化及珠蛋白基因表达的过程中扮演着不同的角色。推测GATA-1可能有促进β-珠蛋白基因的表达,使HEL细胞趋向终末分化的作用,GATA-2则可能与胚胎型珠蛋白基因表达有关,并有抑制红细胞向终末分化的功能。     

香蕉果实特异性ACC合酶基因启动子区的克隆及其功能初探

根据本实验室所获得的香蕉果实特异性ACC合酶cDNA序列,以改进的接头连接PCR方法通过两次步行从香蕉基因组中分别扩增并克隆了其基因5′旁侧区近端1.2kb和远端1.6kb的片段。通过拼接,构建出含有2505bp启动子区和转录起始位点下游86bp的共2591bp的基因5′旁侧区片段;其启发性动子区中34至28为推测的TATA盒序列,158至146为推测的CCAAT盒,与其它植物基因启动子结构相类似。将2.5kb启动子片段与β-葡糖苷酸酶(GUS)基因编码序列融合,用基因枪法将构建的嵌合基因转入香蕉叶、根和果实的细胞后,只在果实细胞中观察到报告基因的瞬时表达,从功能上证明了此25kb的启动子片段具有指导报告基因在香蕉果实中特异性表达的作用。同时构建5个含不同5′端缺失启动子与GUS融合基因的表达载体。瞬时表达结果表明可能负责果实特异性表达的调控区存在于转录起始位点至-1111的启动子区中,而在-1111至-608区间可能存在一个正控制区。     

DNaseI sensitivity of the rat albumin and alpha-fetoprotein genes.

We have analyzed the DNaseI sensitivity of chromatin from the rat albumin and alpha-fetoprotein genes in the fetal liver (which synthesizes albumin and alpha-fetoprotein), adult liver (which synthesizes albumin), fetal yolk sac (which synthesizes alpha-fetoprotein), and adult kidney (which synthesizes neither). Active genes were much more sensitive than their kidney counterparts, and the adult liver alpha-fetoprotein and fetal yolk sac albumin genes showed intermediate levels of sensitivity. Sensitivity was analyzed as a function of the extent of DNaseI digestion. Rate constants were calculated for the degradation of individual DNA hybridization bands and normalized to the intrinsic rate constants of the same bands degraded in purified DNA. This enabled us to eliminate the inconsistencies that otherwise result from comparing chromatin sensitivity of different DNA sequences, or chromatin sensitivity in different nuclear environments.     

Adsorption of DNA on clay minerals: protection against DNaseI and influence on gene transfer

Abstract Numerous authors have investigated DNA relationships with sandy soil. A model composed of various DNAs adsorbed on montmorillonite clay was developed to assay enzyme (DNaseI) activity on clay-adsorbed nucleic acids. The extent of DNA adsorption was affected by the concentration and valency of the cations used (Mg²⁺, Ca²⁺, Na⁺), indicating a charge-dependent process. Calf thymus DNA was found to be highly adsorbed by smectite (up to 30 mg g⁻¹ of dry clay). Adsorbed DNA was shown to be more resistant to degradation by DNaseI than free DNA. Experimental data with plasmid and short linear amplified (through polymerase chain reaction) DNA showed that protection against nucleases was only partial. Nevertheless, clay-adsorbed DNA was found to be still able, even after a strong DNaseI treatment, to artificially transform competent Escherichia coli cells. The results show that persistance of DNA and gene transfer by genetic transformation may occur in soil.     

Differential sensitivity of constitutive and facultative heterochromatin in orthopteran chromosomes to digestion by DNaseI

In situ pancreatic DNaseI digestions were used as probes to study the structural organization of facultative and constitutive heterochromatin during both mitotic and meiotic divisions. Three different types of heterochromatic regions from three insect species were chosen for this study. These regions had been previously characterized by in situ treatments with restriction endonucleases (AT and GC rich DNA sequences). Progressive increase in DNaseI concentration (from 10 to 200 ng/ml) or in incubation time (from 5 to 30 min) revealed a specific pattern of sequential digestion of the constitutive heterochromatic regions, the centromeric ones (AT-rich DNA) being the most resistant to DNaseI action. The interstitial C-bands (with AT or GC-rich DNA) were more sensitive to DNaseI, and the band 4.4 from Baetica ustalata was the most resistant of the non-centromeric bands. Similar results were obtained during meiosis, but increased accessibility to DNAseI was observed compared to mitosis. DNA methylation in the non-centromeric band 4.4 of B. ustulata could be responsible for its differential digestion with respect to the remaining intercalar heterochromatin. Facultatively heterochromatic regions (X chromosomes) were found to exhibit a differential response to DNaseI attack from mitosis to meiosis. While they behaved as cuchromatin during mitosis, they were the most resistant together with centromeric heterochromatin regions, during metaphase I and II. The different responses to digestion of the X chromosome and X-derived regions between somatic and meiotic divisions are probably a consequence of the changes in the organization of this chromosome during the facultative heterochromatinization process.     

siRNA介导基因沉默的分子机器、作用模式和应用前景

siRNA(small interfering RNA)介导的基因沉默是细胞内监控寄生的遗传物质、沉默无用的信息模板和调节自然的时空转换的一种分子机制。它与体液免疫和细胞免疫一起形成了动物和人类机体中免疫系统的三大支柱,从不同水平上来对抗体内外有害物质的干扰和侵犯。现已清楚,大约22个核苷酸长的双链RNA能够通过不同途径,以序列特异的方式来高效地沉默含有同源序列的靶RNA分子。这一古老而又迷人的系统现已被公认为是鉴定基因功能、调控基因表达和改变基因表型的简单而有效的方法。可以预计,这一新颖的技术在不远的将来必将形成一条研究功能基因组学、探索信号通路和创造遗传缺陷模型的高速公路,并将开创一条预防和治疗人类疾病的新途径。