SV40 PolyA序列及其AATAAA信号对上游GFP基因表达及转录终止的影响

SV40 PolyA(猴空泡病毒PolyA,简称PolyA)序列是有转录终止作用和使转录的mRNA添加PolyA尾的DNA序列(240 bp),含有AATAAA六核苷酸多腺苷化信号(Polyadenylation signal)。在pEGFP-C1质粒的GFP基因下游插入14个同向串联的Alu序列(Alu14),构建pAlu14质粒,瞬时转染HeLa细胞,用Northern blot检测和荧光显微镜观察GFP RNA和GFP蛋白表达,发现Alu串联序列强烈抑制GFP基因表达,该序列没有转录终止作用产生高分子量GFP融合RNA。又在pAlu14质粒GFP基因和Alu串联序列之间按正、反方向插入PolyA序列及去除AATAAA信号的PolyA序列,插入的这些PolyA序列均能部分解除Alu14对GFP基因的抑制作用;去除AATAAA信号的PolyA正、反序列仍然引起转录终止。将PolyA反序(PolyAas)分为4段每段60 bp,中间的2段分别称为2F2R和3F3R,将2F2R或3F3R插在pAlu14质粒的Alu串联序列的上游,随着插入2F2R片段拷贝数的增加转录的GFP融合RNA的分子量增加;2F2R的下游如果依然是2F2R那么2F2R可以支持转录延伸,如果2F2R下游是Alu串联序列则2F2R导致转录终止。无论插入一个3F3R或插入64个3F3R,均产生低分子量GFP RNA。     

Effect of alcohol consumption on peripheral blood Alu methylation in Korean men

Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence, which create a substantial public health problem worldwide. To date, no therapeutic can effectively solve these problems. They are complex diseases characterized by both genetic and environmental factors. DNA methylation can act as a downstream effector of environmental signals and account for multi-factorial nature of the disease. Global DNA methylation of peripheral blood cells has recently been proposed as a potential biomarker for disease risk. Alu elements host one-quarter of CpG dinucelotides in the genome to function as proxies for global DNA methylation. In this study, we evaluated the Alu methylation in the peripheral blood DNA of healthy volunteers and AUD patients using the pyrosequencing technology. The Alu methylation level is significantly higher in AUD compared to healthy controls (23.4?±?1.6 versus 22.1?±?1.0, t?=?7.83, p?<?0.0001). Moreover, significant correlation was found between Alu methylation and alcohol use disorders identification test score (r?=?0.250, p?<?0.0001), alcohol problem (r?=?0.294, p?<?0.0001), and life position (r?=?–0.205, p?=?0.0005). Overall, these novel findings indicate that alcohol-related increase in Alu methylation might play a complex role in the etiology and pathogenesis of AUD. Further studies are required to elucidate the mechanisms underlying this relationship.     

Impact of copy number of distinct SV40PolyA segments on expression of a GFP reporter gene

The presence of Alu repeats downregulates the expression of the green fluorescent protein(GFP) gene.We found that SV40PolyA(PolyA,240 bp),in either orientation,eliminated the inhibition of GFP gene expression induced by Alu repeats when it was placed between the GFP gene and the Alu repeats.In this study,4 different segments(each 60 bp) were amplified from antisense PolyA(PolyAas) by PCR,and inserted upstream of Alu14 in pAlu14 plasmid(14 Alu repeats inserted downstream of the GFP gene in vector pEGFP-C1 in...     

Evolution of alu family repeats since the divergence of human and chimpanzee

Summary The DNA sequences of three members of the Alu family of repeated sequences located 5 to the chimpanzee 2 gene have been determined. The base sequences of the three corresponding human Alu family repeats have been previously determined, permitting the comparison of identical Alu family members in human and chimpanzee. Here we compare the sequences of seven pairs of chimpanzee and human Alu repeats. In each case, with the exception of minor sequence differences, the identical Alu repeat is located at identical sites in the human and chimpanzee genomes. The Alu repeats diverge at the rate expected for nonselected sequences. Sequence conversion has not replaced any of these 14 Alu family members since the divergence between chimpanzee and human.     

人肺癌基因组DNA对NIH／3T3细胞的转化作用

从未用过抗癌细胞毒药物的非小细胞肺癌(NSCLC)患者的手术标本(鳞状上皮癌)提取癌细胞基因组总DNA。对小鼠成纤维(NIH/3T3)细胞行转染实验。获二轮转化细胞,发现二轮转化率是一轮的2.7倍。在转染过程中转化灶出现的多少,与所用DNA的量有一定关系。 二轮转化细胞能在软琼脂上存活生长,接种裸鼠能长出肿瘤,分离肿瘤组织细胞,体外培养传代存活。表明该二轮转化细胞具有肿瘤细胞的特性。 取一轮、二轮转化细胞和裸鼠肿瘤细胞的DNA分别与放射性~(32)P标记的人体特有的Alu重复序列和ras家族基因探针进行Southern印迹转移和分子杂交。结果在三者细胞的DNA中都见有与Alu杂交的条带。这表明在转染过 程中人体特有的Alu重复序列已整合到转化细胞的基因组中。并确定了转化细胞中的转化基因之一的属性为Ha-ras癌基因。本工作提示吸烟可能是人肺鳞癌发生和Ha-ras活化的重要因素。     

The age of Alu subfamilies

Using Kimura's distance measure we have calculated the average age of all major Alu subfamilies based on the most recent available data. We conclude that AluJ sequences are some 26 Myr older than previously thought. Furthermore, the origin of the FLA (Free Left Arm) Alu family can be traced back to the very beginning of the mammalian radiation.One new minor subfamily is reported and discussed in the context of sequence diversity in major Alu subfamilies. Correspondence to: J. Jurka