Transcriptional silencing and developmental reactivation of cry1Ab gene in transgenic rice

One transgenic rice line lacking CrylAb expression product was screened in the progenies of Agrobacterium-transformed transgenic rice variety Zhong 8215 with a cry1Ab gene under field releasing conditions by using GUS histochemical assay and Western blot. Molecular hybridization results revealed that the crylAb gene was silenced in the transgenic rice variety Zhong 8215 and two copies of ubiquitin promoter were integrated into the rice genome. The silencing of crylAb gene in transgenic rice was found to be due to the methylation of the ubiquitin promoter as revealed by methylation analysis. Meanwhile, different concentrations of demethylation reagent 5-azacytidine combining with different treatment time were employed to treat the silenced transgenic rice seeds. The results indicated that 5-azacytidine could reactivate 8%-30% of the silenced transgenic rice plants and the expression level of the reactivated cry1Ab transgene could reach as high as 0.147% of the total soluble protein. Treatment with low con     

Deletional analysis of functional regions of complementary sense promoter from cotton leaf curl virus

Complementary sense promoter from cotton leaf curl virus (CLCuV) is a novel plant promoter for genetic engineering that could drive high-level foreign gene expression in plant. To determine the optimal promoter sequence for gene expression, CLCuV promoter was deleted from its 5' end to form promoter fragments with five different lengths, and chimeric gus genes were constructed using the promoterdeletion. These vectors were delivered into Agrobacterium and tobacco (Nicotiana tabacum L cv. Xanthi) plants which were transformed by leaf discs method. GUS activity of transgenic plants was measured. The results showed that GUS activities with the promoter deleted to -287 and -271 from the translation initiation site were respectively about five and three times that of full-length promoter. There exists a c/s-element which is important for the expressing activity in phloem from -271 to -176. Deletion from -176 to -141 resulted in a 20-30-fold reduction in GUS activity in leaves with weak activity in leaves and     

Regulation of purine biosynthetic genes expression in Salmonella typhimurium Ⅳ O~c mutation site of purG and its function analysis

Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded by purG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu-tamine and ATP for the de novo purine nucleotide biosynthesis. purG gene is negatively regulated by a repressor-oper-ator system. The O purG and OC purG were cloned respectively in vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O ) and pLBG-2 (OC) were carried out. The hybrid plasmids pLB1933 (O ) and pLB1927 (OC) containing 5 control region of purG were constructed and the DNA sequences were determined respectively. DNA se-quences data showed that Oc mutation of purG occurred at the 3rd position of 16 bp PUR box in the 5' control region ( G→A). Gel retardation experiment indicated that the repressor bound well with O PUR box, but not with Oc PUR box. The result strongly supported the idea that PUR box is the binding region of represser protein and the 3rd posi-tion base G of PUR bo     

Methylation patterns of the human apoA-I/C-III/A-IV gene cluster in adult and embryonic tissues suggest dynamic changes in methylation during development.

We describe here a detailed analysis of the methylation patterns of the apoC-III and apoA-IV genes in adult and embryonic tissues. Together with previously reported data on the human apoA-I gene (4), the results presented here constitute a comprehensive study on the methylation pattern of the apoA-I/C-III/A-IV gene cluster. The two genes (apoC-III and apoA-IV) display tissue-specific methylation patterns that correlate with their activity. This gene-specific methylation pattern indicates that the apoA-I/C-III/A-IV gene cluster is not one entity with respect to methylation. The cluster is almost entirely methylated in tissues that do not express any of the genes; however, individual gene regions are unmethylated in the tissue of expression. A comparison of the observed methylation patterns in adult tissues with those in embryonic tissues suggests that the mature tissue-specific methylation patterns are a result of an interplay between demethylation and de novo methylation events in the embryo. These changes in DNA methylation include demethylation in the early embryo followed by de novo methylation at later stages. A second round of tissue-specific demethylation and methylation de novo occurs in the late embryo as well. Evidence presented here supports the idea that CpG islands are protected in general from methylation de novo by a built-in signal and not by CpG density per se.     

DNA methylation pattern of CALCA in preterm neonates with bacterial sepsis as a putative epigenetic biomarker

Diagnosis of bacterial sepsis in preterm neonates can be difficult when using serum markers that rely on physiological changes because these changes may not necessarily be the result of bacterial infections alone. This retrospective investigation explores the potential use of the DNA methylation pattern of CpG sites in the promoter region of the calcitonin-related polypeptide α (CALCA) gene as an epigenetic biomarker for bacterial sepsis in preterm newborns. Four novel changes in the DNA methylation of eight CpG sites were detected in this gene and are present only in neonates with bacterial sepsis: (1) partial methylation at -769 CpG in gram-negative or gram-positive early onset sepsis (EOS) and late onset sepsis (LOS) episodes; (2) demethylation of 8 CpGs in gram-negative EOS followed by LOS (ELS) and in gram-negative EOS; (3) demethylation of 7 CpGs in gram-positive ELS and gram-positive EOS; (4) -771 C:G > T:A; 5′ de novo -778 CpG mutation on both alleles in EOS. These changes were not detected in birth weight and gestational age matched controls or in newborns with isolated infections. Our results indicate that the DNA methylation pattern of the promoter region of the CALCA gene varies in different types of bacterial preterm sepsis, thus suggesting a potential use as an epigenetic biomarker. A prospective confirmation of these results is essential.     

高半胱氨酸在平滑肌细胞中介导DNA甲基化及机制的研究

高同型半胱氨酸血症是引起动脉粥样硬化一个重要独立的危险因子,可以引起基因DNA甲基化表型改变和蛋白质表达失调,但是基因甲基化表型改变的特点和动脉粥样硬化是否有关及其机制,到目前为止还没有研究清楚.在平滑肌细胞培养的基础上研究高同型半胱氨酸血症对DNA甲基化的影响,高半胱氨酸诱导DNA甲基化表型改变的特征及潜在的机制.高半胱氨酸加入人脐静脉平滑肌培养24h后,高效液相检测SAM和SAH的浓度,实时RT-PCR和蛋白质印迹检测SAH水解酶mRNA和蛋白质表达.通过内源性DNA甲基转移酶活性变化、基因组DNA接受甲基的能力、甲基化限制性内切酶分析检测DNA甲基化水平的变化.结果显示,随着高半胱氨酸浓度的增加,SAH水平增加,SAM和SAM/SAH比率下降,SAH水解酶水平下降,但DNA甲基转移酶活性增加,用不同甲基化限制性内切酶分析发现C↓CGG序列更容易甲基化.由此可以推测,不同剂量的高半胱氨酸引起细胞损害效应的机制也不同,在低、中度高同型半胱氨酸血症,高半胱氨酸主要通过干扰高同型半胱氨酸的代谢途径影响基因表达表型修饰,在高度高同型半胱氨酸血症可能氧化应激、凋亡、炎症等发挥了更重要的作用.     

Targeting of De Novo DNA Methylation Throughout the Oct-4 Gene Regulatory Region in Differentiating Embryonic Stem Cells

Differentiation of embryonic stem (ES) cells is accompanied by silencing of the Oct-4 gene and de novo DNA methylation of its regulatory region. Previous studies have focused on the requirements for promoter region methylation. We therefore undertook to analyse the progression of DNA methylation of the ∼2000 base pair regulatory region of Oct-4 in ES cells that are wildtype or deficient for key proteins. We find that de novo methylation is initially seeded at two discrete sites, the proximal enhancer and distal promoter, spreading later to neighboring regions, including the remainder of the promoter. De novo methyltransferases Dnmt3a and Dnmt3b cooperate in the initial targeted stage of de novo methylation. Efficient completion of the pattern requires Dnmt3a and Dnmt1, but not Dnmt3b. Methylation of the Oct-4 promoter depends on the histone H3 lysine 9 methyltransferase G9a, as shown previously, but CpG methylation throughout most of the regulatory region accumulates even in the absence of G9a. Analysis of the Oct-4 regulatory domain as a whole has allowed us to detect targeted de novo methylation and to refine our understanding the roles of key protein components in this process.     

Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer.

Cytosine methylation at CpG dinucleotides is thought to cause more than one-third of all transition mutations responsible for human genetic diseases and cancer. We investigated the methylation status of the CpG dinucleotide at codon 248 in exon 7 of the p53 gene because this codon is a hot spot for inactivating mutations in the germ line and in most human somatic tissues examined. Codon 248 is contained within an HpaII site (CCGG), and the methylation status of this and flanking CpG sites was analyzed by using the methylation-sensitive enzymes CfoI (GCGC) and HpaII. Codon 248 and the CfoI and HpaII sites in the flanking introns were methylated in every tissue and cell line examined, indicating extensive methylation of this region in the p53 gene. Exhaustive treatment of an osteogenic sarcoma cell line, TE85, with the hypomethylating drug 5-aza-2'-deoxycytidine did not demethylate codon 248 or the CfoI sites in intron 6, although considerable global demethylation of the p53 gene was induced. Constructs containing either exon 7 alone or exon 7 and the flanking introns were transfected into TE85 cells to determine whether de novo methylation would occur. The presence of exon 7 alone caused some de novo methylation to occur at codon 248. More extensive de novo methylation of the CfoI sites in intron 6, which contains an Alu sequence, occurred in cells transfected with a vector containing exon 7 and flanking introns. With longer time in culture, there was increased methylation at the CfoI sites, and de novo methylation of codon 248 and its flanking HpaII sites was observed. These de novo-methylated sites were also resistant to 5-aza-2'-deoxycytidine-induced demethylation. The frequent methylation of codon 248 and adjacent Alu sequence may explain the enhanced mutability of this site as a result of the deamination of the 5-methylcytosine.