Size reversion of a truncated DNAbeta associated with Tobacco curly shoot virus

Our previous results demonstrated that DNAbeta associated with Tobacco curly shoot virus (TbCSV) is not necessary for infection but intensifies symptoms in some hosts. To better understand the function of DNAbeta in virus infection, a betaC1 deleted infectious clone of the TbCSV DNAbeta was constructed. Agroinoculation showed that the truncated DNAbeta (DNADeltaC1beta) was trans-replicated by TbCSV in tobacco and Petunia hybrida plants. However, PCR and Southern blot analysis demonstrated that the truncated DNAbeta reverted to near wild type component size in some Nicotiana benthamiana, N. glutinosa, N. tabacum Samsun and P. hybrida plants co-inoculated with TbCSV and DNADeltaC1beta. Sequence analysis of four DNADeltaC1beta derivatives revealed that the wild type size DNAbeta molecules were recombinants between TbCSV DNAbeta and the pBinPLUS vector in which dimeric constructs were produced for inoculation. The significance of these findings is discussed with respect to the constraints imposed on begomovirus genome size.     

A modified viral satellite DNA-based gene silencing vector is effective in association with heterologous begomoviruses

We have previously reported effective gene silencing of a transgene and endogenous plant genes in tobacco and tomato plants using a modified viral satellite DNA associated with Tomato yellow leaf curl China virus (TYLCCNV). In this study, we constructed a similar gene silencing vector (DNADeltaC12beta) based on the satellite DNAbeta associated with Tobacco curly shoot virus (TbCSV) by replacing its betaC1 gene with a multiple cloning site. Strong and stable silencing of cognate genes was achieved when this vector, carrying a fragment of the green fluorescent protein (GFP) transgene or a sulfur (Su) endogenous gene encoding one unit of the chloroplast enzyme magnesium chelatase required for chlorophyll II production, was co-agroinoculated with TbCSV used as a helper virus. GFP silenced transgenic Nicotiana benthamiana plants appear red under UV illumination due to loss of green fluorescence, while the Su silenced plants appear white as a result of failure to synthesize chlorophyll. Our results show that the efficiency of Su silencing is independent of the insert orientation in both N. benthamiana and N. glutinosa plants. Most significant however, is the observation that in association with heterologous begomoviruses, such as TYLCCNV or Malvastrum yellow vein virus, the DNADeltaC12beta vector could still effectively induce transgene and endogenous gene silencing in tobacco plants. These observations suggest that the modified viral satellite DNA vector can be applied as a reverse genetics tool for the study, analysis and discovery of gene function in more plants.     

Pathogenicity and stability of a truncated DNAbeta associated with Tomato yellow leaf curl China virus

DNAbeta molecules are single-stranded satellite DNA associated with monopartite begomoviruses (family Geminiviridae). DNAbeta possesses a C1 gene on the complementary strand, which has a conserved position and size. To better understand the function of C1 gene in virus infection, a C1 deletion DNAbeta associated with a Tomato yellow leaf curl China virus (TYLCCNV) isolate was constructed. Co-agroinoculation with TYLCCNV showed the truncated DNAbeta was infectious in Nicotiana benthamiana and N. glutinosa plants but not in N. tabacum Samsun, N. tabacum and Lycopersicon esculentum plants. The wild-type TYLCCNV DNAbeta co-agroinoculated with TYLCCNV caused systemic infection in all the above hosts. Results of Southern blot analysis indicate that C1 gene is not required for TYLCCNV and DNAbeta replication. However, the presence of C1 gene in DNAbeta can increase both TYLCCNV and DNAbeta accumulation in infected plants. The truncated TYLCCNV DNAbeta was stable in N. benthamiana and N. glutinosa plants.     

Characterization of the promoter region of the human PARG gene and its response to PU.1 during differentiation of HL‐60 cells

The metabolism of poly(ADP‐ribose) plays important roles in the nuclear function of mammalian cells. Previously, we analyzed expression of the poly(ADP‐ribose) glycohydrolase (PARG) gene during HL‐60 cell differentiation and found that expression was greatly reduced by 4 h after 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) treatment and returned to the initial level within 20 h. In the present study, a 2.1‐kb fragment of the 5′‐flanking (promoter) region of the human PARG gene was isolated from the HL‐60 genome by polymerase chain reaction and ligated into a luciferase‐expression vector, pGL3, to generate the pPARG‐Luc#2 reporter plasmid. Deletion analysis revealed that a 75‐nt sequence is required for basal promoter activity and TPA responsiveness. Mutations in this 75‐nt sequence reduced promoter activity and the TPA response of HL‐60 cells. TFSEARCH analysis revealed that Ets family binding motifs are located in the 75‐nt sequence. Chromatin immunoprecipitation assay, electrophoretic mobility shift assay and competition analysis indicated that PU.1 (Spi‐1) binds to the 75‐nt sequence. Moreover, co‐transfection of HL‐60 cells with a PU.1 expression plasmid and pPARG‐Luc indicated that PU.1 down‐regulate the PARG promoter. These results suggest that PARG gene expression is modulated by PU.1 during TPA‐induced differentiation of HL‐60 cells.     

人NKX3.1基因启动子的克隆及在不同肿瘤细胞系中启动子活性的测定(英)

目的：克隆NKX3.1基因启动子并检测其启动子活性，为研究NKX3.1 基因转录调控的基本机制奠定基础。 方法： 采用PCR方法从人基因组中扩增NKX3.1基因上游 1.04 kb 启动子片段并分别克隆到报道基因载体pGL3-basic和 pEGFP-1中，通过转染细胞、荧光素酶活性测定及荧光显微镜下观察绿色荧光蛋白的表达，检测其启动子活性。 结果： DNA测序结果证实克隆的 1.04 kb 启动子片段序列正确；pGL3-1.04 kb 启动子转染LNCaP细胞后，双荧光素酶活性测定M1/M2=2.7, 为pGL3-control 活性的1.5倍，为pGL3-basic活性的50倍。表明克隆的人NKX3.1基因上游 1.04 kb 片段具有较强的启动子活性。为检测此 1.04 kb 启动子在不同组织细胞中的活性，分别将pGL3-1.04 kb 启动子和pEGFP-1.04 kb启动子转染入不同的肿瘤细胞系，检测荧光素酶和绿色荧光蛋白的表达。结果显示 1.04 kb 启动子在前列腺癌细胞LNCaP中活性最高。采用TRANSFAC数据库分析，发现在 1 040 bp 片段内含有多种顺式作用元件，它们的功能性将需要进一步实验证明。 结论： 克隆的人NKX3.1基因上游 1.04 kb 片段具有较强的启动子活性，并且在前列腺癌细胞LNCaP中活性最高。     

The minimal transforming fragment of HSV-2 mtrIII can function as a complex promoter element

The minimal transforming fragment (486 TF) of HSV-2 mtrIII (0.567-0.570 map units) is composed of two distinct and non-overlapping promoter elements when linked to bacterial CAT genes. A 230-nucleotide fragment of 486 TF, Sal1-Hpa1, was active as a promoter element in primate cells but not rodent cells. A 173-nucleotide fragment, Sma1-Pst1, was active in both primate and rodent cells. The 486 TF did not compete for limiting cellular factors required to drive the CAT gene under control of the SV40 early promoter/enhancer. However, gel-retardation assays suggest that unique factors exist in cells transformed by HSV-2 which specifically recognized regions of 486 TF. These results are discussed with respect to HSV-2-mediated transformation.