家蚕丝素蛋白轻链基因（fib-L）启动子序列的克隆及其活性分析

家蚕Bombyx mori丝素蛋白轻链（fibroin light-chain, fib-L）基因fib-L具有在后部丝腺组织专一性、高效性表达的特点。为了利用其启动子构建能够表达外源基因的丝腺生物工厂,本实验对fib-L启动子活性进行了研究。通过PCR法克隆了fib-L启动子元件,序列分析显示fib-L启动子由位于-33 ～ -25处的TATA盒元件和位于－128～－121处的特征性序列GTCAATTT共同组成。用fib-L启动子控制报告基因DsRed进行家蚕BmN细胞和蚕体内的瞬时表达研究,结果表明fib-L启动子可以驱动DsRed报告基因在BmN细胞和家蚕后部丝腺组织中瞬时表达。     

Transcriptional repression of the human p53 gene by hepatitis B viral core protein (HBc) in human liver cells

Hepatitis B virus (HBV) is a causative agent of chronic and acute hepatitis, and is associated with the development of hepatocellular carcinoma (HCC). We demonstrate here that the Hepatitis B viral core protein (HBc) functions as a repressor on the promoter activity of the human p53 gene. The functional analyses of the promoter of the p53 gene by serial deletion, site-directed mutagenesis, and the heterologous promoter system revealed that the promoter activity was repressed through the E2F1-binding site (nucleotides -28 to -8) by HBc. An electrophoretic mobility shift assay (EMSA) showed that the HBc reduced the DNA-binding ability of E2F1 to the binding site of the p53 promoter. The interaction of HBc with E2F1 was also observed by glutathione S-transferase (GST) fusion protein binding assay. Furthermore, HBc represses the expression of the p53 gene in the human liver cell line HepG2. Finally, HBc and HBx synergistically repress both the promoter activity and the expression of the p53 gene in HepG2 cells. These results, together with our previous study, strongly suggest that HBc, like HBx, represses the expression of the human p53 tumor suppressor gene.     

脱水应答转录因子CBF1的克隆与转基因小麦的分子检测

根据已发表的小麦(T.aestivum)转录因子CBF1基因序列(GenBank Accession No.AF376136),设计引物从小麦品种‘京花1号’叶片中克隆出该基因,用拟南芥RD29B基因为启动子构建含CBF1基因的逆境诱导表达载体pBAC127F(6 967 bp),以‘99-92’、‘5-98’、‘104’和‘轮选987’等冬小麦品种(系)的幼穗和幼胚为材料,基因枪转化该表达载体。经筛选与植株再生,共获得14株转基因植株及其后代株系。这14个株系经PCR分析和点杂交检测,最终确认了5-98-40、5-98-41这2个株系为转基因株系,结果表明拟南芥RD29B启动子调控下的转录因子CBF1基因已稳定整合到转基因植株中。     

The murine lymphotoxin gene promoter. Characterization and negative regulation

Murine lymphotoxin (LT; TNF-beta) gene upstream regulatory elements were identified by linking fragments of 5' DNA to the chloramphenicol acetyl transferase gene. Fragment LT1 (-293 to +77 in relation to the proximal cap site) exhibited promoter activity which drove CAT expression in transfected murine fibroblasts and T lymphomas. Primer extension analysis of endogenous LT message confirmed that LT1 contained the necessary elements required for promoter function. Promoter activity was not observed when LT2 (-662 to +77), LT3 (-1186 to +77), or LT3 delta AX (-1186 to +77 (delta-662/-269)) were ligated to the chloramphenicol acetyl transferase gene and transfected into fibroblasts or T lymphomas. At least one upstream repressor element is postulated to account for this promoter inhibition. In contrast to the results obtained with fibroblast and T cell transfectants, LT1 was inactive in the B cell transfectants A20 and P3X63. This suggests that some B cells express a repressor factor that inhibits the LT promoter and/or they lack the necessary positive regulatory factors.     

Promoter methylation regulates estrogen receptor 2 in human endometrium and endometriosis

Steroid receptors in the stromal cells of endometrium and its disease counterpart tissue endometriosis play critical physiologic roles. We found that mRNA and protein levels of estrogen receptor 2 (ESR2) were strikingly higher, whereas levels of estrogen receptor 1 (ESR1), total progesterone receptor (PGR), and progesterone receptor B (PGR B) were significantly lower in endometriotic versus endometrial stromal cells. Because ESR2 displayed the most striking levels of differential expression between endometriotic and endometrial cells, and the mechanisms for this difference are unknown, we tested the hypothesis that alteration in DNA methylation is a mechanism responsible for severely increased ESR2 mRNA levels in endometriotic cells. We identified a CpG island occupying the promoter region (-197/+359) of the ESR2 gene. Bisulfite sequencing of this region showed significantly higher methylation in primary endometrial cells (n = 8 subjects) versus endometriotic cells (n = 8 subjects). The demethylating agent 5-aza-2'-deoxycytidine significantly increased ESR2 mRNA levels in endometrial cells. Mechanistically, we employed serial deletion mutants of the ESR2 promoter fused to the luciferase reporter gene and transiently transfected into both endometriotic and endometrial cells. We demonstrated that the critical region (-197/+372) that confers promoter activity also bears the CpG island, and the activity of the ESR2 promoter was strongly inactivated by in vitro methylation. Taken together, methylation of a CpG island at the ESR2 promoter region is a primary mechanism responsible for differential expression of ESR2 in endometriosis and endometrium. These findings may be applied to a number of areas ranging from diagnosis to the treatment of endometriosis.     

Similarities and differences in the function of regulatory elements at the 5' end of the human apolipoprotein B gene in cultured hepatoma (HepG2) and colon carcinoma (CaCo-2) cells.

The arrangement of regulatory elements along the apolipoprotein B promoter region (positions -898 to +1) has been examined in transient transfection experiments performed in HepG2 and Hep3B (hepatic) and CaCo-2 (intestinal) cell lines, all of which express the apoB gene, and also in Chinese hamster ovary cells, which do not express the gene. The overall distribution of positive and negative regulatory segments was very similar in the two hepatoma cell lines (HepG2 and Hep3B) but different from that observed in the colon carcinoma cells (CaCo-2). Thus, whereas 260 base pairs of 5'-flanking sequence were sufficient for maximal expression of the promoter in HepG2 cells, only 139 nucleotides were required for maximal expression in CaCo-2 cells. Promoter activity in Chinese hamster ovary cells was exhibited by short constructs, with maximal activity for the -85 construct. DNase I footprinting of the apolipoprotein B promoter region using hepatic and intestinal extracts revealed multiple sites of interaction between the DNA and nuclear proteins. Gel retention experiments using the region from -262 to -88 (the region of greatest contrast between HepG2 and CaCo-2 cells) revealed interesting variations in the relative abundance of various nuclear proteins between the two cell types. A major functional difference between HepG2 and CaCo-2 cells was localized to the region between -111 and -88, which harbors the sequence TGTTTGCT, a motif present in the promoter region of several liver-specific genes. The molecular basis for the functional differences between these two cell types may be attributable to a difference in the relative abundance of three proteins that bind to sequences between -111 and -88.     

The gene S promoter of hepatitis B virus confers constitutive gene expression.

The properties of the promoter of the hepatitis B surface antigen (HBsAg) were studied using recombinants containing either this promoter or the SV40 early promoter. Mouse L cells were transfected with these recombinants and the levels of gene expression obtained with the two promoters were compared. The level of expression of a cellular gene, the human fibroblast interferon gene, obtained with the HBsAg promoter was comparable to that obtained with the SV40 early promoter. Similarly when the HBsAg gene was controlled by the SV40 early promoter the level of HBsAg synthesis is in the same range as that observed with its own promoter. Together these results suggest that although the HBsAg gene codes for a structural viral protein, its expression is constitutive as for an early gene. The implications of these observations on the synthesis of HBV particles in vivo are discussed.     

Regulation of BN115, a low-temperature-responsive gene from winter Brassica napus.

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5' regulatory region (from bp -1107 to +100) was fused to the beta-glucuronidase (GUS) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic B. napus plants only after incubation at 2 degrees C. No expression was observed after incubation at 22 degrees C, either in the presence or the absence of ABA. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low-temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35S promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature- and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.