Integrated regulation of the photosynthetic gene family from Arabidopsis thaliana in transformed tobacco cells

Summary Expression of the three chlorophyll a/b binding protein (cab) genes of Arabidopsis thaliana was studied in transformed tobacco tissues. For each cab gene, approximately 1000 bp of the promoter region plus a portion of the structural gene was inserted into a promoter-expression vector such that a translational fusion between the cab gene and the promoter-less chloramphenicol acetyltransferase (cat) gene was formed. The constructed molecules were introduced into either cultured tobacco cells or tobacco leaves and the promoter activity was monitored as chloramphenicol acetyltransferase activity. The light-grown tissues exhibited 1.5- to 60-fold greater promoter activity than did dark-grown tissues. Expression of the cab promoters was tissue specific: activities were much stronger in green leaves than other tissues. The cab promoters were almost equally active in transformed calli or shoots derived from leaves. However, in cultured tobacco cells, one promoter was two to three times stronger than the other two. The chimeric gene fusion, cab-cat, segregated in the F1 generation as a dominant Mendelian trait.     

Sequence analysis of the 5′-flanking region of the gene encoding human N-acetylglucosaminyltransferase III

We have isolated and characterized the immediate (1651 bp) 5′-flanking region of the gene (GnT-III) encoding N-acetylglucosaminyltransferase III (GnT-III) from a human placental genomic library. Analysis of promoter elements shows a similarity to the 5′-flanking region of murine 1,4-galactosyltransferase. The sequence lacks obvious TATA elements and CCAAT boxes; however, putative regulatory sites, including 2 potential cAMP-response regulatory elements (CRE), 11 insulin-response element consensus sequences (IRE), 7 potential AP-2-binding sites, 2 SP1 consensus sequences (GC boxes) and 2 sequences similar to the half-palindromic glucocorticoid-responsive element (GRE), are present.     

Differential promoter activities of functional haplotypes in the 5′‐flanking region of human Sulfotransferase 1A1

Previously, we reported five common single nucleotide polymorphisms (SNPs), ?624G>C, ?396G>A, ?358A>C, ?341C>G, and ?294T>C, and six common haplotypes (CGACT, GAACT, GGAGC, GGACC, CAACT, and GAACC) in the 5′‐flanking region of the SULT1A1 gene that were associated with altered enzymatic activity. In the present study, we performed in vitro assays to determine the functional impact of these genetic variations on the promoter activity. Dual luciferase reporter assays revealed that these SNPs are located in a negative regulatory fragment of the SULT1A1 gene. Further experiments demonstrated that these SNPs and haplotypes affected promoter activities of SULT1A1. Electrophoretic mobility shift assays showed distinctive binding patterns for the SNPs ‐396G>A and ‐294T>C, due to differential binding affinities of the G/A alleles and the T/C alleles to nuclear proteins extracted from the liver carcinoma cell lines, HepG2 and Huh7. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:422–428, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21437     

Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection.     

利用强启动功能片段提高麦迪霉素4”，-羟基丙酰化酶基因的表达

利用PCR技术将本室克隆到的强启动功能片段取代麦迪霉素丙酰化酶基因(mpt)的启动子或与mpt基因自身启动子串连,获得含mPt重组质粒pCHFPE3和pCHFPE2。用含有这两个质粒的Streptomyces lividans TK24对螺旋霉素进行微生物转化,结果表明,与含有原启动子的mpt.S.lividans TK24(p.WFPE)相比,丙酰螺旋霉素的组分比例分别提高了89.02％和58.53％。含重组质粒pCHFPE2的螺旋霉素产生菌S.spiramyceticus发酵产物中丙酰螺旋霉素的组分也有较大辐度的提高。说明利用该强启动功能片段可以提高麦迪霉素丙酰化酶基因的表达。     

启动子CMV和EF1α对人胰岛素基因在BHK细胞中表达的影响

目的　构建人胰岛素基因真核高效表达载体 ,为胰岛素转基因研究奠定基础。方法　用限制性内切酶SpeⅠ和HindⅢ消化pEF1α GFP ,回收 1 2kbEF1α启动子 ,插入到pCMV mINS的NruⅠ和HindⅢ位点中 ,获得重组质粒pEF1α mINS ;将pCMV mINS和pEF1α mINS分别转染BHK细胞 ,用G418筛选 ,阳性克隆传至 2 0代后 ,分别用放免方法和免疫组化法分析胰岛素和 或胰岛素原在BHK细胞中的表达情况。结果　经放免测定 ,pCMV mINS和pEF1α mINS在BHK细胞中胰岛素和 或胰岛素原的表达量分别为 4 0 77μIU ml和 6 897μIU ml。经免疫组化分析 ,pCMV mINS在BHK细胞质中胰岛素表达水平的灰度值为 190 0± 19 5 6 ;pEF1α mINS在BHK细胞质中胰岛素表达水平的灰度值为 181 4± 18 45 ,在BHK细胞核中表达水平的灰度值为 15 5 4± 11 6 6。结论　在BHK细胞中启动子EF1α启动胰岛素基因表达的活性比启动子CMV高。     

Molecular cloning and expression analysis of the replacement histone H3 gene of Italian ryegrass (Lolium multiflorum)

The replacement histone H3 gene and its 5'-flanking sequence were isolated from Italian ryegrass by polymerase chain reaction and inverse polymerase chain reaction, respectively. Expression analysis showed that this gene is constitutively expressed in the entire plant. The expression level in leaves was found to be significantly low when compared with that in other tissues. However, the gene expression level in leaves was increased by the treatment with abscisic acid and abiotic stresses such as cold, heat and high-salinity (NaCl). The motif search of the 5'-flanking sequence of the replacement histone H3 gene revealed the presence of several potential cis-acting elements that could respond to the above-mentioned abiotic stresses. In addition to defence-related elements, we also found type I and II-/III-like elements, which are highly conserved motifs in the 5'-regulatory sequence of plant histone genes that are expressed specifically during the S-phase. Experiments using transgenic Italian ryegrass plants proved that the isolated 5'-flanking sequence of the replacement histone H3 gene, which was fused to a beta-glucuronidase reporter gene, was fully functional for inducing gene expression under various abiotic stress conditions.