CS3纤毛抗原表达调控机理的研究

ＣＳ３是某些肠毒素大肠杆菌菌体表面上的多聚物,它能使病原菌粘附于宿主的小肠上皮细胞上,是致病的重要因素．为了探索ＣＳ３菌毛抗原基因的表达调控机制,根据ＣＳ３亚基结构基因的核苷酸序列分析表明,在其翻译起始位点的上游存在着ｒｂｓ位点及原核启动子的－１０区和－３５区ＤＮＡ序列．采用基因重组技术将ＣＳ３结构基因上游１２０ｂｐ的ＤＮＡ片段亚克隆进缺乏启动子而只含报告基因ｌａｃＺ的质粒ｐＣＢ２６７中．凝胶滞留和启动报告基因表达的实验证明了ＣＳ３亚基结构基因具有自身的启动子（Ｐｓ）．将该启动子上游区域不同长度的核苷酸片段克隆进ｐＣＢ２６７中,报告基因表达结果表明ＣＳ３结构基因的表达受其上游区域的抑制．核苷酸序列分析发现,在Ｐｓ－３５区上游５５０ｂｐ和８４０ｂｐ处各存在一个富Ａ－Ｔ簇．结合原核启动子的一般作用规律推知,ＣＳ３的表达可能受ＤＮＡ结合蛋白型的正向调节因子的作用．用ＣＦＡ／１菌毛抗原基因的正向调节基因ｃｆａＤ对ＣＳ３基因进行的互补表达试验表明ｃｆａＤ基因不仅可消除上游区对Ｐｓ的抑制,而且可大幅度地提高Ｐｓ的启动能力．在分析表达调控的基础上获得ＣＳ３重组高效表达．同时提出了其表达调控模型．     

Sequence Requirements for Myosin Gene Expression and Regulation in Caenorhabditis Elegans

Four Caenorhabditis elegans genes encode muscle-type specific myosin heavy chain isoforms: myo-1 and myo-2 are expressed in the pharyngeal muscles; unc-54 and myo-3 are expressed in body wall muscles. We have used transformation-rescue and lacZ fusion assays to determine sequence requirements for regulated myosin gene expression during development. Multiple tissue-specific activation elements are present for all four genes. For each of the four genes, sequences upstream of the coding region are tissue-specific promoters, as shown by their ability to drive expression of a reporter gene (lacZ) in the appropriate muscle type. Each gene contains at least one additional tissue-specific regulatory element, as defined by the ability to enhance expression of a heterologous promoter in the appropriate muscle type. In rescue experiments with unc-54, two further requirements apparently independent of tissue specificity were found: sequences within the 3' non-coding region are essential for activity while an intron near the 5' end augments expression levels. The general intron stimulation is apparently independent of intron sequence, indicating a mechanistic effect of splicing. To further characterize the myosin gene promoters and to examine the types of enhancer sequences in the genome, we have initiated a screen of C. elegans genomic DNA for fragments capable of enhancing the myo-2 promoter. The properties of enhancers recovered from this screen suggest that the promoter is limited to muscle cells in its ability to respond to enhancers.     

Autogenous Regulation of the Rega Gene of Bacteriophage T4: Derepression of Translation

The regA gene of phage T4 encodes a translational repressor that inhibits utilization of its own mRNA as well as the translation of a number of other phage-induced mRNAs. In recombinant plasmids, autogenous translational repression limits production of the RegA protein when the cloned structural gene is expressed under control of a strong, plasmid-borne promoter (lambda PL). We have found that a genetic fusion which places the regA ribosome binding domain in proximity to active translation leads to partial derepression of wild-type RegA protein synthesis. The derepression is not due to increased synthesis of regA RNA, suggesting that it occurs at the translational level. Derepressed clones of the wild-type regA gene were used to overproduce and purify the repressor. In an in vitro assay the wild-type target was sensitive and a mutant target was resistant to inhibition by the added protein. The results suggest that the sensitivity of a regA-regulated cistron to translational repression may depend on the competition between ribosomes and RegA protein for overlapping recognition sequences in the translation initiation domain of the mRNA.     

Interactions of the cbbII promoter-operator region with CbbR and RegA (PrrA) regulators indicate distinct mechanisms to control expression of the two cbb operons of Rhodobacter sphaeroides

In a previous study (Dubbs, J. M., Bird, T. H., Bauer, C. E., and Tabita, F. R. (2000) J. Biol. Chem. 275, 19224-19230), it was demonstrated that the regulators CbbR and RegA (PrrA) interacted with both promoter proximal and promoter distal regions of the form I (cbb(I)) promoter operon specifying genes of the Calvin-Benson-Bassham cycle of Rhodobacter sphaeroides. To determine how these regulators interact with the form II (cbb(II)) promoter, three cbbF(II)::lacZ translational fusion plasmids were constructed containing various lengths of sequence 5' to the cbb(II) operon of R. sphaeroides CAC. Expression of beta-galactosidase was monitored under a variety of growth conditions in both the parental strain and knock-out strains that contain mutations that affect synthesis of CbbR and RegA. The binding sites for both CbbR and RegA were determined by DNase I footprinting. A region of the cbb(II) promoter from +38 to -227 bp contained a CbbR binding site and conferred low level regulated cbb(II) expression. The region from -227 to -1025 bp contained six RegA binding sites and conferred enhanced cbb(II) expression under all growth conditions. Unlike the cbb(I) operon, the region between -227 and -545 bp that contains one RegA binding site, was responsible for the majority of the observed enhancement. Both RegA and CbbR were required for maximal cbb(II) expression. Two potentially novel and specific cbb(II) promoter-binding proteins that did not interact with the cbb(I) promoter region were detected in crude extracts of R. sphaeroides. These results, combined with the observation that chemoautotrophic expression of the cbb(I) operon is RegA independent, indicated that the mechanisms controlling cbb(I) and cbb(II) operon expression during chemoautotrophic growth are quite different.     

Up-promoter mutations in the lpp gene of Escherichia coli.

The promoter of the gene for the major outer membrane lipoprotein, the most abundant protein in Escherichia coli, is considered to be one of the strongest promoters in E. coli. The nucleotide sequences of the -10 and the -35 regions of the lpp promoter were altered in a step-wise manner to conform to their respective consensus sequences by synthetic oligonucleotide-directed site-specific mutagenesis. The mutated promoters were then fused to the lacZ gene to measure promoter activity. The beta-galactosidase activity increased approximately 1.9 and 2.4 fold when the -10 region (AATACT) was altered to TATACT(P1) and TATAAT (consensus sequence; P2), respectively. Similarly, it increased approximately 1.2 and 4.2 fold, when the -35 region (TTCTCA) was altered to TTCACA(R1) and TTGACA (consensus sequence; R2), respectively. When the mutations at the -10 and -35 regions were combined, the overall improvement of the promoter activity for R2-P1 was 4.0 fold over that of the wild-type promoter, while it was only 2.5 fold for R2-P2. These results indicate that substantial improvement of the promoter activity can be achieved by changing either of the two key regions to their respective consensus sequences. However, the complete conformity to consensus sequences at both regions does not necessarily result in the highest activity. With use of the improved lpp promoter in an expression cloning vehicle pIN-III-ompA, staphylococcal nuclease A was produced at a level of approximately 47% of the total cellular protein.     

Genetic and biochemical analysis of the MetR activator-binding site in the metE metR control region of Salmonella typhimurium

The Salmonella typhimurium metE and metR genes share a common control region, with overlapping, divergently transcribed promoters. A double gene fusion was constructed in which the metE promoter directs expression of the Escherichia coli lacZ gene and the metR promoter directs expression of the E. coli galK gene. By using an E. coli strain lysogenized with a lambda bacteriophage carrying the metE-lacZ metR-galK double fusion (lambda Elac.Rgal), two classes of cis-acting mutations were isolated that increase metR-galK expression. The first class of mutations causes a simultaneous decrease in metE-lacZ expression by disrupting the normal MetR-mediated activation of the metE promoter. The mutations are located within a region extending from 17 to 34 base pairs upstream of the -35 region of the metE promoter. Gel mobility shift assays and DNaseI protection experiments demonstrated that the MetR protein specifically binds to a 24-base-pair region encompassing these mutations. The second class of mutations increases metR-galK expression by directly altering the promoter consensus sequences of the metE and metR promoters.     

A Transgenic Mouse Model to Study Transsynaptic Regulation of Tyrosine Hydroxylase Gene Expression

Abstract: Previous studies demonstrated that 9 kb of the rat tyrosine hydroxylase (TH) 5' flanking sequence directed appropriate spatiotemporal expression of a lacZ reporter gene to catecholaminergic cells in the CNS of transgenic mice. In the present study, specificity of transgene expression was further extended to demonstrate cell type-specific functional regulation of lacZ expression using manipulations known to alter endogenous TH expression. Alterations in lacZ reporter expression should parallel changes in endogenous TH levels if the DNA elements mediating these functional changes of TH expression in vivo reside within the 9 kb of the TH promoter region. Naris closure induced an activity-dependent decrease of TH expression in dopaminergic periglomerular cells in the olfactory bulb that was paralleled by down-regulation of lacZ expression in the transgenic mice. Densitometry and image analysis were used to quantify lacZ expression following acute reserpine administration (5 mg/kg, s.c.), which up-regulates endogenous TH. At 48 h postinjection, analysis of OD values indicated a significant increase of X-gal staining in the locus coeruleus and ventral tegmental area but not in the substantia nigra or olfactory bulb of reserpine-treated transgenic animals. These data showed that the 9-kb sequence also mediates cell type-specific transsynaptic regulation of reporter gene expression. Analysis of this transgenic animal offers a useful model system to study in vivo regulation of TH gene expression.     

Molecular analysis of the promoter region of the Escherichia coli K-12 phoE gene. Identification of an element, upstream from the promoter, required for efficient expression of phoE protein

The phoE gene of Escherichia coli codes for an outer membrane pore protein whose expression is induced under phosphate limitation. The promoter of this gene contains a 17 base-pair fragment, designated a pho box, which is present also in other phosphate-controlled promoters. The mRNA start site was determined and found to be located downstream from the pho box, such that this element is located in the -35 region of the phoE promoter. A set of promoter deletions was generated in vitro and analysis of these deletions revealed that sequences upstream from the pho box are required for the efficient expression of phoE. The required upstream region is located (in part) between positions -106 and -121 relative to the mRNA start site, and contains sequences homologous to a pho box and a correctly spaced Pribnow box, but in the reversed orientation relative to the regular -35 and -10 regions. A proper spacing between this upstream region and the -35 region appears to be important, since an oligonucleotide insertion in the intervening region interferes with phoE expression. By cloning the upstream region in a lacZ operon fusion vector, a weak phosphate limitation-inducible promoter activity could be detected.     

麻疯树MIPS基因启动子的分离及在烟草原生质体中瞬时表达活性分析

根据麻疯树MIPS基因序列,设计特异性的巢式引物,运用TAIL-PCR法两次步移得到MIPS基因5＇端侧翼序列,序列分析显示含有多个胁迫应答相关元件,如ABRE、HSE等。以该序列为基础,PCR扩增得到5个5＇端不同长度的缺失片段,分别插入pBI221载体置换CaMV35S启动子,构建的表达载体在PEG介导下转入烟草叶片原生质体进行瞬时表达,检测GUS报告基因的活性。经GUS活性荧光定量检测发现,分离到的MIPS基因侧翼序列5＇端不同缺失片段都能启动GUS报告基因表达,启动活性最高的是WQ1区（-565bp）,核心区位于-565～-449bp。在100μmol·L-1ABA诱导下启动活性增强,但不同区段的增长幅度不同。WQ1区增长幅度最大,比未处理时提高41.4%。     

Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system

Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The A. oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A. oryzae glaA encoding glucoamylase and the agdA encoding alpha-glucosidase. To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A. nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter. Introduction of the sequence between -290 to -233 (the number indicates the distance in base pairs from the translation initiation point (+1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose. The sequence between -377 to -290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III. The sequence between -233 to -181 containing Region II had no effect on the expression. These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression.