鸟分枝杆菌PhoP功能分析及其基因突变株的构建

【目的】对鸟分枝杆菌PhoP的功能进行分析及构建PhoP基因突变株,为深入研究PhoP的调控机制打下基础。【方法】利用PCR扩增出鸟分枝杆菌PhoP DNA结合区(PhoPC)编码序列,与表达载体p GEX-4T-3连接后,转化入大肠杆菌BL21(DE3)中表达GST-PhoPC融合蛋白。用凝血酶去除GST标签,制备PhoPC蛋白;利用PCR扩增出鸟分枝杆菌PhoP基因及其下游基因MAV0127、PhoU和Amt的启动子片段,采用凝胶迁移率移动试验(EMSA)分别检测PhoPC与PhoP、MAV0127、PhoU和Amt的启动子结合的情况。通过PCR扩增PhoP基因上、下游片段,构建PhoP基因缺失性同源核苷酸片段,与自杀质粒p GMB151连接后,通过电转化导入鸟分枝杆菌进行同源交换,利用PCR筛选出PhoP基因缺失突变株。【结果】EMSA结果显示,鸟分枝杆菌PhoP能与PhoP、MAV0127及Amt基因启动子结合,不能与PhoU结合。通过PCR和序列分析证实基因突变株的PhoP基因缺失了309个碱基。【结论】PhoP不仅可调控其下游基因MAV0127和Amt的转录水平,还可调控其自身基因的转录,但不参与调节PhoU二元调控系统。构建了PhoP基因缺失突变株,为进一步研究其在鸟分枝杆菌的调控功能奠定了基础。     

Expression of the rpsO and pnp genes: structural analysis of a DNA fragment carrying their control regions.

Precise physical mapping of the genes rpsO and pnp coding respectively for ribosomal protein S15 and polynucleotide phosphorylase together with regions involved in the regulation of their expression has been obtained by the analysis of in vitro deletion mutants. The results suggest that each gene has its own promotor, but that there is coexpression of rpsO and pnp. The nucleotide sequence of rpsO and of the beginning of pnp is presented and includes the presumed regulatory regions of these genes. Several features of the sequence support the mapping experiments and are discussed in relation to the expression of the ribosomal and pnp genes.     

Nucleotide sequence of the regulatory region of malB operons in E. coli

The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the promoter regions of the malB divergent operons was determined. The region of the proximal gene, malE of the malEFG operon, was identified on the basis of the known amino acid sequence of the precursor molecule of maltose-binding protein. The region of malK, the proximal gene of the malKlamB operon, was deduced from the observation that a cloned segment contains an amino-terminal portion of the malK gene. The non-coding region between malE and malK is 299 base pairs long and contains two long GC clusters. Another feature of this region that may be related to the regulation of gene expression is the presence of two palindromic structures between the GC clusters. The DNA regions binding to cyclic AMP binding protein were determined by a method using polyacrylamide gel electrophoresis. The sites are thought to be located close to GC clusters.     

Recognition of a Gene Involved in the Regulation of Nicotinamide Adenine Dinucleotide Biosynthesis

Mutations affecting the biosynthesis of quinolinic acid, a precursor of nicotinamide adenine dinucleotide (NAD) in Escherichia coli K-12, are either near min 17 (nadA mutants) or near min 49 on the chromosome. These nad mutants all exhibit a phenotypic requirement for NAD or one of its immediate precursors. The mutants with lesions near min 49 can be separated into two groups based on in vitro complementation analysis. One group (nadB) exhibits complementation with nadA mutants, whereas the other group fails to do so. The latter group is tentatively designated nadR based on its regulation of the unlinked nadA gene. The nadR gene maps adjacent to nadB between purI and tyrA.     

Proximal and distal sequences control UV cone pigment gene expression in transgenic zebrafish

The molecular basis of cone photoreceptor-specific gene expression is largely unknown. In this study, we define cis-acting DNA sequences that control the cell type-specific expression of the zebrafish UV cone pigment gene by transient expression of green fluorescent protein transgenes following their injection into zebrafish embryos. These experiments show that 4.8 kb of 5'-flanking sequences from the zebrafish UV pigment gene direct expression specifically to UV cones and that this activity requires both distal and proximal sequences. In addition, we demonstrate that a proximal region located between -215 and -110 bp (with respect to the initiator methionine codon) can function in the context of a zebrafish rhodopsin promotor to convert its specificity from rod-only expression to rod and UV cone expression. These experiments demonstrate the power of transient transgenesis in zebrafish to efficiently define cis-acting regulatory sequences in an intact vertebrate.     

46,XY女性患者SRY基因启动子区域的突变分析

大约15%的46,XY女性患者中发现SRY基因编码区突变,其他患者可能是SRY基因的调节区, 包括启动子区域发生了突变,或者其他相关基因发生突变所致。本文采用限制性酶切、PCR-SSCP及银染检测技术,对7例患者SRY基因的启动子区域进行了突变筛查, 结果未发现异常,提示这些患者的病因与SRY基因启动子区域本身无关,结合对患者SRY基因HMG基序DNA的突变分析结果,表明除SRY基因异常外还存在其他导致46,XY女性性反转综合征的遗传机制。 Abstract:Using restriction endonuelease digestion and PCR-SSCP with silver staining,we analyzed the promotor region of SRY gene in seven 46,XY femalcs.The results showed no abnormality,thus ruling out the mutations in the promotor region of the SRY gene as a possible cause of sex reversal in these XY females.In view with the absence of the mutations in the HMG regions of the SRY genes of several patients,it is suggested that SRY gene is not the only gene responsible for testicular development but is one of many hierarchical genes involved in a genetic cascade for sexual differentiation.     

Structural and functional organization of the colicin operon in the ColD-CA23 plasmid

The organization of the genes involved in colicin D synthesis was studied. These are colicin, immunity and lysis genes. The nucleotide sequence of the immunity gene, its structural and regulatory regions were determined. This gene was shown to be located next to the colicin gene on the same strand and followed by the lysis gene. When colicin synthesis is induced with mitomycin C the immunity gene is transcribed from the general SOS-dependent promotor as a part of the colicin operon. However it has its own SOS-independent promotor in normal growth conditions. A high homology in amino acid sequences of Co1D lysis protein and that of Co1E1, Co1E2, Co1E3, Co1DF13, Co1A was revealed. A detailed scheme of Co1D-CA23 colicin operon structural organization is suggested.     

Activin regulation of the follicle-stimulating hormone beta-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1

FSH is critical for normal reproductive function in both males and females. Activin, a member of the TGFbeta family of growth factors, is an important regulator of FSH expression, but little is known about the molecular mechanisms through which it acts. We used transient transfections into the immortalized gonadotrope cell line LbetaT2 to identify three regions (at -973/-962, -167, and -134) of the ovine FSH beta-subunit gene that are required for full activin response. All three regions contain homology to consensus binding sites for Smad proteins, the intracellular mediators of TGFbeta family signaling. Mutation of the distal site reduces activin responsiveness, whereas mutation of either proximal site profoundly disrupts activin regulation of the FSHbeta gene. These sites specifically bind LbetaT2 nuclear proteins in EMSAs, and the -973/-962 site binds Smad4 protein. Interestingly, the protein complex binding to the -134 site contains Smad4 in association with the homeodomain proteins Pbx1 and Prep1. Using glutathione S-transferase interaction assays, we demonstrate that Pbx1 and Prep1 interact with Smads 2 and 3 as well. The two proximal activin response elements are well conserved across species, and Pbx1 and Prep1 proteins bind to the mouse gene in vivo. Furthermore, mutation of either proximal site abrogates activin responsiveness of a mouse FSHbeta reporter gene as well, confirming their functional conservation. Our studies provide a basis for understanding activin regulation of FSHbeta gene expression and identify Pbx1 and Prep1 as Smad partners and novel mediators of activin action.     

Creation of artificial hybrid operons with partially overlapping genes to achieve an expression of heterologous genes in Escherichia coli cells

A new method of optimization of foreign gene expression in E. coli, based on the construction of hybrid operons with partially overlapping genes is described. The partial overlapping of the translation termination and initiation sites in the formed operon must provide translational coupling of appropriate gene product synthesis. Such an approach has provided the synthesis of human interferon alpha F in E. coli cells under the control of the lacUV5-promotor up to about (3-4).10(7) units per liter of bacterial culture. The reinitiation of the distal gene translation is shown to take place in the intercistronic region. Substitution of the lacUV5 promotor by the more efficient tac one allowed to increase the synthesis level of interferon alpha F to (1-2).10(8) units per liter. The conclusion is made about the equimolarity of distal and proximal to the promotor genes products syntheses when the intercistronic region of E. coli trpE-trpD genes are used for translational coupling.     

The role of hypoxia-inducible factor family (HIF) proteins in the regulation of cells physiologic responses to hypoxia

The review is devoted to the role of hypoxia-inducible factors (HIF) in the regulation of oxygen-dependent gene signalling. Structural features of HIF alpha and beta subunits as well as involvement of hydroxylation in the regulation of HIF stability and activity are described. Special attention is given to the role of phosphorylation and other post-translational modifications in the regulation of HIF expression and activity. The survey considers the involvement of hormones, cytokines and growth factors in HIF stimulation under normoxia. HIF target genes and promotor/enhancer sequences, responsible for oxygen-dependent gene regulation, are also described.     

Determining the extremes of the cellular NAD(H) level by using an Escherichia coli NAD(+)-auxotrophic mutant

NAD (NAD(+)) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD(+) level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD(+) auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD(+) de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD(+). We then constructed the NAD(+)-auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD(+) biosynthesis in cells harboring the ntt4 gene. The minimal NAD(+) level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD(+), while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD(+) was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD(+) concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.     

二元调控系统PhoP-PhoQ在细菌中的调控作用

PhoP-PhoQ是一种二元调控系统,它调控着细菌的毒力,参与细菌对Mg2 限制性生长环境的适应,而且还调节几种革兰阴性细菌的许多细胞活性。本文对PhoP-PhoQ感知Mg2 浓度的变化、与靶基因的启动子区结合引起靶基因的上调和下调,尤其是对脂质A的结构修饰和细菌毒力的调控作用作一介绍。