Tetracycline-Inducible System for Regulation of Skeletal Muscle-Specific Gene Expression in Transgenic Mice

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.     

Aquaporin Expression and Freeze Tolerance in Candida albicans

Aquaporins are members of the major intrinsic protein superfamily of integral membrane proteins which enable the transport of water, glycerol, and other solutes across membranes in various organisms. In microorganisms, the physiological role of aquaporins is not yet defined. We found a clear correlation between expression of the Candida albicans aquaporin-encoding gene AQY1 and freeze tolerance. A connection with the function for the aquaporin in the natural environment of C. albicans is, however, not obvious.     

白念珠菌CaCdc37的表达及功能分析

CDC37基因编码的产物是一个参与蛋白激酶折叠成熟的分子伴侣蛋白,存在于多种真核生物中。在利用酵母双杂交系统筛选白念珠菌蛋白激酶Crk1相互作用蛋白时,获得一个CDC37同源基因。该基因编码区全长1524bp,编码一含508个氨基酸的蛋白质。其氨基酸序列与酿酒酵母Cdc37蛋白的序列同源性达41%。该基因在酿酒酵母中的表达能回复cdc37-1突变株的温度敏感表型,表明它能互补ScCDC37的功能。该基因命名为CaCDC37。Northern杂交显示,该基因在白念珠菌中呈组成型表达,转录水平不受形态转变和生长条件的影响;在crk1缺失株和CRK1高表达菌株中或者在cph1efg1双缺失株中,CaCDC37基因的转录水平没有明显变化。利用酵母双杂交系统分析CaCdc37与另外两个预测的白念珠菌分子伴侣蛋白CaSti1和CaHsp90的相互作用,结果表明CaCdc37能与CaSti1相互作用,而与CaHsp90的相互作用未能检测到。根据这些结果推测了CaCdc37可能的作用机制。     

Overlapping Functions between SWR1 Deletion and H3K56 Acetylation in Candida albicans

Nucleosome destabilization by histone variants and modifications has been implicated in the epigenetic regulation of gene expression, with the histone variant H2A.Z and acetylation of H3K56 (H3K56ac) being two examples. Here we find that deletion of SWR1, the major subunit of the SWR1 complex depositing H2A.Z into chromatin in exchange for H2A, promotes epigenetic white-opaque switching in Candida albicans. We demonstrate through nucleosome mapping that SWR1 is required for proper nucleosome positioning on the promoter of WOR1, the master regulator of switching, and that its effects differ in white and opaque cells. Furthermore, we find that H2A.Z is enriched adjacent to nucleosome-free regions at the WOR1 promoter in white cells, suggesting a role in the stabilization of a repressive chromatin state. Deletion of YNG2, a subunit of the NuA4 H4 histone acetyltransferase (HAT) that targets SWR1 activity through histone acetylation, produces a switching phenotype similar to that of swr1, and both may act downstream of the GlcNAc signaling pathway. We further uncovered a genetic interaction between swr1 and elevated H3K56ac with the discovery that the swr1 deletion mutant is highly sensitive to nicotinamide. Our results suggest that the interaction of H2A.Z and H3K56ac regulates epigenetic switching at the nucleosome level, as well as having global effects.     

Kl LAC4用作白色念珠菌的报告基因

由于ｌａｃＺ基因在白色念珠菌中不能工作。将克氏酵母的β－半乳糖苷酶基因Ｋｌ ＬＡＣ４构建了能在白色念珠菌中工作的报告基因。Ｋｌ ＬＡＣ４基因融合到白色念珠菌乙醇脱氢酶基因（ＡＤＨ１）的启动子后面,在ＡＤＨ１终止子的共同控制下构建Ｋｌ ＬＡＣ４的表达质粒ｐＹＰＢ１－ＬＡＣ４。ＰＹＯＢ１－ＬＡＣ４转化白色念珠菌并测定了在固体培养基中的β－半乳糖苷酶活性以及在液体增减基的β－半乳糖苷酶活力。结果表明Ｋｌ     

Development of a Tetracycline-Inducible Gene Expression System for the Study of Helicobacter pylori Pathogenesis

Deletion mutants and animal models have been instrumental in the study of Helicobacter pylori pathogenesis. Conditional mutants, however, would enable the study of the temporal gene requirement during H. pylori colonization and chronic infection. To achieve this goal, we adapted the Escherichia coli Tn10-derived tetracycline-inducible expression system for use in H. pylori. The ureA promoter was modified by inserting one or two tet operators to generate tetracycline-responsive promoters, named uPtetO, and these promoters were then fused to the reporter gfpmut2 and inserted into different loci. The expression of the tetracycline repressor (tetR) was placed under the control of one of three promoters and inserted into the chromosome. Conditional expression of green fluorescent protein (GFP) in strains harboring tetR and uPtetO-GFP was characterized by measuring GFP activity and by immunoblotting. The two tet-responsive uPtetO promoters differ in strength, and induction of these promoters was inducer concentration and time dependent, with maximum expression achieved after induction for 8 to 16 h. Furthermore, the chromosomal location of the uPtetO-GFP construct and the nature of the promoter driving expression of tetR influenced the strength of the uPtetO promoters upon induction. Integration of uPtetO-GFP and tetR constructs at different genomic loci was stable in vivo and did not affect colonization. Finally, we demonstrate tetracycline-dependent induction of GFP expression in vivo during chronic infection. These results open new experimental avenues for dissecting H. pylori pathogenesis using animal models and for testing the roles of specific genes in colonization of, adaptation to, and persistence in the host.     

Gluconeogenesis in Candida albicans

According to different metabolic situations in various stages of Candida albicans pathogenesis the regulation of carbohydrate metabolism was investigated. We report the genetic characterization of all major C. albicans gluconeogenic and glyoxylate cycle genes (fructose-1,6-bisphosphatase, PEP carboxykinase, malate synthase and isocitrate lyase) which were isolated after functional complementation of the corresponding Saccharomyces cerevisiae deletion mutants. Remarkably, the regulation of the heterologously expressed C. albicans gluconeogenic and glyoxylate cycle genes was similar to that of the homologous S. cerevisiae genes. A C. albicans DeltaCafbp1 deletion strain failed to utilize non-fermentable carbon sources but hyphal growth was not affected. Our results show that regulation of gluconeogenesis in C. albicans is similar to that of S. cerevisiae and that the current knowledge on how gluconeogenesis is regulated will facilitate the physiological understanding of C. albicans.     

Molecular Cloning and Functional Expression of Alternative Oxidase from Candida albicans

The AOX1 gene, which encodes an alternative oxidase, was isolated from the genomic DNA library of Candida albicans. The gene encodes a polypeptide consisting of 379 amino acids with a calculated molecular mass of 43,975 Da. The aox1/aox1 mutant strain did not show cyanide-resistant respiration under normal conditions but could still induce cyanide-resistant respiration when treated with antimycin A. The measurement of respiratory activity and Western blot analysis suggested the presence of another AOX. When C. albicans AOX1 was expressed in alternative oxidase-deficient Saccharomyces cerevisiae, it could confer cyanide-resistant respiration on S. cerevisiae.     

沙门氏菌中dnd基因簇的缺失与异源表达

沙门氏菌Salmonella enterica serovar Cerro 87是一株从鸡场分离到的菌株, 其DNA骨架上的磷硫酰化导致了在高压脉冲电泳过程中DNA降解(DNA degradation, Dnd表型)。本研究采用SacB所介导的负筛选系统, 在该菌株中成功缺失了dnd基因簇, 构建了突变株XTG103, 该突变株不再具有Dnd表型。通过异丙基-b-D硫代半乳糖苷(IPTG)诱导启动子PlacZ的转录可以调控DNA磷硫酰化修饰的dnd基因簇的异源表达。     

Gene disruption in Candida albicans using a synthetic, codon-optimised Cre-loxP system

The development of the molecular toolbox for the fungal pathogen Candida albicans has been hampered by its lack of an exploitable sexual cycle, its diploid nature, and its non-canonical genetic code. We describe the adaptation of the Cre-loxP site-specific recombination system as a tool for the efficient and controlled disruption of C. albicans genes. We have validated this system by disrupting two C. albicans loci: ADE2 and MET15. Ade2 and met15 null mutants were made using loxP-flanked ARG4- and HIS1-based disruption cassettes. These markers were then resolved from the C. albicans genome using a synthetic codon-optimised cre recombinase gene, with near 100% efficiency. Finally, CIp plasmids containing the URA3, HIS1, and ARG4 markers were generated for the reintegration of markers and target genes in control strains. This system allows multiple and sequential genetic manipulations, which will facilitate the functional analysis of multigene families in C. albicans.     

Plasmalemmasomes and lomasomes in Candida albicans

Candida albicans possesses plasmalemmasomes and lomasomes whose origin, structure and distribution are described. It is suggested that plasmalemmasomes become lomasomes during cell wall synthesis. The endoplasmic reticulum and lomasomes may be regarded as the functional equivalent to the Golgi apparatus in C. albicans and this view is discussed.     

Dimorphism and virulence in Candida albicans

Two regulatory pathways govern filamentation in the pathogenic fungus Candida albicans. Recent virulence studies of filamentation regulatory mutants argue that both yeast and filamentous forms have roles in infection. Filamentation control pathways seem closely related in C. albicans and in Saccharomyces cerevisiae, thus permitting speculation about C. albicans filamentation genes not yet discovered.