A female-specific desaturase gene responsible for diene hydrocarbon biosynthesis and courtship behaviour in Drosophila melanogaster

Drosophila melanogaster shows sexually dimorphic cuticular hydrocarbons, with monoenes produced in males and dienes produced in females. Here we describe a female-specific desaturase gene, desatF. RNAi knock-down led to a dramatic decrease in female dienes and increase in monoenes paralleled with an increase in copulation latency and a decrease in courtship index and copulation attempts by the males. The desatF gene was also expressed in females from D. sechellia, rich in dienes, but not D. simulans, which produce only monoenes. When hydrocarbons were feminized in D. melanogaster males by targeted expression of the transformer gene, the expression of desatF occurred. These results strongly suggest that desatF is a crucial enzyme for female pheromone biosynthesis and courtship behaviour in D. melanogaster.     

Divergent pathways mediate the induction of ANF transgenes in neonatal and hypertrophic ventricular myocardium.

To determine whether similar or divergent pathways mediate atrial natriuretic factor (ANF) induction in neonatal and hypertrophied adult ventricular myocardium, and to assess whether studies using an in vitro model system of hypertrophy have fidelity to the in vivo context during pressure overload hypertrophy, we generated transgenic mice which harbor either 638 or 3,003 bp of the rat ANF 5' flanking region ligated upstream from a luciferase reporter. Luciferase activity in the ventricles of day 1 transgenic neonates was 8-24-fold higher than the levels expressed in the ventricles of adult mice. Adult mice expressed the luciferase reporter in an appropriate tissue-specific manner. Transverse aortic constriction of adult mice harboring ANF reporter transgenes demonstrated no significant increase in reporter activity in the ventricle. These findings demonstrate that distinct regions of the ANF 5'-flanking region are required for inducible expression of the ANF gene in the hypertrophic adult ventricle compared with those required for atrial-specific and developmentally appropriate expression in the intact neonatal heart. Furthermore, the cis regulatory elements necessary for induction of ANF expression in endothelin-1 or alpha 1-adrenergically stimulated cultured neonatal ventricular myocytes are not sufficient for induction in the in vivo context of pressure overload hypertrophy.     

调控组织因子基因表达的药物筛选研究

本研究建立TF启动子转录活性的荧光素酶基因稳定细胞株,应用该细胞模型筛选调控TF基因表达的药物,并为深入研究其分子机制打下基础。构建TF启动子的一系列5′端截短型的荧光素酶报告基因质粒(包括-2174 bp~+128 bp,-684 bp~+128 bp,-247 bp~+128 bp,-201 bp~+128 bp),将质粒电转染至U937细胞中,建立表达荧光素酶报告基因的稳定细胞株。应用ATRA验证该细胞株的功能;应用bortezomib、尿多酸肽(CDA-II)等药物处理该细胞株24小时,分析荧光素酶基因活性,筛选出能够调控TF基因表达的药物。结果发现,5 nmol/L bortezomib能激活其转录活性,上调TF转录本表达水平;1 mg/ml CDA-Ⅱ抑制TF启动子的转录活性,下调TF转录本的表达水平。TF启动子逐步截短功能分析发现,bortezomib及CDA-ⅡII调控TF启动子转录活性的区域位于-201 bp—0 bp之间。结论:本研究建立了表达TF启动子荧光素酶活性的U937稳定细胞株,并筛选出能够调控TF基因转录的药物CDA-II及bortezomib,为将来筛选新药物及深入研究其分子机...     

The Bactrocera tryoni homologue of the Drosophila melanogaster sex-determination gene doublesex

A homologue of the bifunctional sex-determining gene, doublesex (dsx), has been identified in the tephritid fruit fly, Bactrocera tryoni, and has been found to be expressed in a sex-specific manner in adult flies. The male- and female-specific cDNAs are identical at their 5′ ends but differ at their 3′ ends and appear to be the products of alternate splicing. The level of identity of the sex-specific DSX proteins of B. tryoni with the D. melanogaster DSX proteins, across the region corresponding to the DNA binding domain and the oligomerization domains, is greater than 85%. Four sequence motifs which are ten to thirteen bases identical to the TRA/TRA-2 binding sites (thirteen-nucleotide repeat sequences) are present in the female-specific exon of the B. tryoni dsx gene.     

Genetic polymorphisms and functional characterization of the 5'-flanking region of the human CYP2C9 gene: in vitro and in vivo studies

OBJECTIVE: Genetic polymorphisms were identified in the 5'-flanking region of the human CYP2C9 gene, and their effects on the phenotype were evaluated on the basis of the luciferase reporter gene assay and the in vivo pharmacokinetics of phenytoin. METHODS: Genetic polymorphisms were screened by polymerase chain reaction-single-strand conformational polymorphism analysis, following sequencing with DNA samples obtained from 50 healthy volunteers and 133 adult epileptic patients. HepG2 hepatoma cells were cotransfected with various sequence patterns of 5'-flanking region-luciferase reporter gene constructs. Pharmacokinetic parameters of phenytoin in relation to the corresponding sequence patterns were estimated by the Bayesian method, and the results were compared with in vitro activities. RESULTS: Genetic analysis revealed the existence of 7 single nucleotide polymorphisms (SNPs). Allele frequencies of T-->C transition at position -1912 (T-1912C), C-1886G, C-1566T, G-1538A, C-1189T, G-982A, and A-162G were 0.019, 0.019, 0.077, 0.019, 0.579, 0.019, and 0.003, respectively. Some mutations occurred simultaneously, and a total of 6 sequence patterns (patterns 1-6) were observed. The luciferase reporter gene assay indicated that the presence of mutation(s) resulted in a reduction in luciferase activity of 41.4% (pattern 2) to 86.8% (pattern 5) compared with the activity of the wild-type construct. The calculated intrinsic clearance of phenytoin was also lower (up to a 40% reduction for pattern 2) when a mutation(s) was present. CONCLUSION: In addition to the two major mutations in the coding region (CYP2C9*2 and CYP2C9*3 ), mutations in the 5'-flanking region of the human CYP2C9 gene appear to contribute to the large interindividual variability in drug metabolism activity.     

Reassessment of 79B actin gene expression in the abdomen of adult Drosophila melanogaster

The expression pattern of the 79B actin gene in Drosophila melanogaster has been inferred previously by means of a reporter gene in which 79B actin promoter sequences drive the lacZ coding sequences. Although the 79B actin gene is expressed primarily in muscles of the thorax and first abdominal segment of the adults of both sexes, expression in the remaining abdominal segments appears limited to the male genital muscles and the male-specific Muscle of Lawrence (MOL). This reported abdominal expression pattern has been reassessed. By varying parameters of tissue preparation and lacZ reporter gene detection, expression of the 79B actin gene has been revealed in most dorsal abdominal longitudinal fibres and genital muscles of both females and males. These new results suggest that there are differences in the level of 79B actin gene expression among the various abdominal muscles of both sexes, and that abdominal expression is not limited primarily to male sex structures.     

Glucocorticoid-inducible gene expression vectors for use in Drosophila melanogaster Schneider 2 cells

Inducible, vector-based, expression systems that allow fine control of transgene expression are gaining more and more use in fundamental research as well as in therapeutic applications. In an effort to develop a tightly regulated heterologous expression system for Drosophila Schneider 2 cells, three different inducible reporter constructs were compared. These comprised six copies of the glucocorticoid response element fused to one of three distinct types of Drosophila gene promoters: (1) a TATA-box containing, (2) a TATA-less and (3) a bidirectional core sequence. These were fused to a luciferase reporter gene. The promoter constructs displayed different basal as well as agonist-induced activities. The implications of the observations made are discussed in the context of promoter properties and of induction of genes that may be studied in Drosophila.     

PML-RARα对环腺苷酸诱导急性髓系白血病细胞分化的影响

为了进一步探讨环腺苷酸cAMP协同低剂量氧化砷诱导急性早幼粒白血病细胞分化的分子机制，以稳定转染了PML—RARα融合基因的PR9细胞为实验对象，通过观察细胞的生长，并利用形态学实验、流式细胞技术和荧光素酶报告基因转染实验等检测cAMP和／或氧化砷处理前后细胞相关指标的变化，研究PML-RARα在cAMP诱导AML细胞分化过程中的作用。结果显示，虽然cAMP单独能使表达PML-RARα的PR9细胞表面分化抗原CD11b的阳性率有所升高；但细胞形态学分析表明，cAMP单用无法诱导表达PML—RARα融合蛋白的PR9细胞分化，只有联合氧化砷才能使细胞表现出完全分化的特征，并伴有CD11b表达的显著升高。此外，PML—RARα还可以明显抑制含有cAMP反应元件的报告基因的转录。结论：PML—RARα融合蛋白对cAMP诱导AML细胞分化的信号转导途径具有显著的抑制作用。     

Regulation of Na,K-adenosine triphosphatase gene expression by sodium ions in cultured neonatal rat cardiocytes.

Na,K-ATPase (Na,K-pump) plays an important role in the regulation of intracellular ion composition. The purpose of this study is to determine whether Na+ regulates the levels of mRNA coding for Na,K-ATPase alpha and beta subunits in cultured neonatal rat cardiocytes. We measured intracellular Na+ levels ([Na+]i) in cardiocytes using a Na(+)-sensitive fluorescence dye (SBFI). 1 mM ouabain caused a significant increase in [Na+]i in cardiocytes; from 12.8 +/- 0.3 to 28.8 +/- 1.8 mM. Exposure of cardiocytes to 1 mM ouabain resulted in a three- to fourfold increase in alpha 1, alpha 2, and alpha 3 mRNA accumulation, and an approximate two-fold increase in beta 1 mRNA accumulation. A maximum elevation was reached at 60 min in both cases. The ouabain-induced alpha 1 mRNA accumulation was still observed in the Ca(2+)-free culture medium. Exposure of cardiocytes to 10 microM monensin in the absence of extracellular Ca2+ also resulted in a threefold increase in alpha 1 mRNA accumulation. The increased alpha 1 mRNA expression by 1 mM ouabain was associated with a fourfold increase in alpha 1 subunit protein accumulation. Transfection experiments with chimeric plasmids containing 5'-flanking sequences of alpha 1, alpha 2, and alpha 3 isoform genes and a luciferase reporter gene revealed that 1 mM ouabain caused a twofold increase in luciferase activity in each alpha system. These results suggest that Na+ directly regulates Na,K-ATPase gene expression in cardiocytes. The transfection study further supports the premise that Na(+)-responsive elements are located within the 5'-flanking sequences of each alpha isoform gene.     

Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter.

To develop a system for overexpressing genes in the vascular wall, we created transgenic mice using the reporter gene luciferase and the murine preproendothelin-1 promoter. In vitro analysis suggested that the murine 5'-flanking region contained endothelial-specific elements in a 5.9-kb fragment. Five transgenic mice colonies established from independent founders all exhibited the highest level of luciferase activity in the aorta with up to 8,540 light units per microgram of protein. Immunohistochemistry with anti-luciferase antisera revealed high levels of expression in the endothelial cells of both large and small arteries and lower levels of expression in veins and capillaries. Significant expression was also seen in arterial smooth muscle cells and in select epithelial surfaces which is consistent with the known distribution of endothelin-1 in mammals. The further demonstrate the targeting capability of this system, we overexpressed the lipid-peroxidating enzyme, human 15-lipoxygenase, in the vessel wall of transgenic mice. As with luciferase, expression of active enzyme and immunohistochemical localization in vascular cells were documented in transgenic animals. Hence, this new system can be used to direct expression of molecules to the vascular wall for the purpose of examining the biological significance of either overexpression or inhibition of select proteins.