Novel screening method for potential skin-whitening compounds by a luciferase reporter assay

Measurement of the melanin content by using B16 melanoma cells is generally applied to find novel skin-whitening agents. However, this measurement method using B16 melanoma cells has such disadvantages, as the time taken, its sensitivity, and troublesomeness. We therefore attempted in the present study to establish a reporter assay system by measuring the tyrosinase promoter activity to use for convenient, high-throughput screening of new melanogenesis inhibitors. We first confirmed the validity of this reporter assay system by using such known skin-whitening agents, as arbutin, sulforaphane, and theaflavin 3,3'-digallate. We then compared the effect of 56 compounds on the tyrosinase promoter activity to test this reporter assay system. Carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity. Moreover, carnosol and rottlerin decreased melanin synthesis and tyrosinase expression in a dose-dependent manner when using B16 melanoma cells. These results indicate this new luciferase reported assay system to be an effective and convenient method for screening potential skin-whitening compounds.     

Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication

The 4150-bp plasmid pBR329 was constructed by the the insertion into pBR327 of an 877-bp DNA fragment carrying the Cmr gene from pBR328. This new cloning vector does not contain the 482-bp inverted duplication that has been reported to be present in pBR325 and pBR328 (Prentki et al., 1981). In pBR329 the Cmr gene lacks its original promoter but is transcribed counterclockwise toward the Apr gene by a promoter located to the right of the HindIII site in the Tcr gene.     

花生子叶生物反应器-花生产业中一支潜在的新生力量

花生子叶生物反应器具有产品部位特异、浓度高、可直接食用等优点。介绍了花生生物反应器需要的技术及该领域的研究现状和前景。花生生物反应器的关键是转基因载体的构建,花生主要贮藏蛋白的启动子是构建这种载体的最佳选择。大豆的Glycinin基因启动子也可用于构建花生生物反应器的转基因载体。     

The MET3 promoter: a new tool for Candida albicans molecular genetics

A central technique used to investigate the role of a Candida albicans gene is to study the phenotype of a cell in which both copies of the gene have been deleted. To date, such investigations can only be undertaken if the gene is not essential. We describe the use of the Candida albicans MET3 promoter to express conditionally an essential gene, so that the consequences of depletion of the gene product may be investigated. The effects of environmental conditions on its expression were investigated, using GFP as a reporter gene. The promoter showed an approximately 85-fold range of expression, according to the presence or absence of either methionine or cysteine in concentrations in excess of 1 mM. In the presence of either amino acid, expression was reduced to levels that were close to background. We used URA3 as a model to demonstrate that the MET3 promoter could control the expression of an essential gene, provided that a mixture of both methionine and cysteine was used to repress the promoter. We describe an expression vector that may be used to express any gene under the control of the MET3 promoter and a vector that may be used to disrupt a gene and simultaneously place an intact copy under the control of the MET3 promoter. During the course of these experiments, we discovered that directed integration into the RP10 locus gives a high frequency of transformation, providing a means to solve a long-standing problem in this field.     

Analysis of the distribution of the nucleotide sequence and electrostatic potential of the Escherichia coli genome

The oligonucleotide composition of the E. coli genome and its sigma70-specific promoters has been analyzed. The promoter DNA was shown to contain mainly AT-rich hexanucleotides having functionally important physical properties such as the ability to form easily melting sites and induce the bending of the double helix. A comparative analysis of the electrostatic characteristics of hexanucleotides within the whole sequence of the E. coli genome and its promoter regions was made. Hexanucleotides possessing a more electronegative surrounding were found to predominate in the nucleotide sequence of the promoter DNA.     

A potential gene delivery strategy using BK virus

Recombinant BK virus (rBKV) is able to express polypeptides under control of its native BKV late promoter. This ability helps to use this construct as a good reporter since it can infect human cells. In this study, we generate a BKV construct containing Renilla luciferase (Rluc) sequences under control of the BKV late promoter. The activity of Rluc was strongly detected in Vero-76 and Cos-1 cells transfected with rBKV-Rluc-myc-2A-VP2 construct, indicating the production of a functional enzyme driven by the native late promoter. Furthermore, a construct made of rBKV-IL2SP-Rluc-myc-2A-VP2 by introducing human IL2 secretion peptide (IL2 SP) caused secretion of IL2SP-Rluc-myc into the culture medium. As a concluding remark, a potential infectious rBKV that can express foreign antigens such as Rluc was generated successfully. The proposed strategy would be useful to engineer recombinant forms of rBKV with many potential applications including development of antiviral assay for new drugs, human vaccines and gene delivery systems for immunotherapeutic or cell transduction.