Roles of genes 44, 50, and 51 in regulating gene expression and host takeover during infection of Bacillus subtilis by bacteriophage SPO1

We show that the products of SPO1 genes 44, 50, and 51 are required for the normal transition from early to middle gene expression during infection of Bacillus subtilis by bacteriophage SPO1; that they are also required for control of the shutoff of host DNA, RNA, and protein synthesis; and that their effects on host shutoff could be accounted for by their effects on the regulation of gene expression. These three gene products had four distinguishable effects in regulating SPO1 gene expression: (i) gp44-50-51 acted to restrain expression of all SPO1 genes tested, (ii) gp44 and/or gp50-51 caused additional specific repression of immediate-early genes, (iii) gp44 and/or gp50-51 stimulated expression of middle genes, and (iv) gp44 and/or gp50-51 stimulated expression of some delayed-early genes. Shutoff of immediate-early gene expression also required the activity of gp28, the middle-gene-specific sigma factor. Shutoff of host RNA and protein synthesis was accelerated by either the 44- single mutant or the 50(-)51(-) double mutant and more so by the 44(-)50(-)51(-) triple mutant. Shutoff of host DNA synthesis was accelerated by the mutants early in infection but delayed by the 44(-)50(-)51(-) triple mutant at later times. Although gp50 is a very small protein, consisting almost entirely of an apparent membrane-spanning domain, it contributed significantly to each activity tested. We identify SPO1 genes 41 to 51 and 53 to 60 as immediate-early genes; genes 27, 28, and 37 to 40 as delayed-early genes; and gene 52 as a middle gene.     

The varicella-zoster virus-mediated delayed host shutoff: open reading frame 17 has no major function, whereas immediate-early 63 protein represses heterologous gene expression

We reported that varicella-zoster virus (VZV) causes a delayed host shutoff during its replicative cycle. VZV open reading frame 17 (ORF17) is the homologue of the herpes simplex virus (HSV) UL41 gene encoding the virion host shutoff (vhs) protein which is responsible for the shutoff effect observed in HSV-infected cells. In the present study, we demonstrated that ORF17 is expressed as a late protein during the VZV replicative cycle in different infected permissive cell lines which showed a delayed shutoff of cellular RNA. A cell line with stable expression of VZV ORF17 was infected with VZV. In these cells, VZV replication and delayed host shutoff remained unchanged when compared to normal infected cells. ORF17 was not capable of repressing the expression of the beta-gal reporter gene under the control of the human cytomegalovirus immediate-early gene promoter or to inhibit the expression of a CAT reporter gene under the control of the human GAPDH promoter, indicating that ORF17 has no major function in the VZV-mediated delayed host shutoff. To determine whether other viral factors are involved in the host shutoff, a series of cotransfection assays was performed. We found that the immediate-early 63 protein (IE63) was able to downregulate the expression of reporter genes under the control of the two heterologous promoters, indicating that this viral factor can be involved in the VZV-mediated delayed host shutoff. Other factors can be also implicated to modulate the repressing action of IE63 to achieve a precise balance between the viral and cellular gene expression.     

Combinatorial, selective and reversible control of gene expression using oligodeoxynucleotides in a cell-free protein synthesis system

Herein we describe the methods for selective and reversible regulation of gene expression using antisense oligodeoxynucleotides (ODNs) in a cell-free protein synthesis system programmed with multiple DNAs. Either a complete shut down or controlled level of gene expression was attained through the antisense ODN-mediated regulation of mRNA stability in the reaction mixture. In addition to the primary control of gene expression, we also demonstrate that the inhibition of protein synthesis can be reversed by using an anti-antisense ODN sequence that strips the antisense ODN off the target sequence of mRNA. As a result, sequential additions of the antisense and anti-antisense ODNs enabled the stop-and-go expression of protein molecules. Through the on-demand regulation of gene expression, presented results will provide a versatile platform for the analysis and understanding of the complicated networks of biological components.     

携带红色荧光蛋白的RU486可诱导真核表达载体的构建及其表达

在基因治疗中, 实现目的基因的调控表达是非常重要的。然而, 传统基因载体的无调控地持续或不适当的表达会影响治疗效果, 甚至可能带来致命的副作用。在本研究中, 我们构建了一种带有DsRed红色荧光蛋白报告基因并可经RU486诱导的真核表达载体, 并在体外评估了其调控表达作用。利用分子生物学技术, 将DsRed基因和启动子, 以及RU486系统构建成单一的质粒载体PDC-RURED, 为减少RU486调控元件和基因表达元件之间的相互干扰, 在两者之间加入1.6 kb的绝缘子。经PCR检测和限制性酶切分析及序列测定均证实了载体的正确性。在转染HEK293细胞后, 运用荧光显微镜和流式细胞技术证实了该载体的调控能力。没有RU486时, 几乎没有红色荧光蛋白的表达, 而加入诱导剂RU486后, 最高可以实现红色荧光蛋白的40余倍的表达。实验结果表明构建的可经RU486诱导的新型真核表达载体可以实现对目的基因的表达时间和表达水平的调控, 为进一步的基因调控研究和和基因治疗提供了良好的工具。     

Direct and tunable modulation of protein levels in rice and wheat with a synthetic small molecule

Direct control of protein level enables rapid and efficient analyses of gene functions in crops. Previously, we developed the RDDK‐Shield1 (Shld1) system in the model plant Arabidopsis thaliana for direct modulation of protein stabilization using a synthetic small molecule. However, it was unclear whether this system is applicable to economically important crops. In this study, we show that the RDDK‐Shld1 system enables rapid and tunable control of protein levels in rice and wheat. Accumulation of RDDK fusion proteins can be reversibly and spatio‐temporally controlled by the synthetic small‐molecule Shld1. Moreover, RDDK‐Bar and RDDK‐Pid3 fusions confer herbicide and rice blast resistance, respectively, in a Shld1‐dependent manner. Therefore, the RDDK‐Shld1 system provides a reversible and tunable technique for controlling protein functions and conditional expression of transgenes in crops.     

Nanoparticles,molecular biosensors,and multispectral confocal microscopy

Complex, multilayered nanoparticles hold great promise for more sophisticated drug/gene delivery systems to single cells. Outermost layers can include cell targeting and cell-entry facilitating molecules. The next layer can include intracellular targeting molecules for precise delivery of the nanoparticle complex inside the cell of interest. Molecular biosensors can be used to confirm the presence of expected molecules (for example, reactive oxygen species (ROS) as a surrogate molecule for signs of infection, or for activation in radiation damage, etc.) prior to delivery of counter-measure molecules such as drugs or gene therapy. They can also be used as a feedback control mechanism to control the proper amount of drug/gene delivery for each cell. Importantly, the full nanoparticle system can be used to prevent any cells from encountering the drug unless that cell is specifically targeted. Thus, if a cell is initially non-specifically targeted, a secondary check for other molecular targets which must also be present inside the target cell of interest can be used to catch initial targeting mistakes and prevent subsequent delivery of treatment molecules to the wrong cells. The precise intracellular location of nanoparticles within specific regions of a cell can be confirmed by 3D multispectral confocal microscopy. These single cell molecular morphology measurements can be extended from individual cells, to other cells in a tissue in tissue monolayers or tissue sections.