Identification and characterization of the human cytomegalovirus immediate-early region 2 gene that stimulates gene expression from an inducible promoter.

The human cytomegalovirus (HCMV) XbaI E cloned DNA fragment of approximately 20 kilobases can complement an adenovirus mutant (dl312) defective in the E1a viral gene product (D. J. Spector and M. J. Tevethia, Virology 151:329-338, 1986). This viral DNA fragment contains three immediate-early (IE) genes between 0.709 and 0.751 map units (M. F. Stinski, D. R. Thomsen, R. M. Stenberg, and L. C. Goldstein, J. Virol. 46:1-14, 1983). Two of the IE genes, IE1 and IE2, were isolated and tested for a role in regulating viral gene expression. Since HCMV early and late promoters require additional characterization, the chloramphenicol acetyl transferase (cat) gene, driven by the adenovirus E2 promoter, was used as an indicator of gene expression. cat expression from this heterologous viral promoter was shown to be stimulated by HCMV at early times after infection. The IE1 gene product did not function independently in activating this promoter. The IE2 gene products could independently stimulate the expression of a plasmid of a plasmid when the cat gene was placed downstream of the inducible E2 promoter (E2CAT). Five proteins of different sizes have been predicted to originate from IE2, depending on mRNA splicing. The protein products specified by the IE2 gene were characterized with an antibody to a synthetic peptide according to the open reading frame of exon 2. Three of the five proteins are encoded by exon 2. Three viral proteins of 82, 54, and 28 kilodaltons (kDa) were detected. The exons contained in the region designated as IE2a have open reading frames that could code for two of the smaller proteins of 27 and 30 kDa. This region, when driven by the HCMV enhancer, could independently stimulate gene expression from E2CAT to a high level. A plasmid with the HCMV enhancer upstream of exons, that could code for the HCMV IE2 proteins of 48 and 51 kDa, as well as 27- and 30-kDa proteins, also stimulated E2CAT expression but at a lower level. The activity of this plasmid was augmented by the IE1 gene product, despite the fact that the latter gene product alone was inactive. It is proposed that the HCMV IE region 2 gene products are involved in the regulation of viral or host cell promoters either independently or in combination with other HCMV IE proteins.     

Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi

The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi.     

The use of the luciferase reporter system forin planta gene expression studies

The properties of the firefly luciferase (LUC) make it a very good nondestructive reporter to quantify and image transgene promoter activity in plants. The short half-life of the LUC mRNA and protein, and the very limited regeneration of the LUC protein after reacting with luciferin, enables monitoring of changes in gene activity with a high time resolution. However, the ease at which luciferase activity is measuredin planta, using a light sensitive camera system (2D-luminometer), contrasts sharply with the complications that arise from interpreting the results. A variegated pattern of luciferase activity, that is often observed inin planta measurements, might either be caused by differences in influx, availability of the substrates (luciferin, oxygen, ATP) or by local differences in reporter gene activity. Here we tested the possible contribution of differences in the availability of each substrate to the variegatedin planta luciferase activity, and we show whenin planta luciferase activity is measured under substrate equilibrium conditions and can be related to the promoter activity of the reporter gene. Furthermore, we demonstrate the effects of protein stability, apparent half-life of luciferase activity, regeneration of luciferase and pH on thein vivo andin vitro luciferase measurements. The combined results give the prerequisites for the correct utilisation of the luciferase reporter system, especially forin vivo gene expression studies in plant research.     

Bacterial luciferase as a reporter of circadian gene expression in cyanobacteria.

To allow continuous monitoring of the circadian clock in cyanobacteria, we previously created a reporter strain (AMC149) of Synechococcus sp. strain PCC 7942 in which the promoter of the psbAI gene was fused to Vibrio harveyi luciferase structural genes (luxAB) and integrated into the chromosome. Northern (RNA) hybridization and immunoblot analyses were performed to examine changes in abundance of the luxAB mRNA, the native psbAI mRNA, and the luciferase protein to determine whether bioluminescence is an accurate reporter of psbAI promoter activity in AMC149. Under constant light conditions, the mRNA abundances of both luxAB and psbAI oscillated with a period of approximately 24 h for at least 2 days. The expression of these two genes following the same pattern: both mRNAs peaked in the subjective morning, and their troughs occurred near the end of the subjective night. The amount of luciferase protein also oscillated with a period of approximately 24 h, and the protein rhythm is in phase with the bioluminescence rhythm. The rhythm of the luciferase mRNA phase-leads the rhythms of luciferase protein and in vivo bioluminescence by several hours. Comparable results were obtained with a short-period mutant of AMC149. Together, these results indicate that the bioluminescence rhythm in AMC149 is due primarily to circadian oscillation of psbAI promoter activity in this cyanobacterium.     

Firefly luciferase as a reporter of regulated gene expression in higher plants

The firefly luciferase, assayedin vivo with a low-light video camera, acts as a non-invasive, real-time reporter of the temporal and spatial regulation of gene expression in single plants. Furthermore, the sensitivity of the luciferase assay in extracts of transformed plant tissue makes it a particularly useful marker in transient or stable transformation experiments.     

Identification and characterization of follistatin as a novel angiogenin-binding protein

Angiogenin enhances tumorigenesis. However, the mechanisms of angiogenin-induced angiogenesis and cancer cell proliferation remain elusive. In this study, follistatin was identified as a binding partner of angiogenin by a yeast two-hybrid screen and confirmed by a pull-down experiment. The interaction of fluorescently tagged angiogenin and follistatin was monitored in real time by a laser confocal microscope and shown to localize at the sub-nuclear region of HeLa cells. Additional yeast two-hybrid analysis revealed that domains 2 and 3 of follistatin were the minimal structure requirement for angiogenin binding. These findings provide new clues for further studies on the mechanisms of angiogenin-induced angiogenesis or cancer cell growth.     

Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas

The use of luciferases as reporters of gene expression in living cells has been extended to the chloroplast genome. We show that the luciferase from the soft coral Renilla reniformis (Rluc) can be successfully expressed in the chloroplast of Chlamydomonas reinhardtii. Expression of the rluc cDNA was driven by the promoter and 5′ untranslated regions of the atpA gene. Western analysis with an anti-Rluc antibody detected a single polypeptide of 38 kDa in the luminescent cells. This is 3 kDa larger than native Rluc, and suggests that translation of the chimeric mRNA begins at the atpA start codon, 29 codons upstream from the rluc start site. We also show that the luminescence of the transformants was sufficient to enable imaging of colonies using a cooled CCD camera. Received: 12 April 1999 / Accepted: 24 June 1999     

Firefly luciferase as a reporter to study gene expression in Streptococcus mutans.

The utility of firefly luciferase as a reporter was tested in Streptococcus mutans. Under control of an endogenous promoter, the luciferase coding sequence was strongly expressed, while a promoterless version was indistinguishable from the background. Luciferase activity was easily extracted and the assay rapid and reproducible. In addition, the half-life of luciferase activity was found to be comparable to those of other frequently used reporters. Thus, firefly luciferase can readily be used as a reporter in S. mutans, a useful alternative to methods requiring radioactive isotopes.     

基于萤火虫荧光素酶报告基因检测凋亡的系统构建及鉴定

为构建一个能够快速检测凋亡的含荧光素酶报告基因 (Fluc) 的真核表达质粒,采用PCR的方法将Caspase-3的识别基序Asp-Glu-Val-Asp (DEVD) 四个氨基酸引入至Fluc基因的C端和N端之间;同时选取Asp-Glu-Val-Gly (DEVG) 四个氨基酸作为阴性对照。随后重组片段分别克隆至分离型内含肽的N端和C端之间,并命名为pFluc-DEVD和pFluc-DEVG。将该表达质粒与海肾荧光素酶报告质粒 (RLUC) 同时转染至HeLa细胞,通过双荧光素酶报告基因和Western blotting实验检测细胞凋亡水平。双荧光素酶报告基因系统实验结果显示,当发生凋亡时,pFluc-DEVD质粒组表达的萤火虫荧光素酶的含量高出pFluc-DEVG质粒组约3倍。Western blotting检测结果显示,转染pFluc-DEVD质粒的细胞在凋亡药物刺激后,Fluc活化的蛋白显著增多,且Caspase-3 的活化程度与Fluc的表达呈正相关,并有显著的统计学差异。以上结果表明,pFluc-DEVD真核表达质粒表达的萤火虫荧光素酶蛋白能被细胞内的Caspase-3酶裂解,且该质粒能够精确地反映出细胞的凋亡水平,为后续进行凋亡定量检测提供了一个可借鉴的方法。     

Identification and characterization of FHL3 as a novel angiogenin-binding partner

Angiogenin (Ang) is known to induce cell proliferation and inhibit apoptosis by cellular signaling pathways and by direct nuclear functions of Ang, but the mechanism of action for Ang is not yet clear. The aim of present study was to identify novel binding partner of Ang and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, Ang was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Four and a half LIM domains 3 (FHL3) was identified as a novel Ang binding partner. The interaction between Ang and the full length FHL3 was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore, FHL3 was required for Ang-mediated HeLa cell proliferation and nuclear translocation of Ang. These findings suggest that the interaction between Ang and FHL3 may provide some clues to the mechanisms of Ang-regulated cell growth and apoptosis.     

小鼠T-bet启动子荧光素酶报告基因载体的构建及活性鉴定

为了研究T-bet在T细胞中的转录调控机制,并研究其在多发性硬化症中的信号通路,本研究构建小鼠TBX21(编码T-bet)基因启动子区和增强子区萤火虫荧光素酶报告基因载体。在对小鼠TBX21基因5?侧翼区进行详尽生物信息学特征分析后,设计相应引物,用PCR的方法从小鼠基因组中扩增出TBX21基因5?侧翼区–1 000 bp-28 bp片段长为1 028 bp的启动子区(以翻译起始点ATG为+1)和–3 308 bp-–2 000 bp片段长为1308 bp的非编码区保守序列(No-coding conserved sequence,CNS),再用定向克隆的方法将这两个片段定向重组入专门用于启动子活性研究的萤火虫荧光素酶报告基因载体(p GL4.10)中,构建出包含小鼠TBX21基因启动子区和CNS区的萤火虫荧光素酶报告基因载体(p GL4.10-TBX21pr-CNS),电泳与测序鉴定,最后再将p GL4.10-TBX21pr-CNS与内参p RL-TK用lipofectamine 2000共转染293T细胞和Jurkat细胞中,通过双荧光素酶报告基因检测系统鉴定p GL4.10-TBX21pr-CNS的启动子和增强子活性,并用独立样本t检验方法进行统计分析。对照组共转染p GL4.10与内参p RL-TK。结果表明,成功构建出荧光素酶报告基因重组质粒p GL4.10-TBX21pr-CNS。与转染空质粒p RL-TK组相比,293T细胞(P=0.012 2)和Jurkat细胞(P=0.002 2)中转染p GL4.10-TBX21pr-CNS组荧光素酶活性升高。研究结果表明在293T细胞和Jurkat细胞中p GL4.10-TBX21pr-CNS可以表现出启动子活性,为后续小鼠T-bet转录调控研究提供了基本材料。