A calcium ionophore-inducible cellular promoter is highly active and has enhancerlike properties.

We examined the regulatory/promoter sequence of a calcium ionophore-inducible gene isolated from the rat genome. Whereas the promoter of this ubiquitously expressed gene is active under noninduced conditions, after induction by calcium ionophore A23187 this promoter is 10- to 25-fold more active than the simian virus 40 early promoter, as measured by chloramphenicol acetyltransferase activities. Within this regulatory/promoter region, we have identified a DNA fragment with enhancer-like properties immediately 5' to the TATA sequence. This 291-nucleotide fragment acts in cis to enhance expression of the neomycin phosphotransferase (neo) gene driven by the herpes simplex virus thymidine kinase promoter in an orientation-independent manner. In addition, this fragment can confer A23187 inducibility to the neo gene and effectively compete for positive regulatory factors involved in A23187 induction. Sequence analysis of this promoter reveals homology with viral core enhancer sequences, and the apparent organization of direct repeat domains is similar to those observed in viral enhancers.     

Expression from an internal AUG codon of herpes simplex thymidine kinase gene inserted in a retrovirus vector.

We identified structural features that affect the expression of an exogenous gene inserted into a retrovirus vector constructed by using spleen necrosis virus, an avian retrovirus. The thymidine kinase gene from herpes simplex virus type 1 containing deletions in the promoter and terminal sequences of the mRNA was inserted into spleen necrosis virus. We found that synthesis of thymidine kinase by the recovered virus was apparently initiated from internal AUG residues. At least in some cases, however, the level of expression depended on the number of AUGs and the nucleotide sequence around the AUGs that preceded the initiator codon of the thymidine kinase gene.     

Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.     

利用HSV1 α-基因启动子构建真核表达载体phIE

以pcDNA3质粒为骨架,构建由1型单纯疱疹病毒(Herpes simplex virus I,HSVI)立即早期基因启动子指导外源蛋白转录的phIE真核表达载体,该质粒为外源蛋白的表达提供了更为广泛的选择,通过插入us1编码序列,phIE-Us1被用于分析ICP22蛋白在病毒感染早期的功能;在多克隆位点插入氯霉素乙酰转移酶编码基因构建phIE-CAT检测载体,应用于检测HSVI病毒蛋白Vp16及ICP22的功能,明确显示该质粒对于研究单纯疱疹病毒的基因转录调控具有重要的应用价值。     

Regulation of cellular genes transduced by herpes simplex virus.

Previous studies demonstrated that the rabbit beta-globin gene is transcribed from its own promoter and regulated as a herpes simplex virus (HSV) early gene following insertion into the early HSV thymidine kinase gene in the intact viral genome (J. R. Smiley, C. Smibert, and R. D. Everrett, J. Virol. 61:2368-2377, 1987). We report here that the beta-globin promoter remained under early control after insertion into the late HSV gene encoding glycoprotein C. On the basis of these findings, we concluded that the beta-globin promoter is functionally equivalent to an HSV early-control region. We found that a transduced human alpha-globin gene was also regulated as an early HSV gene, while two linked Alu elements mimicked the behavior of HSV late genes. These results demonstrate that certain aspects of HSV temporal regulation can be duplicated by cellular elements and provide strong support for the hypothesis that the regulation of HSV gene expression can occur through mechanisms that do not rely on recognition of virus-specific temporal control signals.     

An expression vector inhibits gene expression in Xenopus embryos by antisense RNA

Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.     

Location and cloning of the herpes simplex virus type 2 thymidine kinase gene.

The herpes simplex virus type 2 thymidine kinase gene has been mapped to a position colinear with the herpes simplex virus type 1 thymidine kinase gene and cloned within a 4.0-kilobase fragment in pBR 322.     

Building a metal-responsive promoter with synthetic regulatory elements.

A fusion gene consisting of the promoter region from the mouse metallothionein-I gene joined to the coding region of the herpes simplex virus thymidine kinase gene is efficiently regulated by zinc in a transient assay when transfected into baby hamster kidney cells. Analysis of similar plasmids in which the metallothionein-I promoter region was mutated indicated the presence of multiple metal regulatory elements (MREs) between -176 and -44 base pairs from the cap site. To further investigate the function of MREs, we inserted a synthetic DNA fragment containing the sequence of MRE-a (the element between -55 and -44 base pairs) into the nonresponsive promoter of the thymidine kinase gene in various positions and configurations. Little or no induction by zinc was observed with single insertions of the regulatory sequence, whereas many different constructions having two copies of MRE-a were inducible. The precise position of the two MREs relative to each other or to the thymidine kinase promoter elements had a relatively small effect on the efficiency of induction, but the inducibility could be further increased by the introduction of more MRE-a sequences. MRE-a can function synergistically with the thymidine kinase distal promoter elements, but in the presence of the TATA box alone it functions as a positive, zinc-dependent promoter element.     

The herpes simplex virus thymidine kinase gene is not transcribed in Saccharomyces cerevisiae

The herpes simplex virus thymidine kinase gene has been cloned into a chimeric yeast plasmid cloning vehicle and transformed into appropriate yeast strains. Plasmids carrying the herpes simplex virus thymidine kinase gene can be propagated as autonomously replicating plasmids, but no RNA specific to the thymidine kinase coding sequence was detected.     

Gene transfer in bovine blastocysts using replication-defective retroviral vectors packaged with gibbon ape leukemia virus envelopes

With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat β-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli β-galactosidase marker gene in bovine target cells. By co-culture of bovine blastocysts and virus-producing cells, or by culture of embryos in the medium harvested from virus-producing cells, we transferred the E. coli β-galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV-based replication-defective retroviruses encapsidated with GaLV envelope proteins. The infection was confirmed by the expression of the E. coli β-galactosidase gene under a β-actin internal promoter. In addition, co-culture of ICM cells with virus-producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes. © 1993 Wiley-Liss, Inc.     

Inhibition of herpes simplex virus type 2-induced biochemical transformation by interferon.

The effect of interferon on the biochemical transformation of thymidine kinase-deficient cells by UV-inactivated herpes simplex virus type 2 has been studied. Transformation was much less sensitive to the action of interferon than virus multiplication. However, the continuous presence of a high dose of interferon (2,000 U) inhibited transformation almost completely. Although we could not differentiate between the effect of interferon on fixation and expression of the virus thymidine kinase gene, data suggest that the inhibitory effect of interferon on transformation might be partially due to the suppression of virus thymidine kinase expression.