首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 0 毫秒
1.
We have determined the complete nucleotide sequence of the thymidine kinase gene of herpes simplex virus (HSV) type 2 strain 333. The sequence of the thymidine kinase gene exhibits an open translational reading frame of 1,128 nucleotides encoding a protein of 376 amino acids. The DNA sequence was compared with that of the HSV type 1 thymidine kinase gene from strain MP (S. L. McKnight, Nucleic Acids Res. 8:5949-5964, 1980) and from strain CL 101 (M. J. Wagner, J. A. Sharp, and W. C. Summers, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981) to assess the extent of intra- and intertypic variation for one viral gene. The nucleotides encoding the structural gene varied 1.7% between the two HSV type 1 strains and 19% between HSV type 1 and HSV type 2, which translated to differences in the amino acid sequence of the two proteins of 1.9 and 27%, respectively. The DNA encoding the 5' regulatory sequences appeared to be more conserved than the DNA coding for the structural gene, and the DNA at the 3' end of the gene was the least homologous.  相似文献   

2.
The herpes simplex 1 virus thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and for imaging intracellular molecular events and cell trafficking in living subjects. Two in vitro methods are available to assay gene expression of HSV1-tk or HSV1-sr39tk in cells or tissues. One method determines the level of HSV1-TK or HSV1-sr39TK enzyme activity in cell or tissue lysates by measuring the amount of the radiolabeled substrates that have been phosphorylated by these enzymes in a fixed amount of cell lysate protein after a fixed incubation time. The other method, called the 'cell-uptake assay', takes into account the natural uptake and efflux characteristics of the radiolabeled substrate by specific cells, in addition to the level of HSV1-TK or HSV1-sr39TK activity. Both of these assays can be used to validate molecular models in cultured cells, prior to studying them in living research subjects. Each of these assays can be completed in one day.  相似文献   

3.
When employed as a transgene reporter, the herpes simplex type 1 virus (HSV1) thymidine kinase gene (tk) is ectopically expressed in mouse testis. The principal testicular mRNA lacks the 5'-end of the tk reading frame. As a result the principal translation products, P2 and P3, are N-terminally truncated. These co-migrate in SDS-PAGE with polypeptides synthesised during HSV1 infection that were previously thought to be initiated at methionine codons ATG46 and ATG60. Prompted by these observations we generated modified tk genes each carrying only one of the first three ATG codons. Transfected cells expressed both full-length enzyme (P1) and P2 when only ATG1 was unmodified, P2 and P3 when only ATG46 was unmodified or P2 and a fourth polypeptide (P4) when only ATG60 was unmodified. Our observations indicate that P3 is initiated at ATG46 rather than ATG60, while P2 is initiated at a non-ATG codon rather than ATG46 and P4 is initiated at ATG60. When either of two putative non-ATG initiation codons was modified P2 was no longer produced. Cells mainly expressing either P1 or P3 exhibited the same sensitivity to Ganciclovir as cells transfected with the unaltered tk gene. P1 and P3 both have TK activity while P4 probably has none.  相似文献   

4.
Herpes simplex virus thymidine kinase is important for reactivation of virus from its latent state and is a target for the antiviral drug acyclovir. Most acyclovir-resistant isolates have mutations in the thymidine kinase gene; however, how these mutations confer clinically relevant resistance is unclear. Reactivation from explanted mouse ganglia was previously observed with a patient-derived drug-resistant isolate carrying a single guanine insertion within a run of guanines in the thymidine kinase gene. Despite this mutation, low levels of active enzyme were synthesized following an unusual ribosomal frameshift. Here we report that a virus, generated from a pretherapy isolate from the same patient, engineered to lack thymidine kinase activity, was competent for reactivation. This suggested that the clinical isolate contains alleles of other genes that permit reactivation in the absence of thymidine kinase. Therefore, to establish whether thymidine kinase synthesized via a ribosomal frameshift was sufficient for reactivation under conditions where reactivation requires this enzyme, we introduced the mutation into the well-characterized strain KOS. This mutant virus reactivated from latency, albeit less efficiently than KOS. Plaque autoradiography revealed three phenotypes of reactivating viruses: uniformly low thymidine kinase activity, mixed high and low activity, and uniformly high activity. We generated a recombinant thymidine kinase-null virus from a reactivating virus expressing uniformly low activity. This virus did not reactivate, confirming that mutations in other genes that would influence reactivation had not arisen. Therefore, in strains that require thymidine kinase for reactivation from latency, low levels of enzyme synthesized via a ribosomal frameshift can suffice.  相似文献   

5.
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.  相似文献   

6.
7.
Biochemical transformation of Ltk- cells with the herpes simplex virus thymidine kinase (tk) gene resulted in numerous TK+ colonies that survived selection in hypoxanthine-aminopterin-thymidine medium. Many of these TK+ cell lines switched phenotypes and reverted to the TK- state. In this report, we describe the biological and biochemical characteristics of three TK- revertant lines. One (K1B5) transiently expressed TK in the presence of bromodeoxyuridine, which selects for the TK- phenotype. Another TK- sibling (K1B6n) expressed TK only after removal from bromodeoxyuridine-containing medium. The last variant (K1B6me) lost the ability to switch to the TK+ phenotype, although it maintained the herpes simplex virus sequences coding for TK. Loss of the ability of K1B6me cells to express TK was correlated with extensive methylation of the sequence recognized by the restriction endonuclease HpaII (pCpCpGpG). After these cells were treated with 5-azacytidine, they regained the ability to clone in hypoxanthine-aminopterin-thymidine medium and reexpressed virus tk mRNA and enzyme. In addition, the HpaII sites that were previously shown to be refractile to enzyme digestion were converted to a sensitive state, demonstrating that they were no longer methylated.  相似文献   

8.
Lu Q  Hwang YT  Hwang CB 《Journal of virology》2002,76(11):5822-5828
To examine whether the exonuclease activity intrinsic to the polymerase (Pol) of herpes simplex virus type 1 can influence the mutational spectra, we applied the denaturing gradient gel electrophoresis (DGGE) system combined with sequencing to characterize thymidine kinase mutants isolated from both the wild-type virus and a mutant deficient in exonuclease activity, Y7. Wild-type viruses produced predominantly frameshift mutations (67%), whereas Y7 replicated a significantly lower proportion of frameshifts (21%; P < 0.005). Furthermore, the majority of substitutions were transitional changes in both groups, although they distributed differently. The implications of these findings are discussed.  相似文献   

9.
10.
INTRODUCTI0NHepatocellularcarcinoma(HCC)is0ne0fthem0stc0mm0nhumanmalignancies,causinganestimatedl,250,OOOdeatht0llperyearworldwide[1].Thep0orprognosisencounteredintreatment0fsuchcarcinomaismainlycausedbylatediagn0sisandinsufficiency0feffectivestrategies,especiallyforadvanced-stagedpatients.However,recentknowledge0fpathogenesisofHCCatm0lecularlevelprovidesanalternativeappr0achwhenc0nsideringgenetherapyastreatmelltf0rHCC.Am0ngthevari0usgenetherapystrategiesincancer)itwaJsrep0rtedthatth…  相似文献   

11.
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.  相似文献   

12.
13.
Thymidine kinase from herpes simplex virus type 1 (ATP:thymidine 5'-phosphotransferase; EC 2.7.1.21) has been purified from an overexpression system and crystallized against ammonium sulfate by using the hanging-drop technique. The tetragonal crystals are of space group P4122 or P4322, and have unit cell dimensions a = b = 84 A, c = 180 A.  相似文献   

14.
Recombinant viruses were constructed to have an Escherichia coli replicon containing a mutagenesis marker, the supF gene, integrated within the thymidine kinase locus (tk) of herpes simplex virus type 1. These viruses expressed either wild-type or mutant DNA polymerase (Pol) and were tested in a mutagenesis assay for the fidelity of their replication of the supF gene. A mutation frequency of approximately 10(-4) was observed for wild-type strain KOS-derived recombinants in their replication of the supF gene. However, recombinants derived from the PAA(r)5 Pol mutant, which has been demonstrated to have an antimutator phenotype in replicating the tk gene, had three- to fourfold increases in supF mutation frequency (P < 0.01), a result similar to that exhibited when the supF gene was induced to replicate as episomal DNA (Y. T. Hwang, B.-Y. Liu, C.-Y. Hong, E. J. Shillitoe, and C. B. C. Hwang, J. Virol. 73:5326-5332, 1999). Thus, the PAA(r)5 Pol mutant had an antimutator function in replicating the tk gene and was less accurate in replicating the supF gene than was the wild-type strain. The spectra of mutations and distributions of substituted bases within the supF genes that replicated as genomic DNA were different from those in the genes that replicated as episomal DNA. Therefore, the differences in sequence contents between the two target genes influenced the accuracy of the Pol during viral replication. Furthermore, the replication mode of the target gene also affected the mutational spectrum.  相似文献   

15.
When thymidine kinase-deficient mouse cells "transformed" by in activated herpes simplex virus and expressing the viral thymidine kinase (TK) are grown in nonselective medium, there is an exponential decay in the proportion of cells that continue to express the viral enzyme. However, the viral TK can be reactivated at a frequency of approximately 1 cell in 10(6) in every population that has lost TK activity. When cells in which the viral TK has been reactivated are grown in nonselective medium, a decay in the expression of the viral enzyme occurs again at the same rate as in the initial transformed population. Studies on the reactivation of viral TK indicate that reappearance of the enzyme is not induced by the selective medium (HAT) used to detect cells in which the enzyme has reappeared. Furthermore, treatments known to induce latent viruses in other systems--eg, exposure of the cells to mutagens or cell fusion--do not affect the frequency with which viral TK is reactivated.  相似文献   

16.
A simple procedure for preparation of an affinity resin with 3''-amino thymidine linked to the carboxyl residues on 6-amino-hexanoic agarose is described. We have used this column for a rapid and simple purification of the thymidine kinase encoded by the herpes simplex virus type 1 genome. This resin has two major advantages over the most widely use used resin made with thymidine-p-nitrophenyl phosphate: first it is easily obtainable, and second, it is not subject to destruction by phosphodiesterases. The two resins are very similar in behavior and the resin made with amino thymidine has allowed us to prepare large quantities of highly purified HSV TK for crystallization studies.  相似文献   

17.
This paper presents the nucleotide sequence of the Herpes Simplex Virus thymidine kinase (tk) gene. The position on the DNA sequence corresponding to the 5' and 3' termini of tk messenger RNA have been mapped. The mRNA termini are separated by slightly more than 1,300 nucleotides. The same 2,300 nucleotide segment of tk coding strand DNA is fully protected from S1 nuclease digestion when hybridized to tk mRNA. The location and size of the mRNA-coding segment corresponds to a region of the viral DNA that is essential for tk gene expression in microinjected frog oocytes. The nucleotide sequence of the HSV tk gene exhibits an open translational reading frame of 376 codons that extends from the methionine codon most proximal to the 5' terminus of tk mRNA to a UGA stop codon approximately 70 nucleotides from the poly-A addition site. The results of these experiments indicate that the tk gene is not interrupted by intervening DNA sequences, and that certain oligonucleotide sequences adjacent to the termini of the tk gene are homologous to similarly positioned sequences common to structural genes of eukaryotic cells.  相似文献   

18.
M Czak  L Mrton 《Plant physiology》1994,104(3):1067-1071
The human herpes simplex virus thymidine kinase type 1 gene (HSVtk) acts as a conditional lethal marker in mammalian cells. The HSVtk-encoded enzyme is able to phosphorylate certain nucleoside analogs (e.g. ganciclovir, an antiherpetic drug), thus converting them to toxic DNA replication inhibitors. The utility of HSVtk as a conditional negative-selection marker was explored in Arabidopsis thaliana (L.) Heynh. HSVtk was introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic plants were morphologically indistinguishable from wild type and exhibited normal fertility. Ganciclovir at 10(-5) to 10(-4) M drastically reduced shoot regeneration on transgenic, HSVtk+ root explants or callus formation on HSVtk+ leaf explants but did not affect the wild-type cultures. There was a 35-fold reduction in shoot regeneration 8 d after transfer to shoot-induction medium. Negative selection against HSVtk activity along with kanamycin selection was also efficient in Agrobacterium-mediated gene transfer experiments. Shoot regeneration was 25 times lower on double-selective (ganciclovir plus kanamycin) plates than in the kanamycin control. This regeneration rate in double-selective plates is in the range of the frequency of shoots normally escaping kanamycin selection in Arabidopsis cultures.  相似文献   

19.
We have reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) fused with green fluorescent protein (GFP) is localized in the nucleus of HSV-1 TK-GFP gene-transfected cells (Degrève et al. (1998) J. Virol. 72, 9535-9543). Deletion of the N-terminal 34 amino acids or selective mutation of the nonapeptide (25)RRTALRPRR(33), located in the N-terminal region of HSV-1 TK, resulted in the loss of the specific nuclear localization of HSV-1 TK. Utilizing information on the crystallographic structure of HSV-1 TK, we have now identified three additional putative nuclear localization signals and evaluated their potential role in the nuclear trafficking of HSV-1 TK by site-directed mutagenesis. We found that the sites containing the amino acids R236-R237 and K317-R318 are absolutely required for specific nuclear targeting of HSV-1 TK. The K317-R318 region, located at the interface between the two monomers in the dimeric HSV-1 TK structure, could act as a nuclear localization signal for monomeric HSV-1 TK. Alternatively, crystallographic data indicate that R318 might be essential for the formation of the TK dimer, and therefore it is required if HSV-1 TK is transported as a dimer.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号