Identification of an SV40 DNA sequence related to the reactivation of silent rRNA genes in human > mouse hybrid cells

The early region of the SV40 genome (hereafter referred to as the A gene), can reactivate silent ribosomal RNA (rRNA) genes in human-mouse hybrid cells which contain the rRNA genes of both species but express only those of the dominant species. The present experiments were carried out to determine whether the necessary information for reactivation of rRNA genes could be localized to a specific region of the A gene. Human > mouse hybrid cells (#55-54) were infected with five different nondefective adenovirus-SV40 hybrid viruses, each containing a different size fragment of the SV40 A gene. Viruses containing SV40 DNA sequences mapping from 0.59 to 0.11 (Ad2⁺ND₄), from 0.44 to 0.11 (Ad2⁺ND₂), and from 0.39 to 0.11 (Ad2⁺ND,₅) reactivated silent rRNA genes. Hybrid viruses mapping from 0.28 to 0.11 (Ad2⁺ND₁) and from 0.18 to 0.11 (Ad2⁺ND₁) and the nonhybrid adenovirus itself, failed to reactivate the silent rRNA genes. Silent gene reactivation also occurred when 55-54 cells were microinjected with fragments of cloned SV40 DNA mapping from 0.99 to 0.14 (94G2), and from 0.73 to 0.27 (HpaIIIPstI A fragment). A fragment mapping from 0.75 to 0.375 (HpaI B fragment) failed to reactivate the silent rRNA genes when microinjected into 55-54 cells, while the PvuII A fragment (0.70-0.32 map units) gave a very weak, positive result. These results indicate that the sequences from 0.67 to 0.39 and from 0.27 to 0.17 of the SV40 A gene are not necessary for the reactivation of silent rRNA genes. It is of interest that the same 600 base pairs involved in this particular function of the SV40 A gene are those which are most highly conserved and have the maximum homology with the DNA of polyoma virus.     

Herpesvirus-associated nuclear antigen(s) in cells biochemically transformed by fragments of herpesvirus DNA and in somatic cell hybrids

Human and mouse cells biochemically transformed by ultraviolet light (UV)-irradiated HSV-1 express HSV-1 thymidine kinase (TK) activity and also express type-specific herpesvirus-associated nuclear antigen(s) (HANA). To identify the HSV-1 DNA sequences coding for HANA and their location on the viral genome, studies were carried out on: (i) somatic cell hybrid clones obtained by fusing mouse [LM(TK^?)] cells with UV-irradiated HSV-1-transformed human [HeLa(BU25)/KOS 8-1] cells; and (ii) LM(TK^?) cells biochemically transformed with restriction endonuclease fragments of DNA which code for HSV-1 TK. Molecular hybridization experiments were also carried out and demonstrated that HSV-1 DNA sequences coding for TK were integrated in the biochemically transformed cells. The human-mouse somatic cell hybrid clones (LH81) which were HSV-1 TK+ were also HANA⁺, while clones counterselected in bromodeoxyuridine which had lost HSV-1 TK activity and DNA sequences likewise lost HANA. Previous studies had shown that the HSV-1 TK gene of LH81 hybrid clones was associated with a marker chromosome, designated M7, which consists of a human chromosome 17 translocated to the short arm of chromosome 3, or a modified M7 chromosome containing a translocation from a mouse chromosome. The present results indicate that at least one HANA gene was integrated in the same chromosome as the HSV-1 TK gene. LM(TK^?) cells biochemically transformed by HSV-1 DNA restriction nuclease fragments of diminishing size, which map in the HpaI-I region (26.2 to 31.7) of the HSV-1 genome, were HANA⁺ as well as TK⁺. The HANA⁺ cells included LM(TK^?)/TF pAGO PP and LM(TK^?)/TF pAGO PS clones. The latter are clones of LM(TK^?) cells biochemically transformed, respectively, by a PvuII fragment (1.35 × 10⁶ daltons) and a PvuII-SmaI fragment (0.9 × 10⁶ daltons; 30.2 to 31.1 map units) of HSV-1 DNA derived from Escherichia coli plasmid, pAGO. Since the PvuII-SmaI DNA fragment has only enough genetic information to code for a polypeptide of about 53,000 daltons and the HSV-1 TK polypeptide is about 40,000 daltons, the findings indicate that the genes for HSV-1 TK and one herpesvirus-associated nuclear antigen are either contiguous or overlapping, or HSV-1 TK and one HANA gene are identical.     

携带双自杀基因且可诱导敲除SV40T的逆转录病毒载体的构建与结构鉴定

目的构建携带双自杀基因且可诱导敲除SV40T的逆转录病毒载体,优化目前的肝细胞永生化。方法去除eGFP终止子taa的pSEB-HUS质粒为逆转录病毒基础质粒。先将SV40T及其启动子hEFH亚克隆至pSEB-HUS,再将LoxP位点插入至pSEB-SV40T质粒的抗性基因Blasticidin上游获得pSEB-LoxP-SV40T质粒。同时将CD自杀基因定向克隆至pSEB-HUS的eGFP下游;然后设计一段HSV-tk自杀基因引物,在上游引物的5′端加入方向一致的LoxP序列。PCR获得TK基因后,定向克隆至CD下游获得pSEB-CD-TK,最后将pSEB-CD-TK上的双自杀基因亚克隆至pSEB-LoxP-SV40T质粒上得到携带双自杀基因及SV40T的质粒。结果PCR及酶切鉴定均证实两个自杀基因及SV40T正确克隆至逆转录病毒质粒中,目的基因序列与GenBank报道一致,两个LoxP位点方向相同。结论成功构建携带双自杀基因且可诱导敲除SV40T的逆转录病毒载体。     

Biochemical transformation of LM(TK-) cells by hybrid plasmids containing the coding region of the herpes simplex virus type 1 thymidine kinase gene

Recombinant plasmid pAGO codes for herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) and consists of a 2-kbp HSV-1 DNA fragment inserted at the unique PvuII cleavage site of plasmid pBR322. A hybrid plasmid, designated pMH110, has been derived from plasmid pAGO by deleting the 1689-bp pBR322 nucleotide sequence of pAGO, which extends from the BamHI to the PvuII cleavage site, and the 250-bp HSV-1 nucleotide sequence of pAGO, which extends from the PvuII to the BglII cleavage site. Plasmid pMH110 biochemically transformed LM(TK^?)cells to the TK⁺ phenotype. The biochemically transformed cell lines had the following properties: (i) they were resistant to the growth-inhibiting effects of 1 mM thymidine; and (ii) they expressed an HSV-1-specific TK activity. This HSV-1 TK activity was purified after labeling biochemically transformed cell lines [LM(TK^?)/TF pMH110 E2 and LM(TK^?)/TF pMH110 Hc2] with [³⁵S]methionine. Immunoprecipitation experiments revealed that the TK polypeptides made in the biochemically transformed cells had molecular weights of about 39,000 to 40,000, which are about the same as the molecular weights of the TK polypeptides previously purified from HSV-1-infected LM(TK^?) cells and other biochemically transformed cell lines. The experiments support the hypothesis that the functional coding region of the HSV-1 TK gene is 3′ to the BglII cleavage site, and they also suggest that the HSV-1 TK messenger RNA may have been initiated in cells transformed by HincII- and EcoRI-cleaved pMH110 DNA at a site in cellular (or plasmid) DNA upstream from the HSV-1 DNA BglII cleavage site.     

Identification,mapping and cloning of the thymidine kinase gene of fish lymphocystis disease virus

The thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) was identified by biochemical transformation of 3T3 TK negative (TK⁻) to 3T3 TK positive (TK⁺) cells using specific viral DNA sequences. DNA fragments of the viral genome used in this study were obtained from a defined gene library of FLDV genome containing the complete viral DNA sequences. The selection of the converted cells was carried out under the condition of the HAT selection procedure. The results of these experiments revealed that the EcoRI FLDV DNA fragment C (11.2 kbp; 0.611 to 0.718 map units) is able to transform 3T3 TK⁻ to 3T3 TK⁺ cells. Additional experiments using the subclones of EcoRI DNA fragment C revealed that DNA sequences of 4.1 kbp size between the coordinates 0.669 to 0.718 of the FLDV genome possessed the ability for biochemical transformation, indicating that the TK gene locus is located in this particular region.     

Cloning of the marmoset herpesvirus thymidine kinase gene and analyses of the boundaries of the coding region

In order to delimit the approximate boundaries of the marmoset herpesvirus (MarHV) thymidine kinase (TK) gene, HindIII and BamHI digests of MarHV DNA were cloned in plasmid pBR322. Several recombinant plasmids which transformed E. coli K12 strain RR1 to ampicillin resistance were isolated. The MarHV DNA inserts in these plasmids accounted for about half of the MarHV genome. One of the plasmids, pMAR4, contained a 9.1-kbp fragment of MarHV DNA (HindIII-G), transformed LM(TK^?) cells to TK⁺, and hybridized to the BamHI-I fragment of MarHV DNA, which had previously been shown to have TK-transforming activity. pMAR4 DNA had little or no homology to the 2-kbp PuvII fragment of HSV-1 DNA, which contains the HSV-1 TK gene. Cleavage with PvuII, SacI, SmaI, and KpnI inactivated the TK-transforming activity of pMAR4, but cleavage with HindIII, PstI, EcoRI, XhoI, XbaI, and BamHI did not. Deletion mutants pMAR401 and pMAR420, which lacked the 2.6-kbp KpnI and the 2.75-kbp EcoRI fragments, respectively, of pMAR4, lost transforming activity, whereas pMAR410, which lacked a 2.9kbp XhoI fragment of pMAR4 did not. Recombinant plasmid pMAR430, which contained a 3-kbp PstI fragment of pMAR4, also transformed LM(TK^?) cells to TK⁺. The results strongly suggest that the coding region of the MarHV TK gene was within a 2.4-kbp pMAR4 sequence extending from the PstI (0.33 kbp) to the EcoRI (2.7 kbp) cleavage sites.     

Human cytomegalovirus major immediate early gene product can induce SV40 DNA replication in human embryonic lung cells

Previously we had reported that human cytomegalovirus (HCMV) induced replication of plasmids containing the SV40 origin of replication in human fibroblasts that were nonpermissive for SV40 and permissive for HCMV DNA replication. The amplification of SV40 origin-containing plasmids was dependent upon the HCMV-induced expression of T-antigen RNA. From previous studies it was determined that cotransfection of cosmids, containing HCMV genomic DNA, could stimulate SV40 DNA replication and T-antigen production. This indicated that the gene products of HCMV responsible for inducing SV40 DNA replication could be determined. In this study we report that the cotransfection of the major IE gene of HCMV alone was sufficient to facilitate the replication of the SV40 origin-containing plasmid. The HCMV IE1 gene product (i) increased expression of T-antigen RNA and protein and (ii) induced SV40 plasmid DNA replication in a T-antigen-dependent manner. The SV40 replication event was not due only to the expression of T-antigen. When the gene coding for T-antigen was placed under control of the Rous sarcoma viral promoter so that T-antigen expression in HEL cells was constitutive, it was not sufficient to replicate the SV40 plasmid in the absence of the HCMV IE1 protein. Therefore, the major IE gene of HCMV was capable of increasing the expression of T-antigen RNA and facilitating the replication of the SV40 origin. We are currently investigating the mechanism responsible for these observations.     

真核表达载体p4CCL20-ZsGreen1-DR的构建与鉴定

背景：抗炎药物高通量筛选体系的建立,可为相关药物的研究提供一个理想的技术平台。 目的：构建以核因子κB顺式作用元件4×CCL20基序为增强子,以SV40为启动子,以ZsGreen1-DR为报告基因的真核表达载体p4CCL20-ZsGreen1-DR。 方法：以PGL2-control质粒为模板,PCR扩增目的片段SV40,两侧引入KpnⅠ/BamHⅠ酶切位点,克隆至pZsGreen1-DR质粒的KpnⅠ/BamHⅠ酶切位点中,构建成pSV40-ZsGreen1-DR载体。将4×CCL20基序双链DNA克隆到pSV40-ZsGreen1- DR载体的BglⅡ和EcoRⅠ酶切位点之间,构建p4CCL20-ZsGreen1-DR重组质粒。 结果与结论：经过DNA测序分析证实p4CCL20-ZsGreen1-DR重组质粒构建成功。该重组质粒可作为抗炎药物高通量筛选体系的基础。     

Expression of the herpes simplex virus type-2 major DNA-binding protein (ICP8) gene in COS-1 cells

By the use of an SV40 origin of replication plasmid vector and the COS-1 cell system, expression of the gene encoding the herpes simplex virus type-2 (HSV-2) major DNA binding protein (ICP8) has been achieved. The HSV-2 4.5 kb BgIII 0 DNA fragment containing the ICP8 coding region was inserted into the plasmid vector pSVOd containing the SV40 origin of replication. Transfection of COS-1 cells by the resultant recombinant plasmid (pSVOd20), and subsequent multiplication of this plasmid, led to the production of the HSV-2 major DNA binding protein in sufficient quantities to allow its detection with either monoclonal or polyclonal antibodies as well as sera taken from HSV-2 patients.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen

Mouse LTK- cells (H-2k) were transfected with a series of recombinant plasmids consisting of the herpes simplex virus type 1 thymidine kinase (TK) gene linked to fragments of SV40 DNA coding for portions of SV40 T antigen in pBR322, and TK+ transformants (LTK+) were selected in HAT medium. The TK+ transformants were analyzed for SV40 transplantation rejection antigen (TrAg) at the cell surface by reacting them with cytotoxic lymphocytes (CTL) generated to SV40 TrAg in C3H/HeJ (H-2k) mice. The results indicated that the cells transformed by pVBETK-1 and synthesizing full size SV40 large T antigen were efficiently lysed by SV40 CTL. In addition, cells transformed by the plasmid pVBt1TK-1 and synthesizing a truncated 33 K T antigen were also found to be susceptible to lysis by the CTL. However, LTK+ cells that were transformed with the plasmid pVBt2TK-1 and which synthesized a truncated T antigen of 12.3 K did not provide a target for SV40 CTL nor did pVBETK-1-transformed cells that did not express any of the SV40 tumor antigens. Only the pVBETK-1-transformed cells that express 94 K T antigen were able to immunize mice against a challenge of syngeneic SV40-transformed cells. These results suggest that the TrAg expression at the cell membranes of transformed cells may be associated with the proximal half of SV40 T antigen.     

Transcriptional activation is not responsible for increased levels of autonomously expressed simian virus 40 T-antigen in herpes simplex virus-infected cells

Herpes simplex virus type 1 (HSV-1) superinfection of CV-1 cells weakly transactivated a plasmid-borne metallothionein 1 (MT-1) promoter, but activated the expression of a marker gene controlled by an authentic HSV-1 promoter to a high level. In contrast, CMT-3 cells, which are CV-1 cells stably transformed with the simian virus 40 (SV40) large T-antigen (T-Ag) gene controlled by the MT-1 promoter, contained high levels of T-Ag following HSV-1 superinfection, but only if cells were preincubated in the presence of heavy-metal ions. This T-Ag was functional in that it could mediate the increase in copy number of a marker plasmid containing the SV40 origin of DNA replication. Pulse and continuous labeling of preinduced CMT-3 cells showed that T-Ag expression was not induced by HSV-1; but rather, HSV-1 superinfection resulted in the stabilization of pre-existing protein.     

Construction of a pigeonpox virus recombinant: expression of the Newcastle disease virus (NDV) fusion glycoprotein and protection of chickens against NDV challenge

Summary A pigeonpox transfer plasmid was constructed by cloning a 2.5 kb DNA fragment containing the viral thymidine kinase (TK) gene in the psp65 plasmid. The vaccinia virus P11K promoter followed by the NDV fusion (F) gene was inserted in the TK gene. The F gene was transferred to the viral genome by homologous recombination in pigeonpox virus infected CEF cells, transfected with the recombinant plasmid. Recombinant viruses were selected with BUdR and screened for their ability to induce fusion between adjacent cells. Because of the unexpected growth advantage of the TK+ WT over the TK– recombinants, viral purification was needed to obtain stable recombinants expressing a glycosylated and cleaved F protein. Vaccination of chickens by the follicular method induced high anti-F antibody titers and good protection against challenge with the virulent Italian NDV strain. Half of the oculonasal vaccinated chickens showed anti F antibodies and also half of them were protected. Although protection seems to be correlated with antibody titers, no neutralizing antibodies were found.     

High efficiency transfection of monkey kidney COS-1 cells

Summary Methods are described for achieving high efficiency transient transfection of COS-1 cells. A transfection solution of vector DNA and DEAE-dextran in phosphate buffered saline is added to the cells, followed by treatment with culture medium containing chloroquine, resulting in a maximum efficiency of about 50%. The high efficiency obtained is primarily dependent on the use of Ca²⁺- or Mg²⁺-free buffer solutions for transfection, because the addition of these ions greatly reduces efficiency. Shocking the cells with dimethylsulfoxide does not increase transfection efficiency. COS-1 cells are monkey kidney cells that have a genomic insertion of the SV40 T antigen gene, allowing plasmid expression vectors bearing an SV40 replication origin to be amplified in these cells. Therefore this transfection method is useful for optimizing transient expression of genes in a mammalian cell system.     

Mapping thymidine kinase-deficient mutants of vaccinia virus by marker rescue with hybrid plasmid DNAs containing portions of the HindIII-J fragment of virus DNA

Five hybrid plasmids were constructed, each containing a portion of the vaccinia virus DNA HindIII-J fragment. These plasmid DNAs were used in marker rescue experiments to map the mutations in the thymidine kinase (TK) gene of three TK- vaccinia virus mutants. The TK gene of each of the three mutants was rescued by DNA from plasmid pPJ701, which contained about one-half of the HindIII-J fragment. Two mutants, 1004B and 1017-1, but not the third, 1016-1, were rescued by DNA of two plasmids, pPJ702 and pPJ703, which contained 16 and 18%, respectively, of one end of the J fragment. Mutant 1016-1 could be rescued by plasmid pPJ705 containing a 1.69-kb fragment of the HindIII-J fragment. The J fragment DNA in plasmid pPJ705 is located adjacent to that and separated by an EcoRI site from pPJ703 in the vaccinia virus genome. These results indicate that the mutation site in the TK gene of 1016-1 differs from that in 1004B or 1017-1 and suggests that the structural gene for the vaccinia virus TK lies near one end of the HindIII-J fragment and spans the EcoRI site.     

Extrachromosomal unequal homologous recombination and gene conversion in simian kidney cells: effects of UV damage

Shuttle plasmid vectors containing the SV40 origin of replication and tandem neo genes with distally placed non-overlapping deletions were used to study the effects of DNA damage on extrachromosomal homologous recombination in simian kidney cells. DNA was introduced into COS7 cells by a lipofectin-mediated transfection procedure and recombination was assessed by analyzing the structure of plasmids. Recombinational events observed included unequal homologous recombination (triplication), gene conversion, double reciprocal recombination, deletion (pop-outs), gene amplification (4–6 copies), and multimerization. Triplication, an event that previously had not been reported in association with extrachromosomal recombination, predominated in experiments with undamaged vectors. The recombination frequency (Neo^R/Amp^R) of vectors randomly damaged by UV irradiation was essentially unchanged; however, the relative number of triplication events decreased significantly. Selective damage in one of the two neo genes increased the relative frequency of gene conversion. The experimental system developed for use in this study detects all major homologous recombination events observed in chromosomal direct repeat sequences in mammalian cells and yeast and should prove valuable for future studies of homologous recombination in mammalian cells.     

Deletion and hypermethylation of thymidine kinase gene in V79 Chinese hamster cells resistant to bromodeoxyuridine

Previous studies on V79 Chinese hamster cells have shown that bromodeoxyuridine (BrdU)-resistant variants deficient in thymidine kinase (TK) activity arise by a multistep process which is initiated by a random event and progresses gradually during serial culture in the presence of the drug. In order to determine the molecular basis for the loss of TK activity in these cells, the TK gene was isolated from a phage library of genomic V79 DNA, using a fragment of the human TK gene as a probe. One phage isolated contained the entire TK gene in a 15-kb insert, as demonstrated by the ability of the phage DNA to transform Ltk^}-mouse cells to the TK⁺ phenotype. Five fragments spanning the entire gene were then subcloned into the plasmid pUC12 for DNA methylation studies. With these probes it was shown by hybridization analysis that the copy number of the TK gene in V79 cells is about four times the copy number in CHO cells and Chinese hamster liver cells. Southern hybridization analysis of the DNA from first-stage variants partially resistant to BrdU indicated that partial resistance was accompanied by deletion of a number of copies of theha TK gene in V79 cells. However, the subsequent gradual transition to full BrdU resistance and full loss of TK activity was correlated with a gradual hypermethylation of sites in the 5 region of the TK gene, with no further change in gene copy number.     

WT1基因增强子构建位点对WT1基因启动子转录活性的影响

目的：以WT1基因启动子和增强子的功能片段为研究对象，探讨增强子构建在质粒的不同位点对基因启动子转录活性的影响。方法: 利用基因重组技术将WT1基因增强子的功能片段分别插入在已含有WT1基因启动子的质粒，pEWP的不同位点（MCS中的BamH I、EGFP 终止密码后的Not I和SV40polyA位点后的AflⅡ）中，将重组质粒转染慢粒白血病红白急变细胞株K562细胞、乳腺癌细胞株MCF-7细胞和人类胚胎肾转化细胞株293细胞，通过检测转基因细胞中EGFP的平均荧光强度来评估增强子对启动子的转录促进作用。结果: 通过基因重组技术分别构建了含有WT1基因启动子的载体，即pEWP，和含有WT1基因启动子和增强子的载体，即pEWPE、pEWPD和pEWPA。流式细胞仪检测EGFP的平均荧光强度，发现pEWPA在293细胞和K562细胞中能够增强WT1启动子的转录活性，而pEWPE和pEWPD则无增强作用。3者均不能在MCF7细胞中增强WT1启动子的转录活性。结论: SV40polyA位点后插入增强子能够有效地增强启动子的转录活性，且具有细胞特异性。     

Nanoparticle phagocytosis and cellular stress: involvement in cellular imaging and in gene therapy against glioma

In gene therapy against glioma, targeting tumoral tissue is not an easy task. We used the tumor infiltrating property of microglia in this study. These cells are well adapted to this therapy since they can phagocyte nanoparticles and allow their visualization by MRI. Indeed, while many studies have used transfected microglia containing a suicide gene and other internalized nanoparticles to visualize microglia, none have combined both approaches during gene therapy. Microglia cells were transfected with the TK‐GFP gene under the control of the HSP₇₀ promoter. First, the possible cellular stress induced by nanoparticle internalization was checked to avoid a non‐specific activation of the suicide gene. Then, MR images were obtained on tubes containing microglia loaded with superparamagnetic nanoparticles (VUSPIO) to characterize their MR properties, as well as their potential to track cells in vivo. VUSPIO were efficiently internalized by microglia, were found non‐toxic and their internalization did not induce any cellular stress. VUSPIO relaxivity r₂ was 224 mM^?1.s^?1. Such results could generate a very high contrast between loaded and unloaded cells on T₂‐weighted images. The intracellular presence of VUSPIO does not prevent suicide gene activity, since TK is expressed in vitro and functional in vivo. It allows MRI detection of gene modified macrophages during cell therapy strategies. Copyright © 2009 John Wiley & Sons, Ltd.