Coli Gpt Gene Research Articles

Generation of Recombinant Capripoxvirus Vectors for Vaccines and Gene Knockout Function Studies.

The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is used to generate capripoxvirus knockout viruses (KO), and is based on the targeting a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses.

Serp2, an inhibitor of the interleukin-1beta-converting enzyme, is critical in the pathobiology of myxoma virus.

Recently, myxoma virus was shown to encode an additional member of the serpin superfamily. The viral gene, called serp2, was cloned, and the Serp2 protein was shown to specifically bind to interleukin-1beta (IL-1beta)-converting enzyme (ICE), thus inhibiting the cleavage of pro-IL-1beta by the protease (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860-5866, 1996). Here, we address the role of Serp2 in the development of myxomatosis, a lethal infectious disease of the European rabbit. A Serp2 mutant myxoma virus was constructed by disruption of the single-copy serp2 gene and insertion of the Escherichia coli gpt gene serving as the selectable marker. A revertant virus was obtained by replacing the E. coli gpt gene by the intact serp2 open reading frame. The Serp2(-) mutant virus replicated with wild-type kinetics both in rabbit fibroblasts and a rabbit CD4(+) T-cell line (RL5). Moderate reduction of cell surface levels of major histocompatibility complex I was observed after infection with wild-type or Serp2(-) mutant myxoma virus, and both produced white pocks on the chorioallantoic membrane of the chick embryo. After the infection of European rabbits, the Serp2(-) mutant virus proved to be highly attenuated compared to wild-type myxoma virus, as demonstrated by the clinical course of myxomatosis and the survival rates of infected animals. Pathohistological examinations revealed that infection with wild-type myxoma virus resulted in a blockade of the inflammatory response at the vascular level. In contrast, rapid inflammatory reactions occurred upon infection with the Serp2(-) mutant virus. Furthermore, lymphocytes in lymph nodes derived from animals inoculated with Serp2 mutant virus were shown to rapidly undergo apoptosis. We postulate that the virulence of myxoma virus in the European rabbit can be partially attributed to an impairment of host inflammatory processes and to the prevention of apoptosis in lymphocytes. The weakening of host defense is directly linked to serp2 gene function and is likely to involve the inhibition of IL-1beta-converting-enzyme-dependent pathways.

DNA sequence variation as a clue to the phylogenesis of orthopoxviruses.

We have sequenced DNA equivalent to the E5R ORF of Copenhagen vaccinia virus from an additional strain of vaccinia and from cowpox (three strains), camelpox (two strains), taterapox and ectromelia viruses. None of these showed the disruptions previously reported in the equivalent region of monkeypox virus. We also constructed a viable recombinant of vaccinia virus strain Dairen in which the E5R sequence was disrupted by a 436 bp deletion and substitution of the E. coli gpt gene. Quantitative analysis of the sequences, including available sequences from monkeypox, variola and vaccinia viruses revealed four main groupings, namely cowpox, ectromelia, monkeypox and a cluster which includes variola, camelpox, taterapox and vaccinia viruses. It was noted that, at over 75 % of the positions which differentiated species. all species but one had a common nucleotide. Although the analysis covers one single gene only, the results accord with what is known of the biology of the viruses.

Localization of the xanthine guanine phosphoribosyl transferase gene (gpt) of E. coli in AS52 metaphase cells by fluorescence in situ hybridization.

The purpose of this study was to localize the xanthine guanine phosphoribosyl transferase gene (gpt) to a specific chromosome to investigate its proposed autosomal location in the AS52 cell line. AS52 cells are hgprt-deficient Chinese hamster ovary (CHO) cells which carry a single functional copy of the E. coli gpt gene. Fluorescence in situ hybridization (FISH) and digoxigenin-labeled probes, as small as 673 bp, were used in an attempt to localize the 456 bp gpt gene to a specific chromosome. Chi-square analysis of 13 metaphases showed significant labeling on autosomal chromosomes 6 or 7, which are indistinguishable without further banding analysis. Furthermore, a majority of the signals were on the q arm, proximal to the centromere. The data collected supports incorporation of the gpt gene into an acrocentric autosome of the AS52 cell line.

Mutagenicity of soluble and insoluble nickel compounds at the gpt locus in G12 chinese hamster cells

Nickel is an established human and animal carcinogen, but efforts to demonstrate its mutagenicity in a number of cell types have not been successful. In this report we describe the mutational response to nickel compounds in the G12 cell line, an hprt deficient V79 cell line containing a single copy of the E. coli gpt gene. This cell line has a low spontaneous background, making it suitable for assessment of mutagenic responses to environmental contaminants. When G12 cells were treated with insoluble particles of crystalline nickel sulfide < 5 microns in diameter, a strong, dose-dependent mutagenic response was observed up to 80 times the spontaneous background. Of 48 mutant gpt(-) clones isolated that were induced by insoluble nickel, all were capable of DNA amplification of the gpt sequences by polymerase chain reaction (PCR). The ability to produce full-length PCR products is an indication that large deletions of gene sequences have not occurred. When G12 cells were treated with soluble nickel sulfate, the mutational response was not significantly increased over the spontaneous background. This difference in mutagenic response reflects a large difference in the mutagenic potential of soluble and insoluble nickel compounds, which reflects the carcinogenic potential of these forms of nickel.

A myxoma virus intergenic transient dominant selection vector.

The purpose of this study was to construct an intergenic transfer vector which can be used for the generation of recombinant myxoma viruses (MVs) expressing a foreign gene insert. Recombinant MVs expressing the Escherichia coli lacZ gene were constructed in vitro by transfection of MV-infected rabbit cells with a transfer expression vector, and isolated under growth conditions selecting for transient expression of the E. coli gpt gene. The effect of inserting foreign DNA sequences between the viral thymidine kinase gene and open reading frame MF8a upon the transcription of these genes was investigated.

Mutagenesis and comutagenesis by lead compounds

We have previously reported that lead(II) is weakly mutagenic to Chinese hamster V79 cells. A transgenic cell line G12 containing a single copy of the E. coli gpt gene was developed in this laboratory from Chinese hamster V79 cells. The gpt locus in the G12 cells is more mutable by radiation and oxidative agents compared with the endogenous hprt locus of wild-type V79 cells. We have investigated the mutagenicity of two lead compounds at the gpt locus in G12 cells. Only at a toxic dose is lead acetate significantly mutagenic to G12 cells. Lead nitrate is not significantly mutagenic at any dose. Although both compounds are water-soluble, lead acetate, but not lead nitrate, forms a fine white insoluble precipitate upon addition to growth medium. A nick translation assay on cells treated with lead compounds and then permeabilized indicated that lead nitrate and, to a greater extent, lead acetate causes the appearance of nicks in chromosomal DNA. Lead ions in the presence of hydrogen peroxide, but not alone, introduced nicks into supercoiled plasmid DNA in vitro, suggesting that lead ions can partake in a Fenton reaction and thereby damage DNA. At lower nonmutagenic concentrations, lead acetate enhances the mutagenicity of MNNG and ultraviolet light. DNA damage by ultraviolet light is not enhanced by lead ions in vitro. Our data support the concept that non-toxic concentrations of lead(II) can inhibit DNA repair. Thus, at biologically relevant doses, lead(II) could act as a comutagen and possibly a cocarcinogen, but is not likely to act as an initiating genotoxic carcinogen.

Sequence analysis, expression, and deletion of a vaccinia virus gene encoding a homolog of profilin, a eukaryotic actin-binding protein

A 4,500-bp BamHI fragment, located within the HindIII A segment of the vaccinia virus genome, was found to contain eight potential coding regions for polypeptides of 78 to 346 amino acids. The open reading frames with 133, 346, and 125 codons were homologous to profilin (an actin-binding protein), 3-beta-hydroxysteroid dehydrogenase, and Cu-Zn superoxide dismutase, respectively. Sequence alignments indicated that the vaccinia virus and mammalian profilins were more closely related to each other than to known profilins of other eukaryotes. The expression and possible role of the profilin homolog in the virus replicative cycle were therefore investigated. Antibody raised to Escherichia coli expressed vaccinia virus profilin was used to demonstrate the synthesis of the 15-kDa polypeptide at late times after vaccinia virus infection of mammalian cells. The protein accumulated in the cytoplasm, but only trace amounts remained associated with highly purified virions. The isolation of vaccinia virus mutants (in strains WR and IHD-J), with nearly the entire profilin gene replaced by the E. coli gpt gene, indicated that the protein is not essential for infectivity. The characteristic vaccinia virus-induced changes in actin fibers, seen by fluorescence microscopy, occurred in cells infected with the mutant. Moreover, the virus-encoded profilin homolog was not required for actin-associated events, including intracellular virus movement to the periphery of the cell, formation of specialized microvilli, or release of mature virions, as shown by electron microscopy and yields of infectious intra- and extracellular virus.

Replication-dependent mutagenesis by 5-bromodeoxyuridine: Identification of base change and sequence effects on mutability

The molecular mechanism of reversion induced by 5-bromodeoxyuridine (BrdU) replication-dependent mutagenesis in mammalian cells was studied. Murine cells with single mutant copies of the E. coli gpt gene integrated chromosomally as part of a shuttle vector were mutagenized with BrdU, and GPT+ revertants were selected. Thirteen mutant cell lines (each of which had a gpt gene that varied from the wild-type gene by a different GC----AT base transition in the coding region) were mutagenized, and only four were found to be effectively reverted. All revertant gpt genes that were analyzed had reverted via AT----GC base transition at the original site of mutation, thus demonstrating that replication-dependent mutagenesis by BrdU causes AT----GC transitions. The nine cell lines that were nonrevertible by BrdU replication-dependent mutagenesis could be mutated by this protocol to ouabain resistance as effectively as the four revertible lines, indicating that the nonrevertible lines were susceptible to such mutagenesis. Thus, differences among the cell lines in frequencies of HATr revertants generated by BrdU replication-dependent mutagenesis could not be attributed to differences in general susceptibility of the lines to the mutagenic protocol. The revertible and nonrevertible lines could not be separated according to the position of the original GC----AT transition in the gpt coding region. However, there was evidence that the DNA base sequence flanking the site of mutation affected the susceptibility of that site to BrdU replication-dependent mutagenesis. For example, six of the cell lines tested had gpt genes in which the mutant T residue was immediately adjacent on its 3' side to an A residue, and all six were found to be nonrevertible by BrdU replication-dependent mutagenesis. Furthermore, a target AT base pair flanked by GC base pairs in opposite orientation and either immediately adjacent to or one base removed from the target site on both the 5' and 3' sides appeared to have an increased susceptibility to BrdU replication-dependent mutagenesis.

Effects of cucl2on UV‐mutagenesis and on DNA damage in a restriction fragment ofE. Coligpt∗

CuCl2 does not cause Trp+ reversion in E. coli WP2. However, when the bacteria are exposed to CuCl2 and UV‐irradiated, a greater than 3‐fold enhancement of mutagenesis (compared to UV alone) is seen at 30 μMCuCl2, and significant enhancement is seen even at 3 μM. The mechanism for this comutagenic effect was studied using a restriction fragment of the E. coli gpt gene. Whereas UV or CuCl2 alone caused few strand breaks, UV + CuCl2 induced breaks at every site. This reaction was blocked by KI, a free radical scavenger. While UV alone induced alkali‐labile sites, UV+ CuCl2 induced many more such sites and altered the sequence specificity. We suggest that at least some of the comutagenic effect might be due to hydroxyl radical formed via a Fenton reaction.

Retroviral shuttle vectors as a tool for the study of mutational specificity (base substitution/deletion/mutational hotspot)

This paper summarizes the use of the retroviral shuttle vector pZipGptNeo for studies of mutational specificity in mammalian cells. This vector was constructed by the introduction of a DNA fragment containing the E. coli gpt gene into the retroviral shuttle vector pZipNeoSV(X)1. The pZipGptNeo vector was then introduced into mouse L cells to construct the A9I2 cell line. Studies utilizing the A9I2 cell line to determine the specificity of spontaneous and chemically-induced mutations are summarized. THe construction of a new retroviral shuttle vector and its introduction into the CHO-K1 cell line is described. Preliminary experiments suggest that spontaneous gpt gene mutations arising in CHO cells are similar to those seen in the mouse L cell.

The rat albumin gene promoter is appropriately regulated in transient but not in stable transfections.

The tissue-specific expression of the liver-specific rat albumin gene promoter was examined after transfer to various hepatic and non-hepatic cell lines. A 402 base pair sequence from the albumin gene 5' flank enabled a fused reporter chloramphenicol acetyltransferase gene to be expressed in rat hepatoma cell lines but not in fibroblast lines or dedifferentiated hepatoma cells. However, when this same construct was analyzed in permanently transfected cell populations, it was expressed equally well in differentiated and dedifferentiated hepatoma cells and in two of three fibroblast lines tested. The inappropriate expression of the albumin promoter was also seen using the HSV tk gene and the E. coli gpt gene as reporters, and when assayed by colony formation in HAT medium (tk gene) or by S1 protection of transcripts in cotransfected populations (tk and gpt genes). These results show that gene regulatory elements can behave differently in transient vs. stable transfections, and suggest that chromosomal integration can provide long range positive influences on gene expression.

The expression of the Escherichia coli uvrA gene in human cells

Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously. The E. coli gpt gene in pSV2 gpt is used as a selection marker for uvr gene transfection into XP cells. The uvr genes were cloned into composite pBR322, SV40 and gpt vectors in which each E. coli gene is flanked by individual SV40 regulatory elements. SV40-transformed XP-A cells were transfected with pSV2 uvrASV2 gpt, gpt + colonies were selected, and cell lines esttablished. Several times were examined in detail. Cell lines 714 and 1511 contain uvrA together with flanking SV40 regulatory elements integrated intact in genomic DNA and express UvrA protein as well as a 95 000-dalton UvrA-related protein. The expression of uvrA was found to be 50–100-fold lower than the expression of gpt. Attempts were made to assay the mammalian UvrA protein for functionality, but endogenous activities interfered with assays for each of the UvrA protein's three activities. The peptide maps derived from partial proteolysis of the “mammalian” UvrA protein are identical to the E. coli UvrA protein. The sub-cellular location of UvrA protein in uvrA + XP cells was investigated by fractionation of cell extracts in which an indirect immunofluorescence method revealed its location as being largely extra-nuclear. Two uvrA + cell lines were examined for their UV_resistant phenotye and not unexpectedly were found not to be reverted to a state of repair proficiency.

Differential repair of DNA damage in specific nucleotide sequences in monkey cells.

An immunological method was developed that isolates DNA fragments containing bromouracil in repair patches from unrepaired DNA using a monoclonal antibody that recognizes bromouracil. Cultured monkey cells were exposed to either UV light or the activated carcinogen aflatoxin B1 and excision repair of damage in DNA fragments containing the integrated and transcribed E. coli gpt gene was compared to that in the genome overall. A more rapid repair, of both UV and AFB1 damage was observed in the DNA fragments containing the E. coli gpt genes. The more efficient repair of UV damage was not due to a difference in the initial level of pyrimidine dimers as determined with a specific UV endonuclease. Consistent with previous observations using different methodology, repair of UV damage in the alpha sequences was found to occur at the same rate as that in the genome overall, while repair of AFB1 damage was deficient in alpha DNA. The preferential repair of damage in the gpt gene may be related to the functional state of the sequence and/or to alterations produced in the chromatin conformation by the integration of plasmid sequences carrying the gene.