Biomedical subjects

J P Capone

Publications and source records attributed to J P Capone.

46 records · Page 3Linked to original sources

PubMed1990

Synthesis of the herpes simplex virus type 1 trans-activator Vmw65 in insect cells using a baculovirus vector.

Vmw65, the Herpes Simplex Virus trans-activator of immediate-early genes, was expressed in insect cells using a recombinant baculovirus expression vector and partially purified. Insect cell-derived Vmw65 was shown to be indistinguishable from authentic Vmw65 present in purified HSV-1 virions based on electrophoretic mobility, immunoreactivity with a monoclonal antibody, and ability to interact with cellular factors to form a protein/DNA complex with oligonucleotides containing a TAATGARAT element.

Animals↗

PubMed1990

Enhanced infectivity of herpes simplex virus type 1 viral DNA in a cell line expressing the trans-inducing factor Vmw65.

Vmw65 is a structural component of herpes simplex virus (HSV) which is involved in transactivating the expression of the viral immediate-early (IE) genes. To gain further insight into the function of this protein, a cell line, BSV65, was established which expresses biologically active Vmw65 under control of the Moloney leukemia virus long terminal repeat. This cell line was shown to specifically activate IE genes as demonstrated by transient transfection assays with reporter genes linked to HSV IE or delayed-early promoter-regulatory regions. Furthermore, by using mobility shift assays, cell extracts were shown to be capable of forming a Vmw65-containing complex with oligonucleotides that contained a TAATGARAT motif, a conserved cis-acting IE regulatory element which is required for Vmw65-mediated trans induction. BSV65 cells were able to complement HSV type 1 in 1814, a mutant which is unable to trans-induce IE gene expression and whose growth is impaired at low multiplicities of infection. Transfection of purified HSV type 1 viral DNA into BSV65 cells resulted in an approximately 200-fold increase in virus production compared with the parental cell line. In addition, in comparison to wild-type cells, infectious virus production occurred sooner and efficiency of plaque formation was higher in BSV65 cells following transfection of viral DNA but not following infection with virus. Northern (RNA) dot blot analysis of cells transfected with viral DNA showed that transcription of the IE gene Vmw175 was approximately 10-fold greater in BSV65 cells compared with wild-type cells. These results indicate that, in the presence of functional Vmw65, there is a greater probability that transfected viral DNA will lead to a productive infection.

Animals↗

PubMed1990

A cellular factor binds to the herpes simplex virus type 1 transactivator Vmw65 and is required for Vmw65-dependent protein-DNA complex assembly with Oct-1.

The herpes simplex virus transactivator Vmw65 assembles into a multicomponent protein-DNA complex along with the octamer binding protein Oct-1. Using affinity chromatography on columns conjugated with purified Vmw65 fusion protein expressed in Escherichia coli, we demonstrate that a cellular factor, distinct from Oct-1, binds to Vmw65 in the absence of target DNA and is necessary for Vmw65-mediated complex assembly with Oct-1.

Base Sequence↗

PubMed1990

Autologous fibroblast implantation. Feasibility and potential problems in gene replacement therapy.

Autologous implants of genetically modified fibroblasts may be an ideal approach to gene replacement therapy. However, primary fibroblasts have limited life span and their use for gene therapy is questionable. We have developed a rat model to evaluate the feasibility of this approach. Primary fibroblasts initiated from rat skin biopsies were transfected with plasmid DNA. The transkaryotic fibroblasts were implanted into the original rat donors. A plasmid-encoded gene product, human growth hormone, was detected in the donor rats' circulation but less than 1% of the expected amount was recovered. Implantation at intraperitoneal, intramuscular or subcutaneous sites were effective in delivering the novel gene product. Fluctuating levels of human growth hormone were detected for over 6 months but their determination was rendered unreliable due to interference by extremely high titers of antibodies developed against the human growth hormone. In some cases, the antibody titer continued rising for more than 6 months, indicative of the continued presence of the antigen. One of the recipients developed two intra-abdominal fibrosarcomas that were shown to be derived from the implanted cells. In conclusion, this model demonstrates that autologous fibroblast implantation is a feasible approach to delivering novel gene products lasting for several months. Strong and prolonged immuno-reaction against the gene product will occur if the recipient's immune system is naive to the replacement product, as would occur in patients with CRM- mutations. The potential for neoplastic development from the implanted fibroblasts should prompt further evaluation of the long-term safety in gene replacement therapy.

Animals↗

PubMed1989

Mutational analysis of the herpes simplex virus trans-inducing factor Vmw65.

Vmw65 is a structural component of herpes simplex virus which, in conjunction with host factors, trans-activates the expression of the viral immediate-early genes. In order to examine the relationship between the primary structure of Vmw65 and its trans-activating function, we generated in-frame insertion, deletion, and nonsense mutations in a cloned copy of the gene. The ability of the mutant polypeptides to function as transcriptional activators was assessed by transient transfection of Vero cells using, as the reporter gene, the Escherichia coli chloramphenicol acetyltransferase (cat) gene linked to the promoter-regulatory region from the HSV-1 immediate-early gene coding for Vmw175. These studies have demonstrated that a highly acidic region near the C-terminus of Vmw65 as well as the structural integrity of several other regions of the polypeptide are essential for its transactivating properties, whereas a small region near the N-terminus of the polypeptide is dispensible for activity. Finally, in vivo competition studies using inactive deletion mutants suggest that a domain of the polypeptide located between amino acids 141 and 185 may be involved in protein-protein interactions.

Chloramphenicol O-Acetyltransferase↗

PubMed1989

Identification of a domain of the herpes simplex virus trans-activator Vmw65 required for protein-DNA complex formation through the use of protein A fusion proteins.

In order to identify structural domains of the herpes simplex virus trans-activator Vmw65 required for protein-DNA complex formation, subfragments of Vmw65 were expressed in Escherichia coli as fusion polypeptides with protein A of Staphylococcus aureus, and the purified hybrids were used in a band shift assay. The results indicate that a region near the amino terminus of Vmw65 between amino acids 141 and 185 is necessary for complex formation.

Base Sequence↗

PubMed1988

Modulation of the phenotypic expression of a human serine tRNA gene by 5'-flanking sequences.

Mammalian nonsense suppressors provide a model system to investigate structural and functional aspects of mammalian tRNAs and their genes in vivo. To assess the role that extragenic flanking sequences may have on the expression of mammalian tRNA genes in vivo, deletion/substitutions ending in the 5'-flanking sequence or 3'-flanking sequence of a cloned human serine amber suppressor tRNA gene were constructed. The phenotypic expression of these mutant genes was examined by transfection in mammalian cells and by measuring the efficiency with which they were able to suppress an amber (UAG) nonsense mutation in the Escherichia coli chloramphenicol acetyl transferase (cat) gene. Deletion of the 5'-flanking region up to nucleotide position -66 with respect to the first nucleotide of the coding region had no effect on levels of nonsense suppression as compared to the wild-type gene; however, deletion to -18 led to a 12-fold reduction in suppressor activity. Deletion up to -1 did not further reduce suppression efficiency. Deletion of the 3'-flanking region up to 9 nucleotides downstream from the consecutive T residue termination site resulted in only a slight reduction in functional tRNA expression. In in vivo competition studies, the -18 deletion clone was less able to compete out the activity of a second suppressor tRNA gene than was the wild-type corresponding gene, suggesting that the upstream region plays a role in the formation of active transcription complexes in vivo. These results imply that the human serine tRNA gene contains an upstream regulatory region located between positions -66 and -18 that plays a positive role in modulating expression of this gene in vivo.

Base Sequence↗

PubMed1987

An inducible mammalian amber suppressor: propagation of a poliovirus mutant.

We describe a general protocol for controlled gene amplification, which allows conditional expression of high levels of amber suppressor activity in monkey kidney cells, and we demonstrate its use in the genetic analysis of animal viruses by the generation and propagation of the first nonsense mutant of poliovirus. A human amber suppressor tRNASer gene linked to the SV40 origin of replication and a second DNA carrying a temperature-sensitive SV40 large T antigen gene were cotransfected into monkey cells. Cell lines having stably integrated the DNAs were isolated. Shifting the cells from the nonpermissive temperature to a lower permissive temperature caused the amplification of the suppressor tRNA gene, which resulted in suppression efficiencies at amber codons of 50%-70%, as measured by suppression of an amber codon in the E. coli chloramphenicol acetyltransferase gene. A mutant of poliovirus, in which a serine codon in the replicase gene was converted to an amber codon, was efficiently propagated on the suppressor-positive cell lines. The mutant virus reverted to wild-type by a single base change to a serine codon at a frequency of approximately 2.5 x 10(-6), surprisingly low for a RNA genome.

Animals↗

PubMed1986

Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells.

We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.

Acetyltransferases↗

PubMed1985

Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes.

Animals↗