PubMed Health⌕ Search

Biomedical subjects

W C Summers

Publications and source records attributed to W C Summers.

At least 19 recordsLinked to original sources
PubMed1992

Identification of the 12-O-tetradecanoylphorbol-13-acetate-responsive enhancer of the MS gene of the Epstein-Barr virus.

We previously located two 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive enhancers, MSTRE-I and MSTRE-II, in the upstream sequence of the MS gene of Epstein-Barr virus (Liu, Q., and Summers, W.C. (1989) J. Virol. 63, 5062-5068). The core sequence of the MSTRE-I enhancer is now determined to be between -718 and -708 of the upstream sequence of the MS gene. The activity of the enhancer is also sensitive to its immediate surrounding sequence on either side. A single copy of a 30-base pair (bp) fragment containing the MSTRE-I sequence was able to confer TPA responsiveness upon the MS promoter even in the absence of an AP-1 binding site. Multiple tandem copies of this 30-bp fragment, regardless of their relative orientations to each other, could function synergistically to enhance the MS promoter activity. At least two copies of the 30-bp fragment were required to bestow TPA induction upon the thymidine kinase gene promoter of herpes simplex virus type 1. The MSTRE-I sequence could also be bound by a Fos-GCN4 chimeric protein but with an affinity much lower than that between the chimeric protein and the AP-1 binding site. This MSTRE-I region has strong homology to one of the TPA-responsive elements (the ZII domain) in the upstream sequence of the EBV BZLF1 gene. In addition, a putative negative regulatory region or silencer was found immediately downstream of the MSTRE-I enhancer. This potential silencer region contains a 14-bp sequence that is homologous to the silencer consensus sequence of the BZLF1 gene. Therefore, the regulation of the MS gene may share the same pathway with the immediate early gene BZLF1.

Autoradiography↗
PubMed1990

Identification of a glucocorticoid-responsive element in Epstein-Barr virus.

Immortalization of B lymphocytes by Epstein-Barr virus (EBV) is complex and poorly understood. However, some evidence suggests that glucocorticoids influence this process. We identified a glucocorticoid-responsive element in the BamHI C fragment of EBV which we call ES-1. In glucocorticoid-treated cells, ES-1 enhanced chloramphenicol acetyltransferase gene expression from the herpes simplex virus thymidine kinase promoter, as well as the EBV Bam-C promoter, from which several latent viral gene products are transcribed. By Northern blot analysis, glucocorticoid treatment enhanced transcription from the Bam-C promoter in Jijoye cells, a Burkitt's lymphoma cell line. In addition, the DNA-binding domain of the glucocorticoid receptor bound specifically to the ES-1 region. These glucocorticoid effects on the Bam-C promoter region may provide some insight into the process of EBV immortalization.

Base Sequence↗
PubMed1989

Post-transcriptional mechanisms of deregulation of MYC following conversion of a human B cell line by Epstein-Barr virus.

By utilizing an Epstein-Barr virus (EBV)-negative Burkitt's lymphoma line (BJAB) and several EBV-positive sub-lines derived from it by in vitro infection, it has been shown previously that the presence of the EBV genome in this B cell line is reversibly associated with deregulation of MYC expression. Specifically, the decline in the level of MYC transcripts observed in the EBV-negative BJAB line as cells approach stationary phase of growth is abrogated in the presence of the virus. In the studies described herein, the mechanism of deregulation of MYC in EBV-converted BJAB cells was examined. It was shown that the presence of EBV in BJAB cells was not associated with changes in the rate of transcription from exons I, II or III of MYC as cells approached stationary phase of growth. In contrast, the stability of MYC mRNA was altered in EBV-positive BJAB cells. Specifically, the half-life of MYC mRNA increased from less than 36 to greater than 70 min in EBV-positive BJAB lines as cells progressed from early to late exponential phase of growth. This alteration in stability of MYC transcripts was reversibly associated with the presence of the virus, since an EBV-negative revertant BJAB line did not display an increase in stability of MYC transcripts in late exponential phase of growth. In addition, progression from early to late exponential phase of growth in two EBV-immortalized lymphoblastoid lines derived from normal human B cells was also associated with prolongation of the half-life of MYC.(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocytes↗
PubMed1989

Physical mapping of the human cytomegalovirus (HCMV) (Towne) DNA polymerase gene: DNA-mediated transfer of a genetic marker for an HCMV gene.

DNA-mediated transfer of a drug resistance marker (phosphonoacetate resistance) for the HCMV (Towne) DNA polymerase (pol) gene has been used to genetically confirm the physical localization of the HCMV (Towne) DNA pol gene. The HCMV (Towne) genomic region homologous to the pol genes of other herpesviruses was first identified by moderate stringency Southern hybridization. Restriction fragments from this region were molecularly cloned from previously characterized phosphonoacetic acid resistant (PAAr) HCMV genomes (R. T. D'Aquilla and W. C. Summers, J. Virol., 61, 1291-1295, 1987). A high frequency of recombinant PAAr virus was found among the progeny of cotransfections of infectious, wild-type HCMV (Towne) DNA only with pol-homologous restriction fragments cloned from PAAr HCMV. The co-transfection technique described here may facilitate further gene mapping in HCMV. The results presented here provide functional proof that the HCMV pol gene is encoded by the sequences previously identified as homologous to other herpesvirus pool genes.

Cytomegalovirus↗
PubMed1989

UV-induced DNA-binding proteins in human cells.

To investigate the response of human cells to DNA-damaging agents such as UV irradiation, we examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m2, the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification.

Cycloheximide↗
PubMed1989

Novel 12-O-tetradecanoylphorbol-13-acetate-responsive elements in the upstream sequence of the MS gene promoter of Epstein-Barr virus.

We have demonstrated that gene expression from the promoter of the Epstein-Barr virus (EBV) MS gene with its upstream sequence is inducible by 12-O-tetradecanoylphorbol-13-acetate (TPA). By transfecting mammalian cells with plasmids in which the MS promoter and its upstream sequence are linked to the bacterial chloramphenicol acetyltransferase gene, we have shown that treatment of the cells with TPA stimulates the expression of chloramphenicol acetyltransferase activity in both EBV-negative and -positive cell lines. This TPA response requires the cis-acting sequence between nucleotides 84440 and 85046 of the EBV genome, located either upstream or downstream of the MS promoter. The TPA induction is at the transcriptional level. When this sequence is linked to the promoter of the human herpesvirus 1 thymidine kinase gene, it can also enhance the expression of, and confer TPA responsiveness on, the thymidine kinase promoter. By constructing and transfecting mutants with 5' and 3' deletions, we have identified two TPA-responsive elements, one located between -726 and -690 and the other located between -603 and -546 relative to the transcription start site. These two sequences do not contain any homology to the previously defined elements for TPA response and may play an important role in EBV induction by TPA.

Animals↗
PubMed1989

HIV-1 reverse transcriptase/ribonuclease H: high level expression in Escherichia coli from a plasmid constructed using the polymerase chain reaction.

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT)/ribonuclease H has been expressed to high levels in Escherichia coli from a recombinant plasmid constructed using the polymerase chain reaction (PCR) for in vitro mutagenesis. Translational initiation and termination codons were introduced by the PCR at points corresponding to sites of cleavage of the RT from the gag-pol precursor polyprotein by the HIV-1 protease; the HIV-1 protease is not expressed from this construct. Most of the RT coding sequences derived from PCR were exchanged for a DNA fragment cloned by standard methods to minimize the possibility that an unwanted mutation was introduced during the in vitro amplification. The RT is expressed in bacteria from this plasmid as 66 and 51 kDa proteins, has both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities, and is indistinguishable from native HIV-1 RT in electrophoretic mobility and immunoreactivity. Peptide sequencing of the amino terminus of the HIV-1 RT purified from bacterial lysates is also presented. A novel activity gel assay was used to confirm that only the 66 kd protein catalyzes the RNase H reaction; this assay will simplify analysis of this catalytic activity. This HIV-1 RT expression plasmid is of interest because of the high level of expression in bacteria and the demonstrated RNase H activity of the enzyme. This plasmid will be distributed for research purposes through the NIH AIDS Repository and will facilitate enzymologic, structural, and immunologic evaluation of reverse transcription and its chemotherapeutic inhibition.

Endoribonucleases↗
PubMed1988

Purification and crystallization of thymidine kinase from herpes simplex virus type 1.

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.

Crystallization↗
PubMed1988

Site-directed mutagenesis of a nucleotide-binding domain in HSV-1 thymidine kinase: effects on catalytic activity.

The thymidine kinase encoded by herpes simplex virus type 1 contains an amino acid sequence homologous to a consensus sequence related to the ATP-binding site in many proteins. We have used site-directed mutagenesis to investigate the importance of the five highly conserved amino acids within this segment. When any one of the three glycines was changed to valine the corresponding mutant enzyme was inactive. The mutation of lysine 63 to isoleucine destroyed the enzymatic activity. When threonine 64 was changed to alanine the mutant enzyme lost its activity. However, when this threonine was changed to serine the enzyme was still active but with different apparent Michaelis constants (Km) for thymidine and ATP. The wild-type thymidine kinase has apparent Km's of 0.5 and 20 microM for thymidine and ATP, respectively, while the mutant enzyme displayed Km's of 2.3 and 60 microM for thymidine and ATP. These results indicate that this homologous segment is essential for the function of the thymidine kinase and is involved in the substrate binding domain of the enzyme.

Adenosine Triphosphate↗
PubMed1987

Amplification and deregulation of MYC following Epstein-Barr virus infection of a human B-cell line.

In Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) the role of EBV in the translocation and deregulation of the MYC oncogene remains unknown. By utilizing an EBV-negative BL (BJAB) and several EBV-positive sublines derived from it by in vitro infection, it was possible to show that the presence of the virus was associated with altered expression and copy number of MYC. In the EBV-negative BJAB line, the level of MYC transcripts declined progressively as cells approached the stationary phase of growth. In contrast, in EBV-infected BJAB cells MYC expression remained elevated as cells entered stationary phase. This effect on MYC expression was reversibly linked to the presence of the virus. Furthermore, following EBV infection of BJAB cells by two different strains of EBV, amplification of MYC in association with the appearance of a homogeneously staining region on chromosome 8 at the mapped location of MYC had occurred. These studies suggest that both the deregulation of MYC transcription and the chromosomal rearrangement in the region of the MYC locus in this B-cell line may have occurred as a result of EBV infection.

Antigens, Viral↗
PubMed1987

Isolation and characterization of phosphonoacetic acid-resistant mutants of human cytomegalovirus.

Mutants of the human cytomegalovirus (HCMV) that were 6- to 13-fold more resistant to phosphonoacetic acid than the wild-type HCMV (Towne) were isolated. Extracts from mycoplasma-free, mutant-infected cells had phosphonoacetate-resistant DNA polymerase activity in vitro. This strongly suggests that the selected mutations are in the HCMV DNA polymerase genes of these viruses.

Ammonium Sulfate↗
PubMed1987

DNA mismatch repair detected in human cell extracts.

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.

Bacterial Proteins↗
PubMed1986

Detection and analysis of UV-induced mutations in mammalian cell DNA using a lambda phage shuttle vector.

In order to study mutagenesis in mammalian cells, stable mouse L-cell lines were established with multiple copies of a lambda phage vector that contains the supF gene of Escherichia coli as a target for mutagenesis. Rescue of viable phage from high molecular weight mouse cell DNA using lambda in vitro packaging extracts was efficient (5 phage per microgram of cell DNA per copy) and yielded a negligible background of mutant phage (0 out of 54,605). From mouse cells exposed to 254-nm ultraviolet light (12J/m2), 78,510 phage were rescued, of which 8 were found to have mutant supF genes. DNA sequence analysis of the mutants suggests that the primary site of UV mutagenesis in mammalian cells is at pyrimidine-cytosine (Y-C) sequences, and that the most frequent mutation at this site is a C----T transition.

Animals↗
PubMed1986

Induction of c-myc expression in human B lymphocytes by B-cell growth factor and anti-immunoglobulin.

Purified human B lymphocytes were examined for transcriptional expression of c-myc in response to mitogenic stimulation by the method of in situ hybridization using 35S-labeled DNA probes. The level of c-myc expression increased 10- to 20-fold within 2 hr after the addition of anti-mu, formalinized Staphylococcus aureus Cowan strain I, or B-cell growth factor, as compared to resting B cells. After 72-96 hr of mitogenic stimulation, c-myc expression remained elevated 5-fold, but expression among individual cells had become more heterogeneous than at early time points. To determine whether c-myc expression in human B lymphocytes is phase specific within the cell cycle, mitogen-stimulated cells were sorted by DNA content into populations of cells in G0/G1, S, and G2/M phases of the cell cycle. Examination of c-myc expression in phase-specific cells revealed that c-myc expression was elevated in all phases of the cell cycle, but it appeared to be maximally expressed in S phase. These studies suggest that c-myc expression in normal human B lymphocytes is cell-cycle dependent and remains elevated in all phases of the cycling cell.

Antibodies, Anti-Idiotypic↗
PubMed1986

The promoter for the late gene encoding Vp5 of herpes simplex virus type 1 is recognized by cell extracts derived from uninfected cells.

The ability of whole-cell extracts from uninfected HeLa cells to recognize the promoter for the herpes simplex virus type 1 late gene encoding the major capsid protein Vp5 was investigated by using both in vitro transcriptional and S1 nuclease protection analysis. This gene promoter was recognized by the cell extracts and produced abundant amounts of transcript in the absence of any other virus-encoded factors. This transcript was shown to arise, in vitro, from specific initiation at or very near the physiological mRNA start site. Thus, it appears that cell extracts from uninfected HeLa cells can efficiently recognize both early- and late-gene promoters.

Capsid↗
PubMed1985

Utilization of internal AUG codons for initiation of protein synthesis directed by mRNAs from normal and mutant genes encoding herpes simplex virus-specified thymidine kinase.

Previous studies (H.S. Marsden, L. Haarr, and C.M. Preston, J. Virol. 46:434-445, 1983) have shown that at least three polypeptides, with molecular weights of 43,000, 39,000, and 38,000, are encoded by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene. It has been suggested that the 39,000- and 38,000-molecular-weight polypeptides arise from preinitiation complexes bypassing the first and second AUG codons before commencement of translation since, according to previous work (M. Kozak, Nucleic Acids Res. 9:5233-5252, 1981), these codons are not of the most efficient structure for initiation. This possibility was investigated by using specific herpes simplex virus mutants with alterations in the TK gene. Mutant TK4 has an amber mutation between the first and second AUG codons, whereas mutant delta 1 has a deletion which removes the first AUG codon but leaves other AUG codons, as well as transcriptional promoter sequences, intact. Both mutants synthesized only the 39,000- and 38,000-molecular-weight polypeptides, and the amounts produced were normal in TK4-infected cells but increased in delta 1-infected cells. Furthermore, the levels of TK produced after infection with the mutant viruses correlated with the amounts of the 39,000- and 38,000-molecular-weight polypeptides synthesized. The 43,000-, 39,000-, and 38,000-molecular-weight polypeptides were shown to be related by their positive reaction with anti-TK serum in both immunoprecipitation and immunoblotting experiments. The production of the 39,000- and 38,000-molecular-weight polypeptides through bypassing of the first AUG codon was examined by hybrid arrest experiments with a DNA fragment complementary to only 50 bases at the 5' terminus of TK mRNA. This fragment arrested the synthesis of the 30,000- and 38,000-molecular-weight polypeptides when annealed to mRNA from wild-type HSV-1- or TK4-infected cells, showing that those polypeptides arise from an mRNA initiated upstream from the first AUG codon. mRNA from cells infected with mutant delta 1, which lacks DNA sequences upstream from the first AUG, was not affected by the 50-base-pair fragment. The data therefore confirm that three polypeptides encoded by the HSV-1 TK gene arise by differential use of in-phase AUG codons for the initiation of protein synthesis. This mechanism for the production of related but distinct polypeptides has not previously been demonstrated in a eucaryotic system, and the implications for the regulation of TK enzyme activities are discussed.

Cell Line↗
PubMed1985

Oncogene expression in isogenic, EBV-positive and -negative Burkitt lymphoma cell lines.

To elucidate the mode of action of Epstein-Barr virus (EBV), we have examined the possibility that EBV infection may lead to a change in oncogene expression. Levels of oncogene-related RNA were compared in the Burkitt lymphoma(BL)-derived, EBV-negative cell line BJAB and in two isogenic, EBV-converted sublines of BJAB whose growth characteristics differ from the EBV-negative parent line. The oncogenes c-myc, Ha-ras, and Blym were each found to be expressed at equally high levels in the three cell lines. This suggests that the mechanism of action of EBV in altering the growth properties of lymphoid cells is not dependent on the induction of cellular oncogenes. In addition, since the 8,14 chromosomal translocation has been related to increased c-myc expression in BL cells, a karyotype analysis was done to confirm the initial report of a normal karyotype in BJAB cells. Chromosomes 8 and 14 were seen to be normal. Thus, BJAB cells are a BL cell line in which the elevated expression of c-myc is independent not only of EBV but also of translocations involving chromosome 8.

Burkitt Lymphoma↗