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M D Summers

Publications and source records attributed to M D Summers.

At least 73 records · Page 4Linked to original sources
PubMed1987

Physical Analysis of the Campoletis sonorensis Virus Multipartite Genome and Identification of a Family of Tandemly Repeated Elements.

This report is an analysis of cross-hybridizing sequences found within the 28 superhelical (SH) DNAs of the multipartite genome of the polydnavirus Campoletis sonorensis virus (CsV). A Southern cross-blot hybridization analysis showed that the majority of CsV EcoRI restriction fragments cross-hybridize to multiple EcoRI fragments. These sequence homologies were analyzed by hybridizing recombinant clones of the CsV SH DNAs B, H, M, and O(1) to Southern blots of undigested CsV DNA, using different hybridization stringencies. The results indicated that homologous regions among the SH DNAs include closely related sequences that are detectable under stringent conditions and related but more diverged sequences which are only detectable under reduced stringencies. A sequence that hybridized to the majority of the CsV SH DNAs was identified and subcloned from the SH DNAs O(1), H, and B. Nucleotide sequence data revealed that these homologous regions contained a family of imperfectly conserved repeated elements. These repeat elements were arranged singly or in direct tandem arrays and had an average length of 540 base pairs. Within the sequenced regions that contained the repeated elements six putative open reading frames were identified. These results show that the CsV genome consists of SH DNAs with complex sequence interrelationships that may have arisen due to multiple recombinational events.

Journal Article↗
PubMed1986

Molecular analysis of Campoletis sonorensis virus DNA in the lepidopteran host Heliothis virescens.

Nucleic acid hybridization techniques were used to analyse the fate of Campoletis sonorensis virus (CsV) DNAs in naturally parasitized and virus-injected Heliothis virescens larvae. Viral DNA persisted in injected H. virescens larvae from 0 to 10 days post-injection but no increase in the amount of viral DNA could be detected. Similarly, no increase in the amount of viral DNA was detected in naturally parasitized H. virescens larvae before the development of the C. sonorensis pupae. However, a dramatic increase of viral DNA was detected in pharate and newly emerged (0 to 48 h) adult C. sonorensis wasps. The results from these in vivo molecular analyses suggest that CsV replication does not occur in naturally parasitized or virus-injected H. virescens and that viral replication may be restricted to tissues of C. sonorensis wasps.

DNA Replication↗
PubMed1986

Identification, Mapping, and In Vitro Translation of Campoletis sonorensis Virus mRNAs from Parasitized Heliothis virescens Larvae.

Expression of Campoletis sonorensis virus (CsV) in parasitized Heliothis virescens larvae was investigated by Northern blot analysis of poly(A)(+) mRNAs isolated from H. virescens larvae at various times after parasitization by C. sonorensis. At least 12 CsV mRNAs were detected in parasitized H. virescens larvae. Injection of nonparasitized H. virescens larvae with purified CsV resulted in a pattern of viral mRNAs similar to that observed in naturally parasitized larvae. With CsV DNA restriction fragments which contained expressed sequences, individual CsV mRNAs were mapped to the superhelical DNAs of the viral genome. Two gene-specific probes, which consisted of cloned S1 nuclease-protected restriction fragments, each hybridized to several CsV superhelical DNAs, suggesting that some CsV genes may be shared on several superhelical DNAs. Cloned restriction fragments containing sequences which flank the expressed sequences also hybridized to numerous CsV superhelical DNAs. Some CsV proteins were identified by in vitro translation of hybrid-selected CsV mRNAs.

Journal Article↗
PubMed1986

Campoletis sonorensis Endoparasitic Wasps Contain Forms of C. sonorensis Virus DNA Suggestive of Integrated and Extrachromosomal Polydnavirus DNAs.

Campoletis sonorensis virus (CsV) (Polydnaviridae) previously was detected only in the calyx epithelial cells and lumen of the oviducts from female C. sonorensis (Ichneumonidae) endoparasitic wasps (Norton et al., Cell Tissue Res. 162:195-208, 1975). Using dot-blot hybridizations, we detected low amounts of CsV DNA in male and female wasp head and thorax tissues and in male abdominal tissues. Low amounts of extrachromosomal viral DNA were detected in Southern blots of undigested male wasp DNA and in male DNA purified by isopycnic centrifugation. High-molecular-weight male wasp DNA digested with any of several restriction endonucleases and hybridized with cloned viral DNAs from CsV superhelices B and Q under stringent conditions contained CsV-specific DNA fragments that differed significantly in size and number from the hybridizing fragments detected in comparably digested viral DNA. Identical offsize restriction fragments were detected in digested female head and thorax DNA. These data suggest that at least CsV DNAs B and Q are integrated in C. sonorensis cellular DNA and that the virus may be transmitted through the germline.

Journal Article↗
PubMed1986

Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene.

The temporal regulation of an early gene of the baculovirus Autographa californica nuclear polyhedrosis virus was examined. We constructed a plasmid (plasmid 39CAT) in which the bacterial gene for chloramphenicol acetyltransferase was placed under the control of the promoter for the gene for a A. californica nuclear polyhedrosis virus 39,000-dalton protein (39K). A transient expression assay of plasmid 39CAT revealed that the 39K gene was expressed in infected cells but not in uninfected cells, indicating that the 39K gene should be classified as a delayed-early gene. The 39K promoter also efficiently directed the synthesis of chloramphenicol acetyltransferase when the plasmid was cotransfected with viral DNA which had been restricted with several restriction enzymes. To map the location of the gene(s) required for the synthesis of 39K, plasmid 39CAT was cotransfected with purified restriction fragments of A. californica nuclear polyhedrosis virus DNA. Fragments which mapped between 90.7 and 100.8 map units induced plasmid 39CAT. Plasmid pEcoRI-B, containing EcoRI fragment B (90 to 100 map units), activated plasmid 39CAT. Functional mapping of plasmid pEcoRI-B indicated that the essential region was located between 95.0 and 97.5 map units. The 5' end of this gene was mapped, and the chloramphenicol acetyltransferase gene was inserted under the control of its promoter. Transient assay experiments indicated that the trans-acting regulatory gene was expressed in uninfected cells and is therefore an immediate-early gene. This gene was named IE-1.

Animals↗
PubMed1986

Interspersed Homologous DNA of Autographa californica Nuclear Polyhedrosis Virus Enhances Delayed-Early Gene Expression.

The five regions of homologous DNA which are interspersed in the genome of the baculovirus Autographa californica nuclear polyhedrosis virus increased the expression of a delayed-early gene of this virus. Although this activity was first observed as a 10-fold trans effect, the homologous region 5 (hr5) enhanced the expression of linked genes 1,000-fold. The hr5 enhancer also exhibited the other characteristics associated with viral enhancer elements, including orientation independence and the abilities to function at a distance from the linked promoter, to regulate heterologous promoters, and to increase the number of RNA polymerase molecules transcribing the linked genes. The expression of the immediate-early regulatory gene was not enhanced by cis-linked hr5, although the enhancer function may require the immediate-early regulatory gene product. The hr5 enhancer was relatively insensitive to competition by an excess of enhancer molecules. The nucleotide sequence of hr5 revealed two different conserved repeats separated by nonhomologous DNA. Deletion analysis of the hr5 enhancer indicated that a 30-base-pair inverted repeat was essential for enhancer function.

Journal Article↗
PubMed1986

Complete Sequence and Enhancer Function of the Homologous DNA Regions of Autographa californica Nuclear Polyhedrosis Virus.

The nucleotide sequence of the five regions of homologous DNA in the genome of Autographa californica nuclear polyhedrosis virus DNA was determined. The homology of repeated sequences within a region was 65 to 87%, and the consensus sequences for each region were 88% homologous to each other. Sequences proximal to the EcoRI sites were most conserved, while the distal sequences were least conserved. The EcoRI sites formed the core of a 28-base-pair imperfect inverted repeat. All homologous regions functioned as enhancers in a transient expression assay. A single EcoRI minifragment located between EcoRI-Q and -L enhanced the expression of 39CAT as efficiently as the regions containing numerous EcoRI repeats did.

Journal Article↗
PubMed1985

Transposon-mediated mutagenesis of a baculovirus.

Spontaneous mutants of insect nuclear polyhedrosis viruses of Autographa californica (AcMNPV) and Galleria mellonella (GmMNPV) were analyzed. These mutants produce few polyhedra in infected cells and have insertions of host cell DNA. All the insertions mapped to two adjacent sites in the genome. The junctions between two host insertions and the viral DNA were sequenced. One of the insertions contained a perfect 7-bp inverted terminal repeat, and had caused a direct duplication of 4 bp of viral DNA at the point of insertion. Therefore, this insertion sequence has properties of a transposon of the host cell Trichoplusia ni.

Animals↗
PubMed1985

Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector.

A cDNA coding for human interleukin 2 (IL-2) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Cells infected with recombinant virus produced high levels of Mr 15,500 IL-2 polypeptide, the majority of which was secreted into the culture medium during infection. The recombinant IL-2 was able to stimulate the growth of an IL-2-dependent cell line. The N-terminal amino acid sequence of the insect-derived IL-2 was identical to that of natural IL-2. Thus, a mammalian signal peptide was recognized and properly removed in insect cells.

Amino Acid Sequence↗
PubMed1985

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.

Animals↗
PubMed1983

Physical Analysis of Autographa californica Nuclear Polyhedrosis Virus Transcripts for Polyhedrin and 10,000-Molecular-Weight Protein.

In Autographa californica nuclear polyhedrosis virus-infected cells, polyhedrin, the major structural polypeptide of the viral occlusions, and a low-molecular-weight viral protein with a molecular weight of approximately 10,000 (10K) accumulated to high levels late in infection. Two polyadenylated RNAs 1,200 and 630 bases in size were the most abundant viral transcripts present in the cytoplasm at 48 h postinfection. Evidence is presented that these RNAs were the transcripts for polyhedrin and 10K proteins. The 630-base RNA and five other major polyadenylated RNAs present at 48 h postinfection mapped within or near A. californica nuclear polyhedrosis virus EcoRI-P. The DNA sequences that code for polyhedrin mRNA were examined by S1 nuclease analysis. The polyhedrin gene contained no detectable introns and mapped at 3,990 to 5,200 base pairs to the right of the origin of the circular A. californica nuclear polyhedrosis virus physical map. Heterogeneity at the 5' end of polyhedrin mRNA was observed by using S1 nuclease analysis with 5' end-labeled DNA probes.

Journal Article↗
PubMed1983

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene.

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.

Journal Article↗
PubMed1983

Acquisition of Host Cell DNA Sequences by Baculoviruses: Relationship Between Host DNA Insertions and FP Mutants of Autographa californica and Galleria mellonella Nuclear Polyhedrosis Viruses.

Mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses, which produce an altered plaque phenotype as a result of reduced numbers of viral occlusions in infected cells, were isolated after passage in Trichoplusia ni (TN-368) cells. These mutants, termed FP (few-polyhedra) mutants, had acquired cell DNA sequences ranging from 0.8 to 2.8 kilobase pairs in size. The insertions of cell DNA occurred in a specific region between 35.0 and 37.7 map units of the A. californica viral genome. A cloned viral fragment containing one of the host DNA inserts was homologous to host DNA inserts in two other mutant viruses and to dispersed, repetitious sequences in T. ni cell DNA. Most of the homology between the cloned insert and cell DNA was contained within a 1,280-base-pair AluI fragment. Marker rescue studies and analysis of infected-cell-specific proteins suggested that the insertion of cell DNA into the viral genomes resulted in the FP plaque phenotype, possibly through the inactivation of a 25,000-molecular-weight protein.

Journal Article↗
PubMed1983

Expression of Campoletis sonorensis Virus in the Parasitized Host, Heliothis virescens.

Polyadenylated mRNA transcripts of the virus of Campoletis sonorensis were detected in Heliothis virescens host larvae 2 h after the larvae had been parasitized by C. sonorensis females and continued to be present in host larvae through 9 days postparasitization while C. sonorensis developed endoparasitically. cDNAs of viral transcripts present in H. virescens hybridized with only certain C. sonorensis viral DNAs.

Journal Article↗
PubMed1983

Production of human beta interferon in insect cells infected with a baculovirus expression vector.

Autographa californica nuclear polyhedrosis virus (AcNPV) was used as an expression vector for human beta interferon. By using specially constructed plasmids, the protein-coding sequences for interferon were linked to the AcNPV promoter for the gene encoding for polyhedrin, the major occlusion protein. The interferon gene was inserted at various locations relative to the AcNPV polyhedrin transcriptional and translational signals, and the interferon-polyhedrin hybrid genes were transferred to infectious AcNPV expression vectors. Biologically active interferon was produced, and greater than 95% was secreted from infected insect cells. A maximum of ca. 5 X 10(6) U of interferon activity was produced by 10(6) infected cells. These results demonstrate that AcNPV should be suitable for use as a eucaryotic expression vector for the production of products from cloned genes.

Amino Acid Sequence↗
PubMed1982

Virus with a Multipartite Superhelical DNA Genome from the Ichneumonid Parasitoid Campoletis sonorensis.

Virus was isolated from the lumen of the calyx region of ovaries in the parasitoid wasp Campoletis sonorensis (Hymenoptera: Ichneumonidae), and the nature of the viral DNA was analyzed. DNA purified from a homogeneous band of virus contained double-stranded superhelical molecules which were polydisperse in molecular weight. At least 25 different covalently closed circles were present, ranging in molecular weight from 4.0 x 10(6) to 13.6 x 10(6). The virus DNA was analyzed with restriction enzymes, and the nature of the genetic complexity was evaluated by Southern blot hybridization of native superhelical and relaxed circular virus DNA and of SalI- and HindIII-digested DNA. The data suggest that most of the variously sized covalently closed DNAs were composed primarily of nonhomologous sequences. The different size classes of covalently closed viral DNAs did not appear to exist in equimolar concentrations. However, there was no evidence from observation of virus particles in the electron microscope or from virus fractionation experiments that a mixture of viruses was present in the calyx fluid. The results from this study suggest' that the virus isolated from C. sonorensis, like those isolated from other endoparasitic hymenoptera, may belong to a new class of DNA viruses in which the genome is multipartite, with each DNA existing as a superhelical molecule.

Journal Article↗
PubMed1982

In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus Early and Late mRNAs.

A preliminary translational map of the Autographa californica genome was constructed. Eighteen viral DNA restriction fragments were either purified from agarose gels or obtained from pBR322 recombinant DNA plasmids to locate specific gene products. The DNAs were immobilized on nitrocellulose filters and used to select viral mRNAs isolated from RNA obtained from the cytoplasm of infected Spodoptera frugiperda cells at 21 h postinfection. The fragment-specific mRNAs were translated in vitro in the presence of l-[(3)H]leucine by using a rabbit reticulocyte lysate system and analyzed on sodium dodecyl sulfate-polyacrylamide gels. The approximate locations of 19 A. californica nuclear polyhedrosis virus (AcMNPV) gene products were mapped. The genes for mRNAs present late in viral infection were mapped to DNA fragments that represent nearly the entire genome. The molecular weights of many of these proteins were similar to those present in purified AcMNPV extracellular virus and to proteins being made in infected cells at 18 to 21 h postinfection. Cytoplasmic RNA was isolated at 4 h postinfection from infected cells, a time early in the viral infection cycle, and hybridized to AcMNPV DNA immobilized on nitrocellulose filters. AcMNPV-specific early RNA was translated in vitro into at least six polypeptides, the most abundant having a molecular weight of 39,000. Viral polypeptides were detected in cells pulse-labeled with l-[(3)H]leucine at 3 to 6 h postinfection, with molecular weights similar to those of polypeptides made in vitro from early AcMNPV mRNA.

Journal Article↗