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M J Fraser

Publications and source records attributed to M J Fraser.

At least 55 records · Page 3Linked to original sources
PubMed1989

A single, phosphate-repressible deoxyribonuclease, DNase A, secreted in Aspergillus nidulans.

High levels of nuclease activities were identified in filtrates of Aspergillus cultures after growth in low-but not in high-phosphate media. Deoxyribonuclease activities, characterized extensively by column chromatography, showed a coincident single peak for ss- and ds-DNase which was distinct from the peak for RNase. Both ss-DNase and ds-DNase are endonucleolytic and showed the highest activity in the presence of Ca2+ and Mn2+ (at pH 8.0). They also showed identical heat sensitivities suggesting that a single, phosphate-repressible DNase was secreted. This enzyme, therefore, corresponds to the well-characterized extracellular DNase A of Neurospora. However, the Aspergillus DNase A did not cross-react with antisera to secreted Neurospora nucleases and showed different chromatographic properties, and active peptides of different sizes were visualized on DNA activity gels. The increasing derepression of Aspergillus DNase A by decreasing phosphate levels was similar to that of secreted alkaline phosphatase and these increases were both abolished by the regulatory mutant palcA.

Aspergillus nidulans↗
PubMed1989

Expression of human plasminogen cDNA in a baculovirus vector-infected insect cell system.

A cDNA that encodes the human plasminogen (HPg) amino acid sequence has been inserted adjacent to the polyhedrin promoter in the genome of the baculovirus, Autographa californica nuclear polyhedrosis virus, which was then used to infect cultured cells of the farm armyworm, Spodoptera frugiperda. Under the conditions of cell growth employed, recombinant (rec)-HPg was secreted into the medium after 24 h postinfection (p.i.), at which point virtually no rec-HPg antigen remained inside the cells. At 48 h p.i., a maximal level of intact rec-HPg was present in the medium, which underwent substantial proteolytic digestion after that time. The rec-HPg produced by this expression system possessed a molecular weight equivalent to that of plasma [Glu1]-plasminogen. In addition, the rec-HPg adsorbed to Sepharose-lysine, and was eluted with epsilon-aminocaproic acid (EACA). The recombinant protein also interacted with polyclonal antibodies generated to plasma HPg, as well as with a monoclonal antibody directed against a distinct region (kringle 1-3) of the plasma HPg molecule. Finally, the insect-expressed rec-HPg was activatable to plasmin (HPm) by urokinase. The results demonstrate that this expression system produces a full-length functional single-chain rec-HPg, which can be isolated intact from the culture medium, with some consideration for the temporal events that occur in secretion and longer-term degradation of the protein. The fact that this rec-HPg was converted to HPm with a plasminogen activator, and that it interacted with anti-plasma HPg polyclonal and monoclonal antibodies, as well as with the ligand, EACA, indicates that the molecule retains many of its important functional properties and is folded in an integral manner.

Animals↗
PubMed1989

Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses.

The transposable IFP2 element of Trichoplusia ni was originally isolated as a host DNA insertion in spontaneous FP mutants of Galleria mellonella or Autographa californica nuclear polyhedrosis viruses (NPVs). The termini of IFP2 insertions from five independently isolated FP mutants were sequenced. In all cases IFP2 is flanked by 13-bp terminal inverted repeats and has additional inverted repeats of 19 bp in length located asymmetrically with respect to the ends of the element. Insertion of IFP2 into the viral genome always generated a duplication of the tetranucleotide target site, TTAA. There was an apparent preference for insertion within a 12-bp A + T-rich imperfect palindromic sequence surrounding the target site. Sequence analysis of three independent IFP2 elements revealed an internal domain of 2.475 kb containing an RNA polymerase II promoter region and two large open reading frames. Primer extension analysis of IFP2-specific mRNA positioned the 5' terminus of the transcript. The element is present in DNA isolated from T. ni cell lines TN-368 and TN-5B1, but is not apparent in DNAs isolated from the TN-R2 cell line or our laboratory colony of T. ni larvae, suggesting IFP2 was recently introduced into the T. ni genome.

Base Sequence↗
PubMed1989

Transfection of cloned Heliothis zea cell lines with the DNA genome of the Heliothis zea nuclear polyhedrosis virus.

Transfection conditions were optimized for the cloned UND-K derivative of the IPLB-HZ 1075 cell line using the calcium-phosphate co-precipitation technique and the DNA genome of the Heliothis zea S-type nuclear polyhedrosis virus. Optimal efficiencies were obtained using supercoiled viral DNA, and by extending the adsorption period for the diluted precipitate to 12 h. Transfection efficiencies ranging from 0.5 to 1.3 x 10(3) plaque forming units per microgram of supercoiled viral DNA were routinely obtained for UND-K cells and HzS-15 viral DNA. Transfection efficiencies were compared for 10 other cloned Heliothis cell strains and the uncloned parental IPLB-HZ 1075 cell line. The cloned cell strains UND-F, L, and U were incapable of transfection, while UND-I and G were 3 and 131 fold (respectively) less efficient than UND-K. The UND-K cells and the calcium phosphate transfection procedure permit relatively efficient in vitro manipulation of the Heliothis zea NPV virus genome.

Animals↗
PubMed1987

Nuclear endo-exonuclease of Neurospora crassa. Evidence for a role in DNA repair.

The major nuclease activity in nuclei of mycelia of Neurospora crassa has been identified as that of endoexonuclease, an enzyme purified and characterized previously from mitochondria and vacuoles which acts endonucleolytically on single-stranded DNA and RNA and possesses highly processive exonuclease activity with double-stranded DNA. Cross-contamination from the other organelles was eliminated as a source of the activity. Endo-exonuclease of nucleoplasm, chromatin, and nuclear matrix showed 80-100% cross-reaction with antisera raised to purified extranuclear endoexonuclease and was also strongly inhibited by 20 microM aurin tricarboxylic acid. In addition, it yielded some of the same-sized polypeptides on activity gel analysis. Nuclei also contained immunochemically cross-reactive trypsin-activable endo-exonuclease activity, a form of enzyme that was shown previously to occur in high amounts in the cytosol and in a tightly bound form associated with the mitochondrial inner membrane. Pretreatment of wild-type mycelia for 1 h with 4-16 micrograms/ml the DNA-damaging agent, 4-nitroquinoline-1-oxide (4-NQO), which caused about 50-80% growth inhibition, resulted in a dose-dependent loss of up to 80% of inactive endo-exonuclease from nuclei. At low doses of 4-NQO, this was accompanied by increases in the level of active enzyme. Nuclei of the DNA repair-deficient uvs-3 mutant were found to contain only 12% of the active enzyme and about 32% of inactive enzyme as that in wild-type nuclei. Mycelial growth of this mutant was 10 times more sensitive to 4-NQO than the wild-type. At a dose which resulted in equivalent growth inhibition, 4-NQO had no effect on the level of active endo-exonuclease in uvs-3 nuclei and caused an increase (over 30%) in the level of inactive enzyme. These data are consistent with a role of endo-exonuclease in the repair of nuclear DNA.

4-Nitroquinoline-1-oxide↗
PubMed1987

Intraspecific and interspecific variation in the sequence and abundance of highly repeated DNA among mosquitoes of the Aedes albopictus subgroup.

Interspecific variation in abundance of highly repeated DNA sequences has been examined in three species of the Aedes scutellaris and three species of the A. albopictus subgroup of the A. scutellaris group. Sequences from a population of A. albopictus were hybridised with whole genomic content from other species and strains. Copy number estimates were determined by dot-blot hybridisation. Variation in sequence abundance between strains of A. albopictus was as great as between it and the other six species. Two clusters were formed by principal components analysis, each of which contains populations of A. albopictus. Copy numbers of highly repeated sequences do not correlate with genome size. The results indicate extensive sequence divergence and rapid evolution. These findings are discussed in relation to the importance of concerted evolution and natural selection in the evolution of the species group.

Aedes↗
PubMed1987

Characterization of genotypic and phenotypic variation in plaque-purified strains of HzSNPV Elkar isolate.

Twenty plaque-purified strains of Heliothis zea S-type nuclear polyhedrosis virus (HzSNPV) were characterized based upon restriction endonuclease digests of viral DNAs, structural protein profiles, and larval melanization response. Each of the 20 strains had a unique genotype. Genomic heterogeneity between strains was localized to 4 regions of the HzSNPV genome. Differences in protein profiles of occluded virus were noted for each strain relative to the wild-type isolate. The strains were separated into 3 groups based upon differences in relative virulence and rate of larval melanization upon death. There was no correlation between differences in pathology and specific alterations in either genotype or structural proteins. The genotype of strain HzS-15 was extensively characterized. Physical maps were constructed for the HzS-15 strain and all of the plaque-purified strains.

Animals↗
PubMed1986

An immunochemical study of Neurospora nucleases.

Nucleases derived from Neurospora crassa mycelia with neutral single-strand (ss) endodeoxyribonuclease activity have been examined by immunochemical techniques and by sodium dodecyl sulfate - DNA gel electrophoresis. All of the intracellular nucleases, which have different divalent metal ion requirements, different strand specificities with single- and double-strand DNA, different modes of action on DNA and RNA, and other distinguishing characteristics, are immunochemically related to Neurospora endo-exonuclease. The evidence indicates that these enzymes are derived from one or more related large, inactive (precursor?) polypeptides that are first converted to 75- to 80-kdalton active polypeptide(s) which are very protease sensitive. Further limited proteolysis results in the production of the various active forms of nuclease studied here. Some proteolytic conversions may occur in a controlled manner in vivo in different cell compartments, but others are very likely artifacts resulting from uncontrolled proteolysis during extraction and isolation. The intracellular forms of Neurospora endo-exonuclease are immunologically cross-active with ss-DNA-binding nucleases isolated from Aspergillus nidulans and Saccharomyces cerevisiae. They are not immunochemically related to two extracellular Neurospora nucleases, the pancreatic DNase-I-like DNase A and a ss-specific exonuclease, and they are also not related to other fungal and plant nucleases with ss-specific endonuclease activity such as the S1 nuclease of Aspergillus oryzae, the P1 nuclease of Penicillium citrinum, and mung bean nuclease.

Antibodies↗
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↗
PubMed1983

Purification and properties of single strand DNA-binding endo-exonuclease of Neurospora crassa.

Single strand DNA-binding endo-exonucleases purified from mitochondria, vacuoles, or a mixture of these organelles had the same high specific single strand DNase activity (910 mumol of nucleotides/min/mg), and each contained a polypeptide of Mr = 31,000-33,000 which was found to be active by sodium dodecyl sulfate-DNA-gel electrophoresis. The properties of the three preparations were identical in all respects tested. The enzyme showed distributive endonuclease activity with single strand DNA, but processive exonuclease activity with double strand DNA. In the former case, 5'-phosphoryl-terminated fragments were released at early times, while in the latter case, short 5'-oligonucleotides (n = 2-4) were released. Both activities were dependent on Mg2+ (or Mn2+), but to different extents. In 0.1 mM Mg2+, superhelical bacteriophage phi X174 (replicative form (RF II) DNA and, at converted to relaxed circular (RF II) DNA and, at higher enzyme concentrations, to unit length linear (RF III) DNA. In 10 mM Mg2+, these same conversions took place rapidly, and the RF III DNA which formed was degraded to pieces shorter than unit length. At very low enzyme concentrations, long single strand tails and gaps were detected in bacteriophage T7 linear double strand DNA molecules.

Chromatography, Affinity↗
PubMed1983

Intracellular localization of Neurospora crassa endo-exonuclease and its putative precursor.

Endo-exonuclease of rapidly growing mycelia of Neurospora crassa was found to be distributed in a ratio of about 1.6:1 in vacuoles and in mitochondria where it is associated with the inner membrane. Although the activity in vacuoles was readily released by osmotic shock, very little of that in mitochondria was released by this method. The mitochondrial activity was partially (60 to 70%) released by sonication, and the remaining activity was solubilized in the presence of Triton X-100. An inactive form of endo-exonuclease, activated in vitro by treatment with trypsin, is present in mycelia at a level over four times that of active enzyme. It was found to be distributed in a ratio of about 2.5:1 in the cytosol and in the inner membrane of mitochondria. The mitochondrial protein was more tightly bound than the active enzyme. Very little of the inactive enzyme was released by sonication, but it was solubilized in the presence of Triton X-100. The intracellular distribution of active and inactive forms of endo-exonuclease differs in a mutagen-sensitive mutant of Neurospora crassa (uvs-3) which shows many pleiotropic effects. The most striking difference in distribution is in the mitochondria where endo-exonuclease is present almost entirely in the inactive form at a level 30% higher than in wild-type mitochondria.

Cell Nucleus↗
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

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

Inactivation of bacteriophage lambda and lambda DNA by nitrogen mustard.

Bacteriophage lambda and lambda DNA were treated with alkylating agents. The survival of phage was assayed by infectivity and that of DNA by infectivity of phage particles assembled from the DNA in vitro. Phage lambda were more sensitive to nitrogen mustard (C1(CH2)2NMe(CH2)2C1; HN2) than was lambda DNA. The inactivation of lambda DNA was biphasic; the second component of the inactivation was sensitive to mutations allelic for recA, polA and uvrB. This behaviour was not shown by pBR322 plasmid DNA treated with HN2 nor by lambda DNA treated with monofunctional alkylating agents (or HN2 if the second alkylation reaction was stopped by addition of a mercaptan). From Arrhenius plots, the activation energy for the reactions with DNA and interact phage were found to be different. The activation energy for the inactivation of intact phage was the same as that (measured independently) for the predominant reaction (or class of reactions) in which HN2 cross-links DNA to protein in lambda particles. From these data we conclude that the inactivation of lambda by HN2 is due, primarily, to DNA-protein cross-linking. The implications for the mode of action of DNA-reactive bifunctional anti-viral and cytotoxic compounds are discussed.

Antiviral Agents↗
PubMed1982

Condensed DNA structures derived from bacteriophage heads.

Bacteriophage lambda particles were rendered osmotically fragile by incubation, spread over hypophase and examined by electron microscopy. When water was used as hypophase, condensed structures were released from the phage heads and treatment of these with cytochrome c or several alternative proteins resulted in the release of free, relaxed DNA. Phage were pretreated with nitrogen mustard, a bifunctional alkylating agent; when the condensed structures from such phage particles were treated with protein, DNA was released in small supercoiled domains. This confirmed a previous finding that bacteriophage DNA has a supercoiled topology and suggests that the winding pattern of DNA in the phage might involve small domains of coiled DNA analogous to nucleosomes. Such a conformation could be consistent with other studies on the arrangement of DNA in phage heads if the domains have parallel axes.

Bacteriophage lambda↗
PubMed1981

Liposome-mediated genetic transformation of Neurospora crassa.

Transformation of Neurospora crassa spheroplasts is reported for three different genes, using uncloned Neurospora DNA, both naked and encapsulated in synthetic phosphatidylserine liposomes. Whereas transformation by naked DNA is DNase-sensitive, that by liposomes is not. Per unit of transforming DNA, liposome transformation is significantly more efficient than that with naked DNA, ranging from 19x for the am gene to 41x for pyr-3. Levels of activity of pyr-3 and am transformants, and segregation data on pyr-3 transformation are given.

DNA, Fungal↗