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M J Fraser

Publications and source records attributed to M J Fraser.

At least 37 records · Page 2Linked to original sources
PubMed1996

Endo-exonuclease of human leukaemic cells: evidence for a role in apoptosis.

Inactive forms of endo-exonuclease, activated in vitro by treatment with trypsin, have been identified in human leukaemic CEM and MOLT-4 cells. They comprise over 95% of the total single-strand DNase activity in nuclei and are mainly bound to chromatin and the nuclear matrix. The activated enzyme had Mg2+(Mn2+)-dependent, Ca(2+)-stimulated activities with single- and double-strand DNAs and RNA (polyriboadenylic acid) and other properties characteristic of endo-exonucleases previously described. At least twice as much inactive endo-exonuclease has also been localised in extranuclear compartments of CEM and MOLT-4 cells, 85% bound to the membranes of the endoplasmic reticulum and 15% free in the cytosol. The soluble cytosolic trypsin-activatable endo-exonuclease was immunoprecipitated by antibodies raised independently to both Neurospora and monkey CV-1 cell endo-exonucleases. The free and bound enzymes of both nuclear and extranuclear compartments also cross-reacted on immunoblots with the antibody raised to Neurospora endo-exonuclease to reveal multiple polypeptides ranging in size from 18 to 145 kDa, many of which exhibited activity on DNA gels. The major species bound to the chromatin/matrix were in the 55-63 kDa range. Limited proteolysis of the large polypeptides to those of 18 to 46 kDa accompanied spontaneous chromatin DNA fragmentation to form DNA "ladders' in an isolated nuclei/cytosol system. When the leukaemic cells were treated in culture with either etoposide or podophyllotoxin to induce apoptosis, the largest polypeptides disappeared and smaller endo-exonuclease-related polypeptides of 18 to 46 kDa were detected in the nuclear extracts. The appearance of these polypeptides also correlated with extensive chromatin DNA fragmentation. In addition, there were correlations between the depletion of the major 55-63 kDa species bound to the membranes of the endoplasmic reticulum, depletion of the extranuclear trypsin-activatable activity and the onset and extent of chromatin DNA fragmentation in both cell lines. The extranuclear 55-63 kDa species may be precursors of the chromatin/matrix bound endo-exonuclease. The results indicate that endo-exonuclease plays a role in chromatin DNA degradation in mammalian cells during apoptosis.

Apoptosis↗
PubMed1995

A novel chemokine, macrophage inflammatory protein-related protein-2, inhibits colony formation of bone marrow myeloid progenitors.

A new member of the beta-chemokine family, macrophage inflammatory protein (MIP)-related protein-2 (MRP-2) was isolated from a murine macrophage cell line, RAW 264.7. MRP-2 is composed of 122 amino acids of which the first 21 residues constitute a putative signal sequence. The putative mature protein is composed of 101 amino acids with a molecular weight of 11,600. MRP-2 is structurally similar to MIP-related protein-1 (MRP-1) (C10) and MIP-1 alpha. MRP-2 shows a 50.8% sequence identity at the protein level to MRP-1 and 46.3% identity to MIP-1 alpha. MRP-2 detects approximately 1.3 kilobase mRNA from monocyte and macrophage cell lines but does not detect the mRNA from T and B cells. The MRP-2 gene termed Scya9 was mapped to the central region of mouse chromosome 11 near the Scya1 and Scya2 genes, which are also members of the beta-chemokine superfamily. The Scya gene cluster was located between neurofibromatosis type 1 (Nf1) and myeloperoxidase (Mpo). rMRP-2 significantly suppressed colony formation by murine and human bone marrow granulocyte-macrophage (CFU-granulocyte-macrophage), erythroid (burst-forming unit-E), and multipotential (CFU-granulocyte-erythroid-macrophage-megakaryocyte) progenitor cells stimulated by combinations of growth factors.

Amino Acid Sequence↗
PubMed1995

Assay for movement of Lepidopteran transposon IFP2 in insect cells using a baculovirus genome as a target DNA.

Mutagenesis of baculoviruses by host mobile elements occurs spontaneously and frequently during propagation of the viruses in Lepidopteran cell cultures. Most of the transposons identified as insertions in baculovirus genomes are relatively small Class II elements that exhibit a remarkable specificity for TTAA target sites. We have developed a transposition assay to analyze the movement of these TTAA-specific Lepidopteran transposons using the baculovirus genome as a target and a lacZ gene under control of the polyhedrin gene promoter as a selective marker for the transposon. This assay provides the first demonstration that a Lepidopteran transposon is capable of transposing while carrying a marker gene in insect cells. The data generated from this assay provide strong evidence that IFP2 encodes a protein that facilitates its own movement. This element may be used in a manner analogous to the P-element to mobilize genes in at least some Lepidopteran insect cells. Transposon tagging within the baculovirus genome identified several known genes and two previously undescribed open reading frames as nonessential to in vitro replication of the virus.

Animals↗
PubMed1994

Endo-exonucleases: enzymes involved in DNA repair and cell death?

Endo-exonucleases from E. coli to man, although very different proteins, are multifunctional enzymes with similar enzymatic activities. They probably have two common but opposing biological roles. On the one hand, they promote survival of the organism by acting in recombination and recombinational DNA repair to diversify and help preserve the genome intact. On the other hand, they degrade the genomic DNA when it is damaged beyond repair. This ensures elimination of heavily mutagenized cells from the population.

Animals↗
PubMed1993

Cloning and sequence analysis of a p40 structural protein gene of Helicoverpa zea nuclear polyhedrosis virus.

A gene encoding an occluded virion structural protein was isolated from an expression library constructed from the Helicoverpa zea S-type nuclear polyhedrosis virus (NPV) isolate HzS-15 using both polyclonal and monoclonal antibody screening. The gene was located within a Pstl-Sall fragment of the HzS-15 genome spanning from 96.5 to 97.3 m.u. Sequencing analyses revealed a long open reading frame of 927 nucleotides that predicted a protein of 37 kDa in size. Immunoblot analyses using the monoclonal antibody ENV409 demonstrated that the gene corresponded to a 40-kDa protein (p40) in SDS-polyacrylamide gels that was present exclusively in enveloped occluded virions but not in extracellular budded virions or envelope-stripped nucleocapsids. A p40 protein-specific transcript was detected at 16 hr postinfection in HzS-15-infected Hz 1075/UND-K cells and remained until 22 hr. Primer extension analyses demonstrated that the p40 protein-specific transcript started at -49 nucleotides from the ATG start codon within an ATAAG consensus pentamer found in late protein genes of Baculoviruses. The deduced amino acid sequence of the HzS-15 p40 protein gene shared 44 and 45% sequence homology with the p40 proteins of Bombyx mori NPV and Autographa californica NPV, respectively.

Amino Acid Sequence↗
PubMed1993

TTAA serves as the target site for TFP3 lepidopteran transposon insertions in both nuclear polyhedrosis virus and Trichoplusia ni genomes.

We have analysed TFP3 transposable elements from five independently isolated FP mutants of the Autographa californica nuclear polyhderosis virus (AcMNPV). We also analysed genomic copies of TFP3 elements amplified from the DNAs of the Trichoplusia ni cell line (TN-368) and T. ni larvae using the polymerase chain reaction (PCR). The sequences of all the newly isolated TFP3 elements closely resemble the previously described TFP3/1 element. Each of the transposons isolated from the virus mutants duplicated a TTAA tetranucleotide target site upon insertion into the viral genome. Four of these TFP3 elements transposed into three different 'TTAA' target sites within the 25 K gene (FP locus, map units 36-37 of AcMNPV). The fifth TFP3 element inserted at a 'TTAA' site within the AcMNPV Hin dIII-E fragment. One genomic TFP3 element, amplified from the TN-368 cell line DNA by an inverse PCR method, duplicated a 'TTAA' tetranucleotide target site that is present only once in the homologous larval DNA sequence. These data suggest that mobilization of TFP3 into both viral and cellular sites is identical in specificity and mechanism.

Animals↗
PubMed1992

Docking of dogs.

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Animal Welfare↗
PubMed1992

Expression of foreign epitopes on recombinant occlusion bodies of baculoviruses.

Recombinant polyhedrin proteins of the baculovirus Autographa californica nuclear polyhedrosis virus were constructed to serve as immunologic carriers of foreign epitopes. Several recombinants containing an influenza haemagglutinin epitope were obtained. Three of the five recombinants formed occlusion bodies (OBs) and two did not. All of the recombinant polyhedrin proteins reacted with a monoclonal antibody (mAb) specific for an influenza epitope in Western blots. Presentation of the foreign epitope on the surface of recombinant OBs was demonstrated by specific immunoprecipitation of the anti-influenza mAb with recombinant OBs. The recombinant polyhedrin-influenza epitope fusion protein stimulated an influenza-specific immune response in rabbits.

Animals↗
PubMed1991

Expression of two forms of carp gonadotropin alpha subunit in insect cells by recombinant baculovirus.

There are two types of cDNA clones (designated alpha 1 and alpha 2) encoding the alpha subunit of carp gonadotropin. These two cDNAs are derived from different genes and encode proteins that differ by seven amino acid residues (three in the signal peptide and four in the mature polypeptide). Expression of these two cDNAs in insect cells by recombinant baculovirus revealed that the alpha 1 subunit, after noncovalent association with the beta subunit, has the same potency as the native alpha subunit purified from the pituitary. In contrast, the alpha 2 subunit can associate with the beta subunit, but only to form an inactive gonadotropin. Competition of the alpha 2 subunit with the alpha 1 subunit for association with the beta subunit decreases the gonadotropin activity of the alpha/beta complex. In addition, both alpha 1 and alpha 2 subunits are secreted into the culture medium by insect cells and have an apparent molecular mass approximately 5 kDa higher than that of the native alpha subunit. These results indicate that the insect cell-derived alpha 1 subunit is biologically active and that those four amino acid changes in the mature of alpha 2 protein affect the biological activity and thus provide valuable clues for the study of the structure-function relationship of the alpha subunit of glycoprotein hormones.

Animals↗
PubMed1991

A functional hepatitis B virus X protein produced in insect cells.

We have constructed a recombinant baculovirus from Autographa californica nuclear polyhedrosis virus, called AcX, that expresses the gene encoding the hepatitis B virus X protein in infected Spodoptera frugiperda (Sf21AE) insect cells. A 16.5-kDa monomer and a 33-kDa dimer of the X protein were detected in extracts from AcX-infected cells on immunoblots using a polyclonal anti-X antibody. The biological activity of the insect cell-produced X protein was assayed by fusing AcX-infected Sf21AE cells with African green monkey kidney (CV-1) cells and then transfecting the fused cells with the reporter plasmid pSV2cat. The expression of the cat gene in CV-1:Sf21AE(AcX) fusions was seven times higher than that derived from CV-1:Sf21AE(AcMNPV) fusions, indicating that the insect cell-produced X protein was functional. The transactivation function of the insect cell-produced X protein was also verified by scrape-loading nuclear extracts of AcX-infected Sf21AE cells into pSV2cat-transfected CV-1 cells. Treatment of the AcX-infected cell nuclear extracts with anti-X antisera prior to scrape-loading eliminated the transactivating activity of the extracts. We conclude that the insect cell-produced X protein was functionally identical to that generated in mammalian cells.

Animals↗
PubMed1991

Direct double-stranded DNA sequencing with baculovirus genomes.

Double-stranded DNA sequencing with the modified T7 DNA polymerase (Sequenase) was performed directly with nuclear polyhedrosis virus DNA genomes. The conditions were optimized to allow for a rapid and unambiguous sequence analysis of nuclear polyhedrosis virus genomes.

Animals↗
PubMed1990

Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells.

A comparison has been made between the Asn289-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed in lepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteins by glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, we find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate (Man9GlcNAc2) and truncated high-mannose oligosaccharide (Man5GlcNAc2, Man4GlcNAc2, and Man3GlcNAc2). Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This is the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. Our data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexosaminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, we conclude that amino acid sequences and/or protein conformational properties can control oligosaccharide processing events.

Animals↗
PubMed1990

Neurospora endo-exonuclease is immunochemically related to the recC gene product of Escherichia coli.

Immunochemical cross-reaction between the endo-exonuclease of Neurospora crassa, an enzyme previously implicated in recombination and recombinational DNA repair, and the recC-encoded polypeptide of Escherichia coli has been detected by immunoblotting extracts of strains of E. coli having a deletion that includes the recBCD genes but carrying multicopy plasmids bearing all three of the recBCD genes or only one or two of these genes. It was predicted that homology would also be found at the amino acid sequence level between the recC polypeptide and both nuclear and mitochondrial endo-exonucleases of Saccharomyces cerevisiae, which cross-react with antibodies raised to the N. crassa endo-exonuclease. Since the gene for the S. cerevisiae mitochondrial enzyme, NUC1, has been cloned and sequenced and the predicted amino acid sequence is known, this sequence was aligned with the predicted amino acid sequence of the recC polypeptide. Extensive homology was found by aligning 306 of the 329 amino acids of the yeast mitochondrial nuclease sequence with the carboxy-terminal one-quarter of the amino acid sequence of the recC polypeptide.

Amino Acid Sequence↗
PubMed1990

Endo-exonuclease of Aspergillus nidulans.

Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5'-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical phi X-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5'-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.

Aspergillus nidulans↗
PubMed1989

Transposon mutagenesis of baculoviruses: analysis of TFP3 lepidopteran transposon insertions at the FP locus of nuclear polyhedrosis viruses.

We report the complete sequences of two representatives of the TFP3 transposable element family of the lepidopteran, Trichoplusia ni. These elements were isolated as insertions mobilized from the Lepidopteran host genome into two closely related nuclear polyhedrosis viruses (NPV) during infection. Both elements inserted within the 500-bp FP locus of the respective viral genomes (map units 36.0 to 37.0), causing a distinctive plaque morphology phenotype and the loss of a 25-kDa viral-specific protein. Both insertions occurred at the identical TTAA target site in the respective genomes, in the same relative orientation, and are flanked by 15-bp imperfect inverted repeats. The inserted elements interrupt the 25K open reading frame (ORF). One of these FP mutants undergoes spontaneous reversion. Sequence analysis at the excision site of a spontaneous revertant demonstrates that the TFP3 elements are capable of precise excision, restoring the expression of the 25-kDa protein. We also compare the sequences of the 25K genes of the Autographa californica and Galleria mellonella viruses (AcMNPV and GmMNPV, respectively). The 25K gene sequences diverge in five areas, resulting in an additional EcoRV and TaqI site within the GmMNPV 25K gene, and extension of the ORF for an additional 2 amino acids at the C-terminus of the predicted GmMNPV 25 kDa protein. The phenomenon of transposon mutagenesis of Baculovirus genomes provides a unique opportunity for analysis of transposon mobility.

Amino Acid Sequence↗
PubMed1989

The actions of Neurospora endo-exonuclease on double strand DNAs.

Neurospora crassa endo-exonuclease, an enzyme implicated in recombinational DNA repair, was found previously to have a distributive endonuclease activity with a high specificity for single strand DNA and a highly processive exonuclease activity. The activities of endo-exonuclease on double strand DNA substrates have been further explored. Endo-exonuclease was shown to have a low bona fide endonuclease activity with completely relaxed covalently closed circular DNA and made site-specific breaks in linear double strand DNA at a low frequency while simultaneously generating a relatively high level of single strand breaks (nicks) in the DNA. Sequencing at nicks induced by endo-exonuclease in pBR322 restriction fragments showed that the highest frequency of nicking occurred at the mid-points of two sites with the common sequence, p-AGCACT-OH. In addition, sequencing revealed less frequent nicking at identical or homologous hexanucleotide sequences in all other 54 cases examined where these sequences either straddled the break site itself or were within a few nucleotides on either side of the break site. The exonucleolytic action of endo-exonuclease on linear DNA showed about 100-fold preference for acting in the 5' to 3' direction. Removal of the 5'-terminal phosphates substantially reduced this activity, internal nicking, and the ability of endo-exonuclease to generate site-specific double strand breaks. On the other hand, nicking of the dephosphorylated double strand DNA with pancreatic DNase I stimulated the exonuclease activity by almost 5-fold, but no stimulation was observed when the DNA was nicked by Micrococcal nuclease. Thus, 5'-p termini either at double strand ends or at nicks in double strand DNA are entry points to the duplex from which endo-exonuclease diffuses linearly or "tracks" in the 5' to 3' direction to initiate its major endo- and exonucleolytic actions. The results are interpreted to show how it is possible for endo-exonuclease to generate single strand DNA for switching into a homologous duplex either at a nick or while remaining bound at a double strand break in the DNA. Such mechanisms are consistent with current models for recombinational double strand break repair in eukaryotes.

Base Composition↗