Peter Bromley - Academia.edu (original) (raw)

Papers by Peter Bromley

Research paper thumbnail of A model structure for Rous sarcoma virus genomic RNA and its implications for various functions of the viral RNA

Molecular Biology Reports, 1981

The sequence and structure of RSV genomic RNA leader and gene junctions have been studied. The se... more The sequence and structure of RSV genomic RNA leader and gene junctions have been studied. The secondary structure of the leader and interactions of the gene junctions with a viral protein may well control the functions of RSV-RNA in reverse transcription, translation and processing.

Research paper thumbnail of A transgenic mouse model for the detection of cellular stress induced by toxic inorganic compounds

Nature Biotechnology, 1997

Research paper thumbnail of DNA structure, comprising an activator stimulated promoter for expression of a desired protein, and processes for expression of the protein using the DNA structure

Research paper thumbnail of Methods and Compositions for Expression of Competent Eukaryotic Gene Products

Research paper thumbnail of Method for the in-vivo production and testing of proteins by recombinant gene expression in selected host-cells

Research paper thumbnail of Recombinant Cell Lines for Stress Reporter Assays

Cell Culture Methods for In Vitro Toxicology, 2001

Research paper thumbnail of Production of Secretable Proteins Using the Passage In Vivo as Tumours of Cells Carrying Heat-Inducible Expression Constructs

Research paper thumbnail of Antibody Formation Against Heat-Induced Gene Products Expressed in Animals

Research paper thumbnail of Induction of the human growth hormone gene placed under human hsp70 promoter control in mouse cells: A quantitative indicator of metal toxicity

Cell Biology and Toxicology, 1993

An in vitro test method for general metal toxicity screening was designed, based on the cellular ... more An in vitro test method for general metal toxicity screening was designed, based on the cellular response to stress. The expression of a transfected human growth hormone gene sequence driven by the human heat-shock protein 70 promoter in NIH/3T3 cells was used as marker of noxious contact with metal compounds. Out of a series of 31 metals, 17 were competent for inducing this stress response system. According to the effective concentration and to the intensity of the response, three different clusters of positive compounds emerged and were ranked as strong, intermediate strength and weak inducers. These results correlated well with data from other in vivo and in vitro metal toxicity studies, including LD50 in mice. Apparently the positive/negative compounds also fitted well with data from genotoxicity and carcinogenesis studies on metal salts.

Research paper thumbnail of New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells

Journal of Virology, 1979

The use of mercurated "strong stop" complementary DNA (complementary to the 5'-term... more The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolati...

Research paper thumbnail of Analysis of the secondary and tertiary structures of Rous sarcoma virus RNA

Nucleic Acids Research, 1980

The secondary and tertiary structures of the 35S RNA of Rous Sarcoma virus have been investigated... more The secondary and tertiary structures of the 35S RNA of Rous Sarcoma virus have been investigated. T-, RNase partial digests have been first resolved into their components by gel electrophoresis under non denaturing conditions and then each component analyzed further by various techniques. More than one hundred structured fragments have thus been isolated and shown to consist of several individual nucleotide sequences located far apart on the genome. On the basis of the results,a cloverleaf model for the structure of RSV 35S RNA is proposed that has implications for the various biological functions of this RNA.

Research paper thumbnail of Characterization of the genomic RNA from a Rous sarcoma virus mutant temperature sensitive for cell transformation

Nucleic Acids Research, 1979

We have found that the LA23 t/s mutant of Rous sarcoma virus (phenotype Prague B), even when pass... more We have found that the LA23 t/s mutant of Rous sarcoma virus (phenotype Prague B), even when passaged repeatedly at high multiplicity of infection, does not give rise to transformation defective deletion mutants comparable to those derived from RSV. In view of this fact and of the high rate of production of this mutant at 410C, we have undertaken a detailed analysis of the genome of this virus by ordering all large T1 oligonucleotides and by determining their nucleotide sequences. The results indicate a high degree of mutation in the onc gene as compared to that of Pr-A or Pr-B.

Research paper thumbnail of Spatial and Temporal Control of Transgene Expression Through Ultrasound-Mediated Induction of the Heat Shock Protein 70B Promoter In Vivo

Human Gene Therapy, 2002

Adenoviral vectors have been constructed that express the transgenes luciferase (Adeno-HSP-Luc) o... more Adenoviral vectors have been constructed that express the transgenes luciferase (Adeno-HSP-Luc) or Fas ligand (Adeno-HSP-FasL) under the control of the heat shock protein 70B (hsp70B) promoter. Cultures infected with Adeno-HSP-Luc transiently expressed high levels of luciferase after heat shock. When cultures infected with Adeno-HSP-FasL were maintained at 37°C, no transgene expression was observed, but when cultures were exposed to heat stress, transgene expression resulted in apoptotic cell death. In vivo, transgene expression was induced by ultrasound-mediated heating of adenovirus-infected tissue. In mice or rats injected with the Adeno-HSP-Luc construct, high levels of localized expression of luciferase activity were observed in regions subjected to ultrasound-mediated irradiation. Adeno-HSP-FasL was administered systemically to mice via the tail vein to evaluate safety. Animals receiving Adeno-HSP-FasL in the absence of ultrasound treatment did not display liver toxicity, whereas animals receiving ultrasound treatment to induce the expression of Fas ligand from the hsp70B promoter had significant increases in serum levels of liver enzymes. These data demonstrate that combining the inducible hsp70B promoter with ultrasound induction allows safe local expression of cytotoxic genes with possible therapeutic utility.

Research paper thumbnail of Molecular weight of RNA subunits of Rous sarcoma virus determined by electron microscopy

Journal of Virology, 1975

Secondary cultures of chicken embryo fibroblasts were infected with the Schmidt Ruppin strain of ... more Secondary cultures of chicken embryo fibroblasts were infected with the Schmidt Ruppin strain of Rous sarcoma virus (RSV). Five days after infection, the medium was replaced at 2-h intervals with phosphate-free Eagle medium containing 50 muCi of [32P]orthophosphate per ml. Virus was collected by centrifugation, and the RNA was extracted and denatured with dimethyl sulfoxide, and the 33S subunit RNA was isolated by sucrose gradient centrifugation. The molecular weight of the RSV subunit RNA was determined by length measurement in the electron microscope, by using bacteriophage MS2 RNA as a length marker. Molecules of between 2.5 and 3.3 mum in length made up over 50% of the subunit RNA preparation. In this paper, we define RSV RNA subunits as that RNA released from the 70S RNA complex by dimethyl sulfoxide treatment, which sediments as a peak at 33S. Assuming the molecular weight of MS2 RNA to be 1.2 times 10-6, we calculate the molecular weight of RSV subunit RNA to be 3.12 times 10...

Research paper thumbnail of Quantitative Determination and Location of Newly Synthesized Virus-Specific Ribonucleic Acid in Chicken Cells Infected with Rous Sarcoma Virus

Journal of Virology

A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively ... more A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively labeled avian tumor virus-specific RNA in infected chicken cells has been developed. In our experiments we made use of the fact that DNA synthesized by virions of avian myeloblastosis virus in the presence of actinomycin D (AMV DNA) is complementary to at least 35% of the sequences of 70 S RNA from the Schmidt-Ruppin strain (SRV) of Rous sarcoma virus. Annealing of radioactive RNA (either SRV RNA or RNA extensively purified from SRV-infected chicken cells) with AMV DNA followed by ribonuclease digestion and Sephadex chromatography yielded products which were characterized as avian tumor virus-specific RNA-DNA hybrids by hybridization competition with unlabeled 70 S AMV RNA, equilibrium density-gradient centrifugation in Cs 2 SO 4 gradients, and by analysis of their ribonucleotide composition. The amount of viral RNA synthesized during pulse labeling with 3 H-uridine could be quantitated ...

Research paper thumbnail of Isolation and functional analysis of a human 70,000-dalton heat shock protein gene segment

Proceedings of the …, 1985

Isolation and functional analysis of a human 70,000-dalton heat shock protein gene segment (DNA s... more Isolation and functional analysis of a human 70,000-dalton heat shock protein gene segment (DNA sequence/transient expression/mammalian cells/Xenopus oocytes/hybrid genes)

Research paper thumbnail of MRI-guided ultrasonic heating allows spatial control of exogenous luciferase in canine prostate

The need for efficient and controlled delivery is one of the major obstacles to clinical use of g... more The need for efficient and controlled delivery is one of the major obstacles to clinical use of gene therapy. In this study, we investigated the use of magnetic resonance imaging-monitored ultrasound (US) to induce expression of luciferase after local injection of the construct Ad-HSP-Luc, an adenoviral vector containing a transgene encoding firefly luciferase under the control of the human hsp70B promoter. The hsp promoter allows induction of the associated transgene only in areas that are subsequently heated after infection. US imaging was used to guide the injection of purified virus into both lobes of the prostates of three beagles. At 48 h after injection, the left lobe of the prostate was heated using a 1.5-MHz US transducer driven by a multichannel radiofrequency system and employing an magnetic resonance imaging guidance system. High levels of luciferase expression were observed only in areas exposed to ultrasonic heating. This study demonstrates the feasibility of using ultrasonic heating to control transgene expression spatially using a minimally-invasive approach.

Research paper thumbnail of A model structure for Rous sarcoma virus genomic RNA and its implications for various functions of the viral RNA

Molecular Biology Reports, 1981

The sequence and structure of RSV genomic RNA leader and gene junctions have been studied. The se... more The sequence and structure of RSV genomic RNA leader and gene junctions have been studied. The secondary structure of the leader and interactions of the gene junctions with a viral protein may well control the functions of RSV-RNA in reverse transcription, translation and processing.

Research paper thumbnail of A transgenic mouse model for the detection of cellular stress induced by toxic inorganic compounds

Nature Biotechnology, 1997

Research paper thumbnail of DNA structure, comprising an activator stimulated promoter for expression of a desired protein, and processes for expression of the protein using the DNA structure

Research paper thumbnail of Methods and Compositions for Expression of Competent Eukaryotic Gene Products

Research paper thumbnail of Method for the in-vivo production and testing of proteins by recombinant gene expression in selected host-cells

Research paper thumbnail of Recombinant Cell Lines for Stress Reporter Assays

Cell Culture Methods for In Vitro Toxicology, 2001

Research paper thumbnail of Production of Secretable Proteins Using the Passage In Vivo as Tumours of Cells Carrying Heat-Inducible Expression Constructs

Research paper thumbnail of Antibody Formation Against Heat-Induced Gene Products Expressed in Animals

Research paper thumbnail of Induction of the human growth hormone gene placed under human hsp70 promoter control in mouse cells: A quantitative indicator of metal toxicity

Cell Biology and Toxicology, 1993

An in vitro test method for general metal toxicity screening was designed, based on the cellular ... more An in vitro test method for general metal toxicity screening was designed, based on the cellular response to stress. The expression of a transfected human growth hormone gene sequence driven by the human heat-shock protein 70 promoter in NIH/3T3 cells was used as marker of noxious contact with metal compounds. Out of a series of 31 metals, 17 were competent for inducing this stress response system. According to the effective concentration and to the intensity of the response, three different clusters of positive compounds emerged and were ranked as strong, intermediate strength and weak inducers. These results correlated well with data from other in vivo and in vitro metal toxicity studies, including LD50 in mice. Apparently the positive/negative compounds also fitted well with data from genotoxicity and carcinogenesis studies on metal salts.

Research paper thumbnail of New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells

Journal of Virology, 1979

The use of mercurated "strong stop" complementary DNA (complementary to the 5'-term... more The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolati...

Research paper thumbnail of Analysis of the secondary and tertiary structures of Rous sarcoma virus RNA

Nucleic Acids Research, 1980

The secondary and tertiary structures of the 35S RNA of Rous Sarcoma virus have been investigated... more The secondary and tertiary structures of the 35S RNA of Rous Sarcoma virus have been investigated. T-, RNase partial digests have been first resolved into their components by gel electrophoresis under non denaturing conditions and then each component analyzed further by various techniques. More than one hundred structured fragments have thus been isolated and shown to consist of several individual nucleotide sequences located far apart on the genome. On the basis of the results,a cloverleaf model for the structure of RSV 35S RNA is proposed that has implications for the various biological functions of this RNA.

Research paper thumbnail of Characterization of the genomic RNA from a Rous sarcoma virus mutant temperature sensitive for cell transformation

Nucleic Acids Research, 1979

We have found that the LA23 t/s mutant of Rous sarcoma virus (phenotype Prague B), even when pass... more We have found that the LA23 t/s mutant of Rous sarcoma virus (phenotype Prague B), even when passaged repeatedly at high multiplicity of infection, does not give rise to transformation defective deletion mutants comparable to those derived from RSV. In view of this fact and of the high rate of production of this mutant at 410C, we have undertaken a detailed analysis of the genome of this virus by ordering all large T1 oligonucleotides and by determining their nucleotide sequences. The results indicate a high degree of mutation in the onc gene as compared to that of Pr-A or Pr-B.

Research paper thumbnail of Spatial and Temporal Control of Transgene Expression Through Ultrasound-Mediated Induction of the Heat Shock Protein 70B Promoter In Vivo

Human Gene Therapy, 2002

Adenoviral vectors have been constructed that express the transgenes luciferase (Adeno-HSP-Luc) o... more Adenoviral vectors have been constructed that express the transgenes luciferase (Adeno-HSP-Luc) or Fas ligand (Adeno-HSP-FasL) under the control of the heat shock protein 70B (hsp70B) promoter. Cultures infected with Adeno-HSP-Luc transiently expressed high levels of luciferase after heat shock. When cultures infected with Adeno-HSP-FasL were maintained at 37°C, no transgene expression was observed, but when cultures were exposed to heat stress, transgene expression resulted in apoptotic cell death. In vivo, transgene expression was induced by ultrasound-mediated heating of adenovirus-infected tissue. In mice or rats injected with the Adeno-HSP-Luc construct, high levels of localized expression of luciferase activity were observed in regions subjected to ultrasound-mediated irradiation. Adeno-HSP-FasL was administered systemically to mice via the tail vein to evaluate safety. Animals receiving Adeno-HSP-FasL in the absence of ultrasound treatment did not display liver toxicity, whereas animals receiving ultrasound treatment to induce the expression of Fas ligand from the hsp70B promoter had significant increases in serum levels of liver enzymes. These data demonstrate that combining the inducible hsp70B promoter with ultrasound induction allows safe local expression of cytotoxic genes with possible therapeutic utility.

Research paper thumbnail of Molecular weight of RNA subunits of Rous sarcoma virus determined by electron microscopy

Journal of Virology, 1975

Secondary cultures of chicken embryo fibroblasts were infected with the Schmidt Ruppin strain of ... more Secondary cultures of chicken embryo fibroblasts were infected with the Schmidt Ruppin strain of Rous sarcoma virus (RSV). Five days after infection, the medium was replaced at 2-h intervals with phosphate-free Eagle medium containing 50 muCi of [32P]orthophosphate per ml. Virus was collected by centrifugation, and the RNA was extracted and denatured with dimethyl sulfoxide, and the 33S subunit RNA was isolated by sucrose gradient centrifugation. The molecular weight of the RSV subunit RNA was determined by length measurement in the electron microscope, by using bacteriophage MS2 RNA as a length marker. Molecules of between 2.5 and 3.3 mum in length made up over 50% of the subunit RNA preparation. In this paper, we define RSV RNA subunits as that RNA released from the 70S RNA complex by dimethyl sulfoxide treatment, which sediments as a peak at 33S. Assuming the molecular weight of MS2 RNA to be 1.2 times 10-6, we calculate the molecular weight of RSV subunit RNA to be 3.12 times 10...

Research paper thumbnail of Quantitative Determination and Location of Newly Synthesized Virus-Specific Ribonucleic Acid in Chicken Cells Infected with Rous Sarcoma Virus

Journal of Virology

A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively ... more A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively labeled avian tumor virus-specific RNA in infected chicken cells has been developed. In our experiments we made use of the fact that DNA synthesized by virions of avian myeloblastosis virus in the presence of actinomycin D (AMV DNA) is complementary to at least 35% of the sequences of 70 S RNA from the Schmidt-Ruppin strain (SRV) of Rous sarcoma virus. Annealing of radioactive RNA (either SRV RNA or RNA extensively purified from SRV-infected chicken cells) with AMV DNA followed by ribonuclease digestion and Sephadex chromatography yielded products which were characterized as avian tumor virus-specific RNA-DNA hybrids by hybridization competition with unlabeled 70 S AMV RNA, equilibrium density-gradient centrifugation in Cs 2 SO 4 gradients, and by analysis of their ribonucleotide composition. The amount of viral RNA synthesized during pulse labeling with 3 H-uridine could be quantitated ...

Research paper thumbnail of Isolation and functional analysis of a human 70,000-dalton heat shock protein gene segment

Proceedings of the …, 1985

Isolation and functional analysis of a human 70,000-dalton heat shock protein gene segment (DNA s... more Isolation and functional analysis of a human 70,000-dalton heat shock protein gene segment (DNA sequence/transient expression/mammalian cells/Xenopus oocytes/hybrid genes)

Research paper thumbnail of MRI-guided ultrasonic heating allows spatial control of exogenous luciferase in canine prostate

The need for efficient and controlled delivery is one of the major obstacles to clinical use of g... more The need for efficient and controlled delivery is one of the major obstacles to clinical use of gene therapy. In this study, we investigated the use of magnetic resonance imaging-monitored ultrasound (US) to induce expression of luciferase after local injection of the construct Ad-HSP-Luc, an adenoviral vector containing a transgene encoding firefly luciferase under the control of the human hsp70B promoter. The hsp promoter allows induction of the associated transgene only in areas that are subsequently heated after infection. US imaging was used to guide the injection of purified virus into both lobes of the prostates of three beagles. At 48 h after injection, the left lobe of the prostate was heated using a 1.5-MHz US transducer driven by a multichannel radiofrequency system and employing an magnetic resonance imaging guidance system. High levels of luciferase expression were observed only in areas exposed to ultrasonic heating. This study demonstrates the feasibility of using ultrasonic heating to control transgene expression spatially using a minimally-invasive approach.