Lee Chatfield - Academia.edu (original) (raw)
Papers by Lee Chatfield
Journal of Bacteriology, Dec 1, 1982
The sog gene of the Inclot group plasmid ColIb is known to encode a DNA primase that can substitu... more The sog gene of the Inclot group plasmid ColIb is known to encode a DNA primase that can substitute for defective host primase in dnaG mutants of Escherichia coli during discontinuous DNA replication. The biological significance of this enzyme was investigated by using sog mutants, constructed from a derivative of ColIb by in vivo recombination of previously defined mutations in a cloned sog gene. The resultant Sogplasmids failed to specify detectable primase activity and were unable to suppress a dnaG lesion. These mutants were maintained stably in E. coli, implying that the enzyme is not involved in vegetative replication of ColIb. However, the Sog-plasmids were partially transfer deficient in E. coli and Salmonella typhimurium matings, consistent with the hypothesis that the normal physiological role of this enzyme is in conjugation. This was confirmed by measurements of conjugal DNA synthesis. Studies of recipient cells have indicated that plasmid primase is required to initiate efficient synthesis of DNA complementary to the transferred strand, with the protein being supplied by the donor parent and probably transmitted between the mating cells. Primase specified by the dnaG gene of the recipient can substitute partially for the mutant enzyme, thus providing an explanation for the partial transfer proficiency of the mutant plasmids. Conjugal DNA synthesis in dnaB donor cells was deficient in the absence of plasmid primase, implying that the enzyme also initiates synthesis of DNA to replace the transferred material. During bacterial conjugation mediated by plasmids of the la incompatibility group, which includes ColIb, R144, and R64, a specific strand of the plasmid is transferred between the mating cells (26). DNA transfer is normally associated with synthesis of a replacement strand in the donor cell and a complementary strand in the recipient. Since DNA polymerases require a primer terminus to initiate DNA synthesis on a template (17), a primer-generating mechanism is required to initiate conjugal DNA synthesis. Until now, studies of conjugation have concentrated on the possible involvement of the defined Escherichia coli priming systems. These involve either RNA polymerase (8, 13) or primase, the rifampin-resistant product of the dnaG gene (22, 27). On certain templates, primase acts in conjunction with the primosome, a multiprotein complex that includes dnaB protein (1). The nature of the primer-generating mechanism required for conjugal synthesis of an la plasmid in the donor cell is unresolved. The 3' terminus of the strand undergoing transfer might be used, as proposed in the original rolling circle model (14). However, initiation of transfer re
T i t l e ROLE OF PLASMID C0LIb-P9 DNA PRIMASE by Lee K. C h a tf ie ld The sog locus of ColIb-P9... more T i t l e ROLE OF PLASMID C0LIb-P9 DNA PRIMASE by Lee K. C h a tf ie ld The sog locus of ColIb-P9 i s known to encode a DNA prim ase th a t g en e ra te s RNA prim ers fo r DNA sy n th e s is on a v a r ie ty o f DNA tem p la tes and i t promotes b a c te r ia l DNA r e p l ic a t io n in p rim ase -d e fec tiv e (dna03) m utants of E sc h e rich ia c o l i K-12. The th e s i s re p o r ts th a t the p h y s io lo g ic a l ro le o f the enzyme i s in co n ju g a tiv e m etabolism of plasm id DNA. D e riv a tiv e s of C olIb-P9drd-1 c a rry in g defin ed sog m utations were c o n s tru c te d by j j i vivo recom bination . The mutant plasm ids were m ain tained s ta b ly , showing th a t the prim ase i s in e s s e n t ia l fo r v e g e ta tiv e DNA r e p l ic a t io n , but they were d e f ic ie n t in tra n sco n ju g a n t fo rm ation during b a c te r ia l m ating. Amounts of co n ju g a tiv e DNA sy n th e s is in m atings in v o lv in g m utants and complementing plasm ids impl...
Forensic Science Policy & Management: An International Journal, 2010
In May 2009, the University of Central Lancashire, UK, launched TRACES (Taphonomic Research in An... more In May 2009, the University of Central Lancashire, UK, launched TRACES (Taphonomic Research in Anthropology-Centre for Experimental Study). This facility uses animal models in taphonomic research. The use of animal models facilitates wider studies of factors influencing decomposition than the low replicate numbers common to human cadaver studies. The establishment of dedicated facilities to carry out taphonomic research is long and complex. Whether the facility uses human cadavers, as in the United States, or animal models, as here in the UK, the issues that arise can be common to both. These include commitment of resources, local community concerns and planning and legal issues. However, the use of animal models also raises additional ethical and legislative concerns. These include environmental impact, animal welfare, bio-security and disposal activities. This article discusses the processes undertaken during the establishment of a taphonomic facility using animal models in the UK and demonstrates the level of commitment, enthusiasm and perseverance required.
Biochemical Society Transactions, 1995
Biochemical Society Transactions, 1995
Mol Gen Genet, 1984
DNA primase of ColIb-P9drd-1 generates RNA primers that are thought to initiate DNA synthesis on ... more DNA primase of ColIb-P9drd-1 generates RNA primers that are thought to initiate DNA synthesis on the conjugatively transferred strand of the plasmid. To examine whether plasmid-specified primase is transferred during conjugation, we exploited the property of the enzyme to promote bacterial DNA replication in dnaG (primase-defective) mutants of Escherichia coli. It was found that dnaG3 recipient cells, treated with rifampicin to inhibit transcription, recovered ability to synthesise bacterial DNA by a process requiring an active plasmid primase gene in donor cells and a functional conjugation system. A non-transferable primase gene in the donor strain complemented a primase-negative derivative of ColIb-P9drd-1, confirming that the enzyme responsible for recovery was supplied by donor cells. The implication is that certain proteins are transmitted from donor cells to promote conjugative metabolism of plasmid DNA in the recipients.
Oncogene, 2002
Activated ras is known to dysregulate TGF-b signaling by altering the expression of TGF-b type II... more Activated ras is known to dysregulate TGF-b signaling by altering the expression of TGF-b type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-b signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 mM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 mM concentrations (P40.004). The TGF-b effector gene p21 waf1/cip1 was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-b responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-b signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT -PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-b signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.
Microbial Ecology, May 1, 1997
Nickel-resistant bacteria were isolated from effluent discharged from a sewage treatment outfall ... more Nickel-resistant bacteria were isolated from effluent discharged from a sewage treatment outfall over an 18-month period. One of these strains, Enterobacter cloacae FBA30, was found to harbor a narrow host range conjugative plasmid, designated pFBA30, which confers nickel resistance on its host. A 10.2-kb SstI restriction fragment was cloned from pFBA30 and was shown to specify inducible nickel resistance, both in its original host and in laboratory strains of Escherichia coli.
Biologist (London, England)
The promise of gene therapy has yet to be realised and a recent death has cast a long shadow over... more The promise of gene therapy has yet to be realised and a recent death has cast a long shadow over clinical trials. Will gene therapy develop into the magic bullet of the 21st Century or is it a technology too complicated to succeed?
Biologist (London, England)
Gene therapy has the potential to cure currently incurable conditions, including some cancers and... more Gene therapy has the potential to cure currently incurable conditions, including some cancers and inherited disorders. It might even be used in the womb to prevent congenital abnormalities. The potential was greeted with great excitement ten years ago, when gene therapy first appeared to be viable, but little progress is perceived. Just how close are we to solving the obstacles in the way of successful gene implantation / replacement?
Journal of Bacteriology
Structural genes for catechol 2,3-oxygenase (C230) were cloned from the TOL plasmids pWW5, pWW14,... more Structural genes for catechol 2,3-oxygenase (C230) were cloned from the TOL plasmids pWW5, pWW14, pWW74, pWW84, and pWW88 isolated fro*n Pseudomonas strains of diverse geographical origins. Each pKT230-based C230+ recombinant plasmid carried a 2.05-kilobase XhoI insert which showed strong homology in Southern hybridizations with the xyLE gene from the archetype TOL plasmid pWWO. Fragments were mapped for restriction endonuclease sites and were classified into two closely related groups on the basis of restriction maps. C230 structural genes were located on cloned fragments by a combination of subcloning and site-specific mutagenesis. All five TOL plasmids examined yielded clones whose maps differed from that of xyLE of pWWO by only a single XbaI site, but in addition plasmids pWW5, pWW74, and pWW88 carried a second, homologous C230 gene with seven further restriction site differences. The remaining plasmids, pWW14 and pWW84, carried a second nonhomologous C230 gene related to the second C230 gene (C2301) of TOL plasmid pWW15 described previously (H. Keil, M. R. Lebens, and P. A. Williams, J. Bacteriol. 163:248&255, 1985). Thus, each naturally occurring TOL plasmid in this study appears to carry genes for two meta cleavage dioxygenases.
Biochemical Society transactions, 1995
Biologist (London, England), 2002
Gene therapy has the potential to cure currently incurable conditions, including some cancers and... more Gene therapy has the potential to cure currently incurable conditions, including some cancers and inherited disorders. It might even be used in the womb to prevent congenital abnormalities. The potential was greeted with great excitement ten years ago, when gene therapy first appeared to be viable, but little progress is perceived. Just how close are we to solving the obstacles in the way of successful gene implantation / replacement?
Plasmids in Bacteria, 1985
Oncogene, 2002
Activated ras is known to dysregulate TGF-b signaling by altering the expression of TGF-b type II... more Activated ras is known to dysregulate TGF-b signaling by altering the expression of TGF-b type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-b signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 mM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 mM concentrations (P40.004). The TGF-b effector gene p21 waf1/cip1 was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-b responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-b signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT -PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-b signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.
MGG Molecular & General Genetics, 1984
DNA primase of ColIb-P9drd-1 generates RNA primers that are thought to initiate DNA synthesis on ... more DNA primase of ColIb-P9drd-1 generates RNA primers that are thought to initiate DNA synthesis on the conjugatively transferred strand of the plasmid. To examine whether plasmid-specified primase is transferred during conjugation, we exploited the property of the enzyme to promote bacterial DNA replication in dnaG (primase-defective) mutants of Escherichia coli. It was found that dnaG3 recipient cells, treated with rifampicin to inhibit transcription, recovered ability to synthesise bacterial DNA by a process requiring an active plasmid primase gene in donor cells and a functional conjugation system. A non-transferable primase gene in the donor strain complemented a primase-negative derivative of ColIb-P9drd-1, confirming that the enzyme responsible for recovery was supplied by donor cells. The implication is that certain proteins are transmitted from donor cells to promote conjugative metabolism of plasmid DNA in the recipients.
Molecular and Cellular Biochemistry, 2014
Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress co... more Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress conditions, such as cancer and hypoxia. This study investigated the level of HSPA gene expression in human cell lines exposed to hypoxic conditions. Three human glioma cell lines were selected for this study, each representing different types of glioma (astrocytoma, oligodendroglioma and glioblastoma), with a normal human astrocyte cell line used as a control. HSPA RNA transcripts and proteins were examined in these samples using qRT-PCR, immunofluorescence and flow cytometry techniques. The average HSPA mRNA copy numbers detected in three glioma cell lines were approximately sixfold higher than in a normal astrocyte cell line. The expression of HSPA was induced in normal cell lines immediately after exposure to hypoxia with 33% of cells exhibiting expression. However, the effects of hypoxia on gene expression were marginal in glioma cells, due to the already increased levels of HSPA with both pre- and post-hypoxia samples showing expression in approximately 90% of cells. These results show that whilst the stress caused by both cancer and hypoxia induce HSPA expression the underlying imprint of tumourgenesis leads to sustained expression.
Molecular and Cellular Biochemistry, 2014
Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress co... more Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress conditions, such as cancer and hypoxia, with production also being linked to tumourigenesis. HSPA mRNA transcripts and proteins were examined in three human glioma cell lines, representing astrocytoma, oligodendroglioma and glioblastoma, plus 18 clinical brain tissue samples. GAPDH was used as a control gene throughout these studies and exhibited a consistent level of expression in a normal astrocyte cell line, tumourous cell lines and tissue samples. In contrast, the average HSPA mRNA copy numbers detected in glioblastoma tissue were between 1.8- and 8.8-fold higher than in lower grade glioma and control tissue, respectively, which is suggestive of a grade-related transcription profile. Similar patterns of grade-related expression were also observed in glioma cell lines. This study indicates for the first time that HSPA expression in glioma cells may possibly be grade related, and hence could have potential as a prognostic marker.
Journal of Bacteriology, Dec 1, 1982
The sog gene of the Inclot group plasmid ColIb is known to encode a DNA primase that can substitu... more The sog gene of the Inclot group plasmid ColIb is known to encode a DNA primase that can substitute for defective host primase in dnaG mutants of Escherichia coli during discontinuous DNA replication. The biological significance of this enzyme was investigated by using sog mutants, constructed from a derivative of ColIb by in vivo recombination of previously defined mutations in a cloned sog gene. The resultant Sogplasmids failed to specify detectable primase activity and were unable to suppress a dnaG lesion. These mutants were maintained stably in E. coli, implying that the enzyme is not involved in vegetative replication of ColIb. However, the Sog-plasmids were partially transfer deficient in E. coli and Salmonella typhimurium matings, consistent with the hypothesis that the normal physiological role of this enzyme is in conjugation. This was confirmed by measurements of conjugal DNA synthesis. Studies of recipient cells have indicated that plasmid primase is required to initiate efficient synthesis of DNA complementary to the transferred strand, with the protein being supplied by the donor parent and probably transmitted between the mating cells. Primase specified by the dnaG gene of the recipient can substitute partially for the mutant enzyme, thus providing an explanation for the partial transfer proficiency of the mutant plasmids. Conjugal DNA synthesis in dnaB donor cells was deficient in the absence of plasmid primase, implying that the enzyme also initiates synthesis of DNA to replace the transferred material. During bacterial conjugation mediated by plasmids of the la incompatibility group, which includes ColIb, R144, and R64, a specific strand of the plasmid is transferred between the mating cells (26). DNA transfer is normally associated with synthesis of a replacement strand in the donor cell and a complementary strand in the recipient. Since DNA polymerases require a primer terminus to initiate DNA synthesis on a template (17), a primer-generating mechanism is required to initiate conjugal DNA synthesis. Until now, studies of conjugation have concentrated on the possible involvement of the defined Escherichia coli priming systems. These involve either RNA polymerase (8, 13) or primase, the rifampin-resistant product of the dnaG gene (22, 27). On certain templates, primase acts in conjunction with the primosome, a multiprotein complex that includes dnaB protein (1). The nature of the primer-generating mechanism required for conjugal synthesis of an la plasmid in the donor cell is unresolved. The 3' terminus of the strand undergoing transfer might be used, as proposed in the original rolling circle model (14). However, initiation of transfer re
T i t l e ROLE OF PLASMID C0LIb-P9 DNA PRIMASE by Lee K. C h a tf ie ld The sog locus of ColIb-P9... more T i t l e ROLE OF PLASMID C0LIb-P9 DNA PRIMASE by Lee K. C h a tf ie ld The sog locus of ColIb-P9 i s known to encode a DNA prim ase th a t g en e ra te s RNA prim ers fo r DNA sy n th e s is on a v a r ie ty o f DNA tem p la tes and i t promotes b a c te r ia l DNA r e p l ic a t io n in p rim ase -d e fec tiv e (dna03) m utants of E sc h e rich ia c o l i K-12. The th e s i s re p o r ts th a t the p h y s io lo g ic a l ro le o f the enzyme i s in co n ju g a tiv e m etabolism of plasm id DNA. D e riv a tiv e s of C olIb-P9drd-1 c a rry in g defin ed sog m utations were c o n s tru c te d by j j i vivo recom bination . The mutant plasm ids were m ain tained s ta b ly , showing th a t the prim ase i s in e s s e n t ia l fo r v e g e ta tiv e DNA r e p l ic a t io n , but they were d e f ic ie n t in tra n sco n ju g a n t fo rm ation during b a c te r ia l m ating. Amounts of co n ju g a tiv e DNA sy n th e s is in m atings in v o lv in g m utants and complementing plasm ids impl...
Forensic Science Policy & Management: An International Journal, 2010
In May 2009, the University of Central Lancashire, UK, launched TRACES (Taphonomic Research in An... more In May 2009, the University of Central Lancashire, UK, launched TRACES (Taphonomic Research in Anthropology-Centre for Experimental Study). This facility uses animal models in taphonomic research. The use of animal models facilitates wider studies of factors influencing decomposition than the low replicate numbers common to human cadaver studies. The establishment of dedicated facilities to carry out taphonomic research is long and complex. Whether the facility uses human cadavers, as in the United States, or animal models, as here in the UK, the issues that arise can be common to both. These include commitment of resources, local community concerns and planning and legal issues. However, the use of animal models also raises additional ethical and legislative concerns. These include environmental impact, animal welfare, bio-security and disposal activities. This article discusses the processes undertaken during the establishment of a taphonomic facility using animal models in the UK and demonstrates the level of commitment, enthusiasm and perseverance required.
Biochemical Society Transactions, 1995
Biochemical Society Transactions, 1995
Mol Gen Genet, 1984
DNA primase of ColIb-P9drd-1 generates RNA primers that are thought to initiate DNA synthesis on ... more DNA primase of ColIb-P9drd-1 generates RNA primers that are thought to initiate DNA synthesis on the conjugatively transferred strand of the plasmid. To examine whether plasmid-specified primase is transferred during conjugation, we exploited the property of the enzyme to promote bacterial DNA replication in dnaG (primase-defective) mutants of Escherichia coli. It was found that dnaG3 recipient cells, treated with rifampicin to inhibit transcription, recovered ability to synthesise bacterial DNA by a process requiring an active plasmid primase gene in donor cells and a functional conjugation system. A non-transferable primase gene in the donor strain complemented a primase-negative derivative of ColIb-P9drd-1, confirming that the enzyme responsible for recovery was supplied by donor cells. The implication is that certain proteins are transmitted from donor cells to promote conjugative metabolism of plasmid DNA in the recipients.
Oncogene, 2002
Activated ras is known to dysregulate TGF-b signaling by altering the expression of TGF-b type II... more Activated ras is known to dysregulate TGF-b signaling by altering the expression of TGF-b type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-b signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 mM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 mM concentrations (P40.004). The TGF-b effector gene p21 waf1/cip1 was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-b responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-b signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT -PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-b signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.
Microbial Ecology, May 1, 1997
Nickel-resistant bacteria were isolated from effluent discharged from a sewage treatment outfall ... more Nickel-resistant bacteria were isolated from effluent discharged from a sewage treatment outfall over an 18-month period. One of these strains, Enterobacter cloacae FBA30, was found to harbor a narrow host range conjugative plasmid, designated pFBA30, which confers nickel resistance on its host. A 10.2-kb SstI restriction fragment was cloned from pFBA30 and was shown to specify inducible nickel resistance, both in its original host and in laboratory strains of Escherichia coli.
Biologist (London, England)
The promise of gene therapy has yet to be realised and a recent death has cast a long shadow over... more The promise of gene therapy has yet to be realised and a recent death has cast a long shadow over clinical trials. Will gene therapy develop into the magic bullet of the 21st Century or is it a technology too complicated to succeed?
Biologist (London, England)
Gene therapy has the potential to cure currently incurable conditions, including some cancers and... more Gene therapy has the potential to cure currently incurable conditions, including some cancers and inherited disorders. It might even be used in the womb to prevent congenital abnormalities. The potential was greeted with great excitement ten years ago, when gene therapy first appeared to be viable, but little progress is perceived. Just how close are we to solving the obstacles in the way of successful gene implantation / replacement?
Journal of Bacteriology
Structural genes for catechol 2,3-oxygenase (C230) were cloned from the TOL plasmids pWW5, pWW14,... more Structural genes for catechol 2,3-oxygenase (C230) were cloned from the TOL plasmids pWW5, pWW14, pWW74, pWW84, and pWW88 isolated fro*n Pseudomonas strains of diverse geographical origins. Each pKT230-based C230+ recombinant plasmid carried a 2.05-kilobase XhoI insert which showed strong homology in Southern hybridizations with the xyLE gene from the archetype TOL plasmid pWWO. Fragments were mapped for restriction endonuclease sites and were classified into two closely related groups on the basis of restriction maps. C230 structural genes were located on cloned fragments by a combination of subcloning and site-specific mutagenesis. All five TOL plasmids examined yielded clones whose maps differed from that of xyLE of pWWO by only a single XbaI site, but in addition plasmids pWW5, pWW74, and pWW88 carried a second, homologous C230 gene with seven further restriction site differences. The remaining plasmids, pWW14 and pWW84, carried a second nonhomologous C230 gene related to the second C230 gene (C2301) of TOL plasmid pWW15 described previously (H. Keil, M. R. Lebens, and P. A. Williams, J. Bacteriol. 163:248&255, 1985). Thus, each naturally occurring TOL plasmid in this study appears to carry genes for two meta cleavage dioxygenases.
Biochemical Society transactions, 1995
Biologist (London, England), 2002
Gene therapy has the potential to cure currently incurable conditions, including some cancers and... more Gene therapy has the potential to cure currently incurable conditions, including some cancers and inherited disorders. It might even be used in the womb to prevent congenital abnormalities. The potential was greeted with great excitement ten years ago, when gene therapy first appeared to be viable, but little progress is perceived. Just how close are we to solving the obstacles in the way of successful gene implantation / replacement?
Plasmids in Bacteria, 1985
Oncogene, 2002
Activated ras is known to dysregulate TGF-b signaling by altering the expression of TGF-b type II... more Activated ras is known to dysregulate TGF-b signaling by altering the expression of TGF-b type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-b signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 mM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 mM concentrations (P40.004). The TGF-b effector gene p21 waf1/cip1 was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-b responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-b signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT -PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-b signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.
MGG Molecular & General Genetics, 1984
DNA primase of ColIb-P9drd-1 generates RNA primers that are thought to initiate DNA synthesis on ... more DNA primase of ColIb-P9drd-1 generates RNA primers that are thought to initiate DNA synthesis on the conjugatively transferred strand of the plasmid. To examine whether plasmid-specified primase is transferred during conjugation, we exploited the property of the enzyme to promote bacterial DNA replication in dnaG (primase-defective) mutants of Escherichia coli. It was found that dnaG3 recipient cells, treated with rifampicin to inhibit transcription, recovered ability to synthesise bacterial DNA by a process requiring an active plasmid primase gene in donor cells and a functional conjugation system. A non-transferable primase gene in the donor strain complemented a primase-negative derivative of ColIb-P9drd-1, confirming that the enzyme responsible for recovery was supplied by donor cells. The implication is that certain proteins are transmitted from donor cells to promote conjugative metabolism of plasmid DNA in the recipients.
Molecular and Cellular Biochemistry, 2014
Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress co... more Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress conditions, such as cancer and hypoxia. This study investigated the level of HSPA gene expression in human cell lines exposed to hypoxic conditions. Three human glioma cell lines were selected for this study, each representing different types of glioma (astrocytoma, oligodendroglioma and glioblastoma), with a normal human astrocyte cell line used as a control. HSPA RNA transcripts and proteins were examined in these samples using qRT-PCR, immunofluorescence and flow cytometry techniques. The average HSPA mRNA copy numbers detected in three glioma cell lines were approximately sixfold higher than in a normal astrocyte cell line. The expression of HSPA was induced in normal cell lines immediately after exposure to hypoxia with 33% of cells exhibiting expression. However, the effects of hypoxia on gene expression were marginal in glioma cells, due to the already increased levels of HSPA with both pre- and post-hypoxia samples showing expression in approximately 90% of cells. These results show that whilst the stress caused by both cancer and hypoxia induce HSPA expression the underlying imprint of tumourgenesis leads to sustained expression.
Molecular and Cellular Biochemistry, 2014
Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress co... more Production of heat shock protein 70 (HSP70/HSPA) is induced by a wide range of cellular stress conditions, such as cancer and hypoxia, with production also being linked to tumourigenesis. HSPA mRNA transcripts and proteins were examined in three human glioma cell lines, representing astrocytoma, oligodendroglioma and glioblastoma, plus 18 clinical brain tissue samples. GAPDH was used as a control gene throughout these studies and exhibited a consistent level of expression in a normal astrocyte cell line, tumourous cell lines and tissue samples. In contrast, the average HSPA mRNA copy numbers detected in glioblastoma tissue were between 1.8- and 8.8-fold higher than in lower grade glioma and control tissue, respectively, which is suggestive of a grade-related transcription profile. Similar patterns of grade-related expression were also observed in glioma cell lines. This study indicates for the first time that HSPA expression in glioma cells may possibly be grade related, and hence could have potential as a prognostic marker.