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Biomedical subjects

J A Fyfe

Publications and source records attributed to J A Fyfe.

At least 19 recordsLinked to original sources
PubMed2000

The genome of Neisseria gonorrhoeae retains the remnants of a two-component regulatory system that once controlled piliation.

An intact activator-binding site upstream of the sigma(54) promoter of the pilin-encoding pilE gene of Neisseria gonorrhoeae suggests gonococci produce a protein capable of binding this sequence. We cloned a chimeric gene, rsp, that has sequence similarity to both the pilS and pilR genes of Pseudomonas aeruginosa encoding a two-component regulatory system that controls piliation. This gene is transcribed in N. gonorrhoeae and indirect evidence suggests that Rsp binds to the activator-binding site of the pilE gene. Despite this, mutation of rsp has no effect on piliation in N. gonorrhoeae, suggesting that the remnants of this regulatory system have persisted in the genome, despite the loss of its original function.

Bacterial Proteins↗
PubMed1998

Neisseria gonorrhoeae contains multiple copies of a gene that may encode a site-specific recombinase and is associated with DNA rearrangements.

A 960-bp ORF potentially encoding a site-specific recombinase has been cloned from Neisseria gonorrhoeae MS11-A. This ORF was designated pivNg on the basis of similarity of the deduced amino acid sequence to the Piv proteins of Moraxella spp. that are site-specific invertases. Southern hybridization and sequence analysis revealed that there were multiple copies of pivNg sequence within the genomes of N. gonorrhoeae strains tested, but not in several other neisserial species. Southern hybridization and sequence analysis further suggested that pivNg sequences may be associated with genomic rearrangements.

Amino Acid Sequence↗
PubMed1998

Cobalamin inhibition of HIV-1 integrase and integration of HIV-1 DNA into cellular DNA.

Our prior studies showed that certain cobalamins inhibit productive HIV-1 infection of primary cultures of blood lymphocytes and monocytes. We demonstrate here that this antiviral activity may be mediated by an inhibition of HIV-1 integrase, an enzyme required for productive infection. Purified recombinant HIV-1 integrase activity was inhibited in vitro by hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl), adenosylcobalamin (Ado-Cbl), and dicyanocobinamide (CN2-Cbi) with IC50 values of approximately 17, 17, 17, and 4 microM, respectively. The agents inhibited HIV-1 infection of cultured monocytes (IC50 values for OH-Cbl, Me-Cbl, Ado-Cbl, and CN2-Cbi of 6, 7, 4, and 1 microM, respectively) and of cultured lymphocytes (IC50 values of 60, 50, 60, and 11 microM, respectively). Experiments using cultured monocytes or lymphocytes demonstrated that OH-Cbl inhibited integration of HIV-1 DNA into cellular DNA. Thus, cobalamins and cobinamides represent novel inhibitors of HIV-1 integrase. These or related agents may be useful as anti-viral treatments that target HIV-1 integrase.

Base Sequence↗
PubMed1998

Absence of an SOS-like system in Neisseria gonorrhoeae.

The DNA repair capacities of Neisseria gonorrhoeae have not been well characterised, however, it is known that the gonococcus possesses an excision repair system. The fact that genes in this system are part of the SOS regulon in Escherichia coli prompted this investigation into the transcriptional regulation of genes involved in DNA repair in N. gonorrhoeae. Northern (RNA-DNA) dot blot hybridisation was used to investigate potential DNA damage-mediated induction of the gonococcal recA, uvrA and uvrB genes. In contrast to the situation in E. coli, transcription of these genes in N. gonorrhoeae was not induced in response to treatment with methyl methanesulfonate (MMS) and UV light. These data indicated that the gonococcus does not possess an SOS-like system that is induced in response to DNA damage.

Adenosine Triphosphatases↗
PubMed1998

The pathogenic neisseriae contain an inactive rpoN gene and do not utilize the pilE sigma54 promoter.

The sigma54 promoter (P3) upstream of the pilE gene in Neisseria gonorrhoeae was shown to be non-functional by transcriptional analysis of a PpilE::lacZ fusion containing only P3. A region on the chromosome of N. gonorrhoeae strain MS11-A was identified that potentially encodes a protein with a significant similarity to the Escherichia coli RpoN protein. However, this region (designated RLS for rpoN-like sequence) does not contain a single open reading frame (ORF) capable of encoding a functional RpoN protein. It appears that RLS may have arisen from an ancestral rpoN homologue that underwent a deletion removing the sequence encoding the essential helix-turn-helix (HTH) motif, and changing the subsequent reading frame. An RLS has been identified in several strains of N. gonorrhoeae and N. meningitidis. A 90-kDa gonococcal protein has previously been shown to react with a monoclonal antibody raised against the RpoN from Salmonella typhimurium. However, mutagenesis and Western blot analysis confirmed that the gene encoding this protein is not contained within RLS.

Amino Acid Sequence↗
PubMed1998

An AT-rich tract containing an integration host factor-binding domain and two UP-like elements enhances transcription from the pilEp1 promoter of Neisseria gonorrhoeae.

The pilE gene of Neisseria gonorrhoeae is transcribed from a sigma70 promoter (pilEp1) with an AT-rich tract extending 65 nucleotides upstream of the -35 box. Within this region is an integration host factor (IHF)-binding core consensus sequence. We have performed a detailed analysis to determine which upstream sequences are required for efficient transcription from pilEp1 in N. gonorrhoeae. Deletion of sequences upstream of the AT-rich tract had no effect on the level of transcription. However, the IHF-binding core consensus sequence and the AT-rich sequence further upstream were both required for enhanced levels of transcription from this promoter in both N. gonorrhoeae and an Escherichia coli strain producing IHF. In addition, an UP-like element positioned between the -35 box and the IHF-binding site was required for maximal transcription. The AT-rich region upstream of the IHF-binding core consensus sequence can also act as an UP-like element when appropriately repositioned upstream of the -35 box.

Adenine↗
PubMed1997

The normally silent sigma54 promoters upstream of the pilE genes of both Neisseria gonorrhoeae and Neisseria meningitidis are functional when transferred to Pseudomonas aeruginosa.

The pilE gene encodes the pilin subunit in Neisseria gonorrhoeae and Neisseria meningitidis. Transcriptional analysis of promoters upstream of pilE in N. gonorrhoeae has been described previously (Fyfe et al. (1995) J. Bacteriol. 177, 3781-3787). Transcription from the sigma54-dependent promoter P3 was detected in Pseudomonas aeruginosa. Here we show that this transcription is dependent on the P. aeruginosa transcriptional activator PilR, and a specific upstream sequence with a high degree of similarity to the PilR-binding site found upstream of the P. aeruginosa pilin gene. This implies there is an upstream activator site (UAS) present 5' of pilE. Sequencing upstream of the N. meningitidis MC58 c2 pilE gene shows this region to be very similar to that in N. gonorrhoeae. P3 and the UAS are conserved, although insertions were noted on either side of the UAS. Transcriptional analysis has shown that the N. meningitidis P3 promoter is used in P. aeruginosa, provided PilR and an upstream region that includes sequence similar to the UAS are present. Transcription from the N. meningitidis PpilE is stronger than from the N. gonorrhoeae equivalent. N. meningitidis uses the sigma70 promoter P1 to transcribe pilE.

Bacterial Adhesion↗
PubMed1997

Cloning, nucleotide sequence and transcriptional analysis of the uvrA gene from Neisseria gonorrhoeae.

A recombinant plasmid capable of restoring UV resistance to an Escherichia coli uvrA mutant was isolated from a genomic library of Neisseria gonorrhoeae. Sequence analysis revealed an open reading frame whose deduced amino acid sequence displayed significant similarity to those of the UvrA proteins of other bacterial species. A second open reading frame (ORF259) was identified upstream from, and in the opposite orientation to the gonococcal uvrA gene. Transcriptional fusions between portions of the gonococcal uvrA upstream region and a reporter gene were used to localise promoter activity in both E. coli and N. gonorrhoeae. The transcriptional starting points of uvrA and ORF259 were mapped in E. coli by primer extension analysis, and corresponding sigma70 promoters were identified. The arrangement of the uvrA-ORF259 intergenic region is similar to that of the gonococcal recA-aroD intergenic region. Both contain inverted copies of the 10 bp neisserial DNA uptake sequence situated between divergently transcribed genes. However, there is no evidence that either the uptake sequence or the proximity of the promoters influences expression of these genes.

Adenosine Triphosphatases↗
PubMed1997

Transcriptional analysis of the groESL operon of Neisseria gonorrhoeae.

The nucleotide sequence upstream of the groEL gene of Neisseria gonorrhoeae has been determined. Upstream of groEL is a homolog of groES and the divergently transcribed frpB gene. The promoter region of groES lacks the inverted repeat sequences (IR) that act as a regulatory element controlling the expression of similar operons in many other bacterial species. This region contains overlapping consensus sequences for sigma 32-dependent and sigma 70-dependent promoters, and an appropriately placed transcription start point was mapped downstream of these promoters. Northern hybridization demonstrated that synthesis of a full-length groES-groEL transcript was induced in heat-stressed cells. These experiments also revealed the presence of a shorter groES-specific transcript, apparently the result of the premature termination of transcription at an IR situated between the groES and groEL genes.

Amino Acid Sequence↗
PubMed1995

Mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine against herpesviruses.

The activity, metabolism, and mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) were studied. Compared to acyclovir (ACV), H2G has superior activity against VZV (50% inhibitory concentration of 2.3 microM) and Epstein-Barr virus (50% inhibitory concentration of 0.9 microM), comparable activity against HSV-1, and weaker activity against HSV-2. The antiviral effect on HSV-1 showed persistence after removal of compound. H2G was metabolized to its mono-, di- and triphosphate derivatives in virus-infected cells, with H2G-triphosphate being the predominant product. Only small amounts of H2G-triphosphate were detected in uninfected cells (1 to 10 pmol/10(6) cells), whereas the level in HSV-1-infected cells reached 1,900 pmol/10(6) cells. H2G was a substrate for all three viral thymidine kinases and could also be phosphorylated by mitochondrial deoxyguanosine kinase. The intracellular half-life of H2G-triphosphate varied in uninfected (2.5 h) and infected (HSV-1, 14 h; VZV, 3.7 h) cells but was always longer than the half-life of ACV-triphosphate (1 to 2 h). H2G-triphosphate inhibited HSV-1, HSV-2, and VZV DNA polymerases competitively with dGTP (Ki of 2.8, 2.2, and 0.3 microM, respectively) but could not replace dGTP as a substrate in a polymerase assay. H2G was not an obligate chain terminator but would only support limited DNA chain extension. Only very small amounts of radioactivity, which were too low to be identified by high-performance liquid chromatography analysis of the digested DNA, could be detected in purified DNA from uninfected cells incubated with [3H]H2G. Thus, H2G acts as an anti-herpesvirus agent, particularly potent against VZV, by formation of high concentrations of relatively stable H2G-triphosphate, which is a potent inhibitor of the viral DNA polymerases.

Antiviral Agents↗
PubMed1995

The pilE gene of Neisseria gonorrhoeae MS11 is transcribed from a sigma 70 promoter during growth in vitro.

Type 4 pili are essential for virulence in Neisseria gonorrhoeae. The gonococcal pilin subunit is encoded by pilE, upstream of which three putative promoter sequences (P1, P2, and P3) have been identified. P1 and P2 are sigma 70-like promoters and are functional when a PpiE::cat transcriptional fusion is expressed in Escherichia coli DH5 alpha. P3 is sigma 54 dependent and overlaps the P1 sequence. Site-directed mutagenesis of the pilE promoters followed by transcriptional analysis in E. coli indicated that in the absence of an appropriate activator protein, binding of RNA polymerase-sigma 54 to P3 inhibits transcription from P1 on the order of 30-fold. Transcription from P3 was undetectable in E. coli. However, PilR-dependent, P3-associated expression was detected in Pseudomonas aeruginosa PAK containing a PpilE::cat fusion, with P3 the only intact promoter. A similar analysis was performed on gonococcal reporter strains containing wild-type and mutated PpilE::cat cassettes recombined into the chromosome. In such piliated gonococcal recombinants cultured in vitro, P1 was responsible for cat expression and almost certainly for transcription of pilE. Transcription from P2 and P3 was not detectable under these conditions. Inhibition of transcription from P1 by sigma 54 binding to P3 was not apparent in N. gonorrhoeae MS11-A, suggesting that sigma 54 was either absent or unable to bind to P3 in these cells.

Bacterial Proteins↗
PubMed1995

A promoter associated with the neisserial repeat can be used to transcribe the uvrB gene from Neisseria gonorrhoeae.

A recombinant plasmid capable of restoring UV resistance to an Escherichia coli uvrB mutant was isolated from a genomic library of Neisseria gonorrhoeae. Sequence analysis revealed an open reading frame whose deduced amino acid sequence displayed significant similarity to those of the UvrB proteins of E. coli, Micrococcus luteus, and Streptococcus pneumoniae. A gonococcal uvrB mutant was constructed and found to be extremely sensitive to UV radiation. Transcriptional fusions between portions of the gonococcal uvrB upstream region and a reporter gene were used to localize promoter activity, and the transcriptional start point of the gonococcal uvrB gene was mapped in E. coli by primer extension. A corresponding sigma 70 promoter was identified within a copy of the 26-bp neisserial repeat, and this identification provided the first evidence of a promoter associated with this repetitive element in N. gonorrhoeae.

Amino Acid Sequence↗
PubMed1993

Varicella-zoster virus thymidine kinase. Characterization and substrate specificity.

The varicella-zoster virus (VZV) thymidine kinase (TK) EC 2.7.2.21) catalyzes the phosphorylation of many anti-VZV nucleosides. Purified, bacterially expressed VZV TK was characterized with regard to N-terminal amino acid sequence, pI value, pH optimum, metal ion requirement, phosphate donor and acceptor specificity, and inhibition by dTTP. Initial velocities of thymidine phosphorylation with variable MgATP concentrations fit a two-site model with apparent Km values for MgATP of 0.10 and 900 microM. dTTP was a noncompetitive inhibitor of thymidine phosphorylation but was competitive with MgATP. Phosphate donor and acceptor specificities of the bacterially expressed enzyme were indistinguishable from those of VZV TK purified from infected cells. Detailed studies of the nucleoside specificity with the bacterially expressed enzyme showed that, for a given sugar moiety, thymine nucleosides were the most efficient substrates followed by nucleosides of cytosine, uracil, adenine, and with some exceptions, guanine. For a given pyrimidine or purine (except guanine), 2'-deoxyribonucleosides were the most efficient substrates, followed by arabinosides, ribonucleosides, 2',3'-dideoxyribonucleosides, and the acyclic moiety of acyclovir.

Adenosine Triphosphate↗
PubMed1993

Control of gonococcal pilin-encoding gene expression in Escherichia coli.

The pilE gene from Neisseria gonorrhoeae, unlike other type-4 pilin-encoding genes, is well expressed in Escherichia coli. Two putative promoters have been implicated in the transcription of this gene. Besides the -24/-12 promoter used to transcribe type-4 pilin-encoding genes in most species, the consensus sequence for a conventional promoter is also present. The two promoters overlap and would have almost identical transcription start points (tsp). Transcription from a -24/-12 promoter should be abolished in an E. coli rpoN mutant. A recombinant plasmid carrying pilE could not be transformed into such a mutant, apparently because the synthesis of the N-terminal hydrophobic domain of pilin is lethal to the rpoN mutant. This suggests that pilE is expressed at a higher level in an rpoN mutant than it is in a wild-type (wt) strain of E. coli. This suggestion was confirmed by constructing fusions between the pilE promoter region and a promoter-less cat gene. We suggest that the conventional promoter is primarily responsible for the transcription of pilE, but that the binding of the RpoN sigma factor partially represses transcription of this gene in wt strains. In an rpoN mutant, the repression is removed and transcription occurs at a level that is lethal to the mutant host.

Bacterial Outer Membrane Proteins↗
PubMed1992

The anti-hepatitis B virus activities, cytotoxicities, and anabolic profiles of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.

The anti-hepatitis B (anti-HBV) activities of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (2'-deoxy-3'-thia-5-fluorocytosine [FTC]) were studied by using an HBV-transfected cell line (HepG2 derivative 2.2.15, subclone P5A). The (-) isomer was found to be a potent inhibitor of viral replication, with an apparent 50% inhibitory concentration of 10 nM, while the (+) isomer was found to be considerably less active. Both isomers showed minimal toxicity to HepG2 cells (50% inhibitory concentration, > 200 microM) and showed minimal toxicity in the human bone marrow progenitor cell assay. In accord with the cellular antiviral activity data, the 5'-triphosphate of (-)-FTC inhibited viral DNA synthesis in an endogenous HBV DNA polymerase assay, while the 5'-triphosphate of the (+) isomer was inactive. Unphosphorylated (-)-FTC did not inhibit product formation in the endogenous assay, suggesting that the antiviral activity of the compound is dependent on anabolism to the 5'-triphosphate. Both (-)- and (+)-FTC were anabolized to the corresponding 5'-triphosphates in chronically HBV-infected HepG2 cells. The rate of accumulation and the steady-state concentration of the 5'-triphosphate of (-)-FTC were greater. Also, (-)-FTC was not a substrate for cytidine deaminase and, therefore, is not subject to deamination and conversion to an inactive uridine analog. The (+) isomer is, however, a good substrate for cytidine deaminase.

Animals↗
PubMed1992

Human immunodeficiency virus type 1 integration protein: DNA sequence requirements for cleaving and joining reactions.

Using purified integration protein (IN) from human immunodeficiency virus (HIV) type 1 and oligonucleotide mimics of viral and target DNA, we have investigated the DNA sequence specificity of the cleaving and joining reactions that take place during retroviral integration. The first reaction in this process is selective endonucleolytic cleaving of the viral DNA terminus that generates a recessed 3' OH group. This 3' OH group is then joined to a 5' phosphoryl group located at a break in the target DNA. We found that the conserved CA located close to the 3' end of the plus strand of the U5 viral terminus (also present on the minus strand of the U3 terminus) was required for both cleaving and joining reactions. Six bases of HIV U5 or U3 DNA at the ends of model substrates were sufficient for nearly maximal levels of selective endonucleolytic cleaving and joining. However, viral sequence elements upstream of the terminal 6 bases could also affect the efficiencies of the cleaving and joining reactions. The penultimate base (C) on the minus strand of HIV U5 was required for optimal joining activity. A synthetic oligonucleotide mimic of the putative in vivo viral "DNA" substrate for HIV IN, a molecule that contained a terminal adenosine 5'-phosphate (rA) on the minus strand, was indistinguishable in the cleaving and joining reactions from the DNA substrate containing deoxyadenosine instead of adenosine 5'-phosphate at the terminal position. Single-stranded DNA served as an in vitro integration target for HIV IN. The DNA sequence specificity of the joining reaction catalyzed in the reverse direction was also investigated.

Acquired Immunodeficiency Syndrome↗
PubMed1991

6-Methoxypurine arabinoside as a selective and potent inhibitor of varicella-zoster virus.

Seven 6-alkoxypurine arabinosides were synthesized and evaluated for in vitro activity against varicella-zoster virus (VZV). The simplest of the series, 6-methoxypurine arabinoside (ara-M), was the most potent, with 50% inhibitory concentrations ranging from 0.5 to 3 microM against eight strains of VZV. This activity was selective. The ability of ara-M to inhibit the growth of a variety of human cell lines was at least 30-fold less (50% effective concentration, greater than 100 microM) than its ability to inhibit the virus. Enzyme studies suggested the molecular basis for these results. Of the seven 6-alkoxypurine arabinosides, ara-M was the most efficient substrate for VZV-encoded thymidine kinase as well as the most potent antiviral agent. In contrast, it was not detectably phosphorylated by any of the three major mammalian nucleoside kinases. Upon direct comparison, ara-M was appreciably more potent against VZV than either acyclovir or adenine arabinoside (ara-A). However, in the presence of an adenosine deaminase inhibitor, the arabinosides of adenine and 6-methoxypurine were equipotent but not equally selective; the adenine congener had a much less favorable in vitro chemotherapeutic index. Again, this result correlated with data from enzyme studies in that ara-A, unlike ara-M, was a substrate for two mammalian nucleoside kinases. Unlike acyclovir and ara-A, ara-M had no appreciable activity against other viruses of the herpes group. The potency and selectivity of ara-M as an anti-VZV agent in vitro justify its further study.

Acyclovir↗