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Biomedical subjects

J Davey

Publications and source records attributed to J Davey.

At least 73 records · Page 4Linked to original sources
PubMed1994

Chemical synthesis of the M-factor mating pheromone from Schizosaccharomyces pombe.

Conjugation in the fission yeast Schizosaccharomyces pombe is controlled by the reciprocal action of mating pheromones. We recently showed that M-factor, the pheromone released by cells of the cellular mating type Minus, is a nonapeptide in which the C-terminal cysteine residue is carboxyl-methylated and S-alkylated, probably with a farnesyl residue (Davey, 1992): Tyr-Thr-Pro-Lys-Val-Pro-Tyr-Met-Cys(S-farnesyl)- OCH3. Here we describe the chemical synthesis of this modified peptide and show that it exhibits all of the properties of the native pheromone. These results confirm the structure of the M-factor while the production of relatively large amounts of pure pheromone will be invaluable for studying the mating response in this yeast.

Amino Acid Sequence↗
PubMed1994

Mating pheromone-induced expression of the mat1-Pm gene of Schizosaccharomyces pombe: identification of signalling components and characterization of upstream controlling elements.

Transcription of the mat1-Pm gene of Schizosaccharomyces pombe controlling entry into meiosis is stimulated by the mating pheromone, M-factor. We have studied its expression by monitoring beta-galactosidase activity in cells carrying a plasmid-borne mat1-Pm/lacZ fusion construct. Stimulation required the M-factor receptor (Map3) and other proteins (Gpa1, Byr1, Byr2 and Spk1) thought to be involved in propagating the pheromone signal within the cell. Mutational activation of gpa1 encoding an alpha subunit of the receptor-coupled heterotrimeric G protein causes full expression of mat1-Pm even in the absence of pheromone, suggesting that Gpa1 is a key signal transmitter. Furthermore, an activated ras1val17 mutant exhibited a much stronger level of induction than wild-type cells, though full expression needs M-factor treatment. Deletion analysis of the mat1-Pm promoter region identified a stretch of 21 bp that is shown to play a critical role in controlling expression. This region lies just upstream of a TATA-like box and contains a TR-box (TTCTTTGTTY) motif which is the recognition site of a putative transcription factor Ste11. Point mutations in the TR-box motif abolished the expression of mat1-Pm/lacZ. Almost no expression of mat1-Pm was detected in a ste11 deletion mutant, whereas overproduction of Ste11 greatly increased the expression.

Base Sequence↗
PubMed1994

Assessment of pheromone production and response in fission yeast by a halo test of induced sporulation.

We describe a rapid, sensitive and semi-quantitative plate assay for monitoring pheromone activity in the fission yeast Schizosaccharomyces pombe. It is based on the observation that meiosis requires stimulation by pheromone and exploits diploid strains that will only sporulate after addition of exogenous pheromone. The tester strains are heterozygous for mating type, are non-switching, and are mutated in one of the early subfunctions (either mat1-Mc or mat1-Pc), so that meiosis is only induced after exposure to exogenous pheromone (M-factor or P-factor, respectively). Pheromone activity is assessed as an iodine-positive halo of sporulation surrounding the pheromone source, and the width of the halo is related to the amount of pheromone being produced. The assay is sufficiently sensitive to monitor the low amount of M-factor produced by an M mam1 strain, and its sensitivity towards P-factor is greatly increased by using a hyper-sensitive tester strain lacking the Sxa2 protease that is believed to degrade this pheromone. We also demonstrate that the production of P-factor is very much stimulated by exposure of P cells to M-factor.

Pheromones↗
PubMed1994

Mutations in cyr1 and pat1 reveal pheromone-induced G1 arrest in the fission yeast Schizosaccharomyces pombe.

Investigations into sexual differentiation and pheromone response in the fission yeast Schizosaccharomyces pombe are complicated by the need to first starve the cells of nitrogen. Most mating-related experiments are therefore performed on non-dividing cells. Here we overcome this problem by using two mutants that bypass the nutritional requirements and respond to the M-factor mating pheromone in rich medium. The first mutant lacks the cyr1 gene which encodes adenylate cyclase and these cells contain no measurable amounts of cAMP. When M-factor is added to a growing h+ cyr1- strain it causes a transient G1 arrest of cell division, transcription of mat1-Pm, and elongation of the cells to form shmoos. The second mutant contains the temperature-sensitive pat1-114 allele. At 30 degrees C this mutant was previously shown not only to bypass the nutritional signal but also to stop growing in a state derepressed for pheromone-controlled functions. We now report that an h+ pat1-114 strain growing mitotically at 23 degrees C responds to M-factor. This shows that the pat1 protein kinase can be tuned to derepress nutritional signalling while repressing the other stages in the differentiation process.

Adenylyl Cyclases↗
PubMed1994

Correlates of ventricular ectopic activity in isolated systolic hypertension. SHEP Cooperative Research Group.

Ventricular ectopic activity was recorded at baseline in 5.6% of the 12-lead electrocardiograms and 8.2% of the 2-minute rhythm strips of 4674 subjects with isolated systolic hypertension (systolic blood pressure 160 to 219 mm Hg, diastolic blood pressure < 90 mm Hg) participating in the Systolic Hypertension in the Elderly Program (SHEP). In this study 1.3% had 6 to 10 ventricular premature beats (VPB), and 0.7% had > 10 VPB on the 2-minute rhythm strip. Correlates of VPB presence on the 12-lead ECG were older-age male sex, presence of Q/QS pattern and higher heart rate. Participants with serum potassium < 3.5 mmol/L had a higher prevalence of VPB. Similarly, the number of VPB on the 2-minute rhythm strip was associated with male sex, increasing age, with lower serum potassium, history of palpitations, and presence of Q/QS patterns.

Aged↗
PubMed1994

Approaching safe and secure health information systems in Europe.

Information systems in health care are being utilised in more and more demanding and more clinical situations where security and safety issues become more significant. These issues are explored within the context of the UK National Health Service and the corresponding activities within the European Union.

Computer Security↗
PubMed1994

Analysis of the structural genes encoding M-factor in the fission yeast Schizosaccharomyces pombe: identification of a third gene, mfm3.

We previously identified two genes, mfm1 and mfm2, with the potential to encode the M-factor mating pheromone of the fission yeast Schizosaccharomyces pombe (J. Davey, EMBO J. 11:951-960, 1992), but further analysis revealed that a mutant strain lacking both genes still produced active M-factor. Here we describe the isolation and characterization of a third M-factor gene, mfm3. A mutant lacking all three genes fails to produce M-factor, indicating that all functional M-factor genes now have been identified. The triple mutant exhibits an absolute mating defect in M cells, a defect that is not rescued by addition of exogenous M-factor. A mutational analysis reveals that all three mfm genes contribute to the production of M-factor. Their transcription is limited to M cells and requires the mat1-Mc and ste11 gene products. Each gene is induced when the cells are starved of nitrogen and further induced by a pheromone signal. Additionally, the signal transduction machinery associated with the pheromone response is required for transcription of the mfm genes in both stimulated and unstimulated cells.

Amino Acid Sequence↗
PubMed1994

Tools and techniques for the development of secure software.

System development projects in health care have not properly addressed the issues of availability, confidentiality and integrity. The safety critical aspects of health care have not been well recognised in the context of information systems, as opposed to medical devices where considerable effort has been made to ensure the 'correctness' of the device before it is authorized for use. Consideration should be given at the start of the development process to the particular requirements of the project and the project should be structured to ensure that the specific functional requirements for security are satisfied. In addition the development process itself must be conducted in a rigorous fashion in order to ensure-and to demonstrate--that the end product has the appropriate degree of 'correctness'. Suitable tools and techniques will be required to assist the efficiency and the integrity of this process.

Computer Security↗
PubMed1993

Thumb in cheek? Sensory reorganization and perceptual plasticity after limb amputation.

We describe a patient who initially suffered from carpal tunnel syndrome that was alleviated by surgery. Subsequently, the patient's right arm was amputated and a phantom limb emerged. Stimuli applied to different areas of the face gave rise to sensations that were referred to the phantom by precise topographic mapping. This perceptual mapping of the (phantom) hand onto the face area modelled the form of prior pathology due to carpal tunnel syndrome. The findings have implications for the relief of phantom pain.

Adult↗
PubMed1993

Schizosaccharomyces pombe map3+ encodes the putative M-factor receptor.

A defect in the map3 gene of the fission yeast Schizosaccharomyces pombe causes h+ mating-type-specific sterility. This gene was cloned by complementation. Nucleotide sequence analysis showed that it has a coding capacity of 365 amino acids. The deduced map3 gene product is a putative seven-transmembrane protein and has 20.0% amino acid identity with the a-factor receptor of Saccharomyces cerevisiae, encoded by STE3. It is also homologous with the Ustilago maydis mating pheromone receptors. The map3 gene is expressed in h+ cells but not in h- cells, and the transcripts are induced in response to nitrogen starvation. h+ cells defective in map3 do not respond to purified M-factor. When map3 is expressed ectopically in h- cells, they apparently acquire the ability to respond to the M-factor produced by themselves. The gpa1 gene, which encodes the alpha-subunit of a G-protein presumed to couple with the mating pheromone receptors, is essential for this function of map3. These observations strongly suggest that map3 encodes the M-factor receptor. Furthermore, this study provides strong support for the notion that pheromone signaling is essential for initiation of meiosis in S. pombe and that either M-factor signaling or P-factor signaling alone is sufficient.

Amino Acid Sequence↗
PubMed1992

Mating pheromones of the fission yeast Schizosaccharomyces pombe: purification and structural characterization of M-factor and isolation and analysis of two genes encoding the pheromone.

Conjugation in the fission yeast Schizosaccharomyces pombe is controlled by the action of mating pheromones. Here I describe the isolation and characterization of M-factor, the pheromone released by M-type cells. M-factor is a nanopeptide in which the carboxy-terminal cysteine residue is carboxy-methylated and S-alkylated, probably with a farnesyl residue: Tyr-Thr-Pro-Lys-Val-Pro-Tyr-Met-Cys(S-farnesyl)-OCH3. Evidence for this structure was obtained by amino acid analysis, mass spectrometry and tandem mass spectrometry of the native M-factor. Two genes encoding the M-factor were also identified and characterized. It appears that M-factor is synthesized as a larger precursor which is post-translationally cleaved and modified to yield the active pheromone. The proposed modifications are consistent with mechanisms known to exist in other yeast and higher eukaryotes.

Amino Acid Sequence↗
PubMed1992

The ras1 function of Schizosaccharomyces pombe mediates pheromone-induced transcription.

Loss of ras1+ function renders fission yeast cells unable to undergo morphological changes in response to mating pheromones, whereas cells carrying activated mutations in ras1 are hyper-responsive. This has led to the suggestion that the ras1 gene product plays a role in mating pheromone signal transduction. Using partially purified M factor we demonstrate that the mat1-Pm gene, which controls entry into meiosis, is transcribed in response to a pheromone signal. Strains mutated in the ras1 gene or in ste6, the fission yeast homologue of Ras protein GDP/GTP exchange factor, are unable to induce transcription of mat1-Pm in response to M factor. Furthermore, an activated ras1val17 mutant exhibits a stronger induction of the mat1-Pm transcript. However, transcription still depends on nitrogen deprivation as well as on the presence of pheromone, showing that activation of the Ras1 protein alone does not substitute for any of these signals. The pat1-114 mutant bypasses the ras1/ste6 checkpoint, suggesting that activation of ras1 contributes to inactivation of the pat1 protein kinase.

Blotting, Northern↗
PubMed1989

Recognition of influenza virus proteins by cytotoxic T lymphocytes.

Recombinant DNA techniques have been used to express the proteins of influenza virus individually. Target cells expressing single viral proteins were then used to identify the molecules recognized by cytotoxic T lymphocytes (CTLS). Results have shown that, contrary to expectation, the majority of the proteins recognized by class I major histocompatibility complex-restricted CTLS are not transmembrane glycoproteins. Experiments with deletion mutants of the nucleoprotein (NP) gene showed that transport of epitopes to the membrane for recognition by CTLS was independent of a definable signal sequence. In addition, the epitopes recognized were contained within short linear sequences of amino acids, and rapid degradation of large NP fragments within the target cell did not prevent recognition by CTLS. These results led to the suggestion that the epitopes recognized by class-I-restricted CTLS resulted from degradation of viral proteins. If so, the epitopes should, like those for class-II-restricted T cells, be replaceable in vitro with short synthetic peptides. Five different epitopes of NP have now been demonstrated that can be defined with short peptides in vitro. Each peptide is recognized with a specific class I molecule (Db, Kk, Kd and HLA B37). This has been extended to the influenza matrix protein, and a peptide epitope defined that is recognized by human CTLS in association with HLA-A2. The question arose as to whether a similar phenomenon would be found with viral proteins which are naturally inserted in the target cell membrane. A mutant haemagglutinin has been produced that lacks a hydrophobic signal sequence. This protein is expressed as a short-lived, unglycosylated, intracellular protein. However, target cells expressing this molecule were recognized efficiently by CTLS raised to the wild-type haemagglutinin and vice versa. These and more recent results with non-viral glycoproteins are consistent with the existence of a mechanism for degrading viral (and perhaps host) proteins and exposing them at the cell surface for recognition by cytotoxic T cells in association with class I molecules of the major histocompatibility complex.

Animals↗