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M D Watson

Publications and source records attributed to M D Watson.

34 records · Page 2Linked to original sources
PubMed1994

Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases.

We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44 degrees C on ampicillin was isolated. Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells. Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells. Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays. The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42,543. The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids. Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E. coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗
PubMed1993

Evidence for a dual osmoregulatory mechanism in the yeast Saccharomyces cerevisiae.

Osmoadaptation in S. cerevisiae occurs through intracellular accumulation of glycerol in response to an increase in osmolarity of the surrounding environment. Analysis of ssv1-2, a strain carrying a mutation in a gene required for vacuole biogenesis, protein-sorting and osmohomeostasis, shows that the strain is terminally inactivated by 1.5 M NaCl within 10 seconds while the isogenic wild type maintains slow growth and accumulates glycerol within 18 hours. This study provides the first evidence that the vacuole participates in an immediate osmoregulatory process permitting survival until the osmoadaptive glycerol accumulation allows growth under osmotically unfavorable conditions.

Magnetic Resonance Spectroscopy↗
PubMed1993

Differential, temporal and spatial expression of genes involved in storage oil and oleosin accumulation in developing rapeseed embryos: implications for the role of oleosins and the mechanisms of oil-body formation.

The temporal and spatial expression of oleosin and delta 9-stearoyl-ACP desaturase genes and their products has been examined in developing embryos of rapeseed, Brassica napus L. var. Topas. Expression of oleosin and stearate desaturase genes was measured by in situ hybridisation at five different stages of development ranging from the torpedo stage to a mature-desiccating embryo. The temporal pattern of gene expression varied dramatically between the two classes of gene. Stearate desaturase gene expression was relatively high, even at the torpedo stage, whereas oleosin gene expression was barely detectable at this stage. By the stage of maximum embryo fresh weight, stearate desaturase gene expression had declined considerably while oleosin gene expression was at its height. In contrast to their differential temporal expression, the in situ labelling of both classes of embryo-specific gene showed similar, relatively uniform patterns of spatial expression throughout the embryo sections. Immunogold labelling of ultra-thin sections from radicle tissue with anti-oleosin antibodies showed similar patterns to sections from cotyledon tissue. However, whereas at least three oleosin isoforms were detectable on western blots of homogenates from cotyledons, only one isoform was found in radicles. This suggests that some of the oleosin isoforms may be expressed differentially in the various types of embryo tissue. The differential timing of stearate desaturase and oleosin gene expression was mirrored by similar differences in the timing of the accumulation of their ultimate products, i.e. storage oil and oleosin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Brassica↗
PubMed1993

Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.).

Oleosins of Brassica napus L. (oilseed rape) synthesized by in-vitro translation were found to be very efficiently targeted to microsomal membranes but only poorly translocated to oil bodies or emulsified oil. The use of other bilayer membranes as controls showed that this interaction was specific. The rate of oleosin synthesis in the presence of microsomes was enhanced about threefold, indicative of the involvement of the signal-recognition particle in the targeting process. There is no evidence for the cleavage of the protein during targeting and the protein sequence reveals no consensus cleavage site for the signal peptide. Protection experiments using Proteinase K revealed that about 6 kDa of the protein is exposed on the cytoplasmic side of the ER but the remainder is protected. Carbonate (pH 11) washing of microsomal membranes after in-vitro translation confirmed that oleosins have a domain which remains inserted in the ER rather than the protein being transported completely into the lumen of the ER. These results indicate that oleosins are transported via the ER prior to their accumulation on oil bodies.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of osmosensitive vacuolar mutants of Saccharomyces cerevisiae.

The yeast vacuole plays an important role in nitrogen metabolism, storage and intracellular macromolecular degradation. Evidence suggests that it is also involved in osmohomeostasis of the cell. We have taken a mutational approach for the analysis of vacuolar function and biogenesis by the isolation of 97 mutants unable to grow if high concentrations of salt are present in the medium. Phenotypic analysis was able to demonstrate that apart from osmosensitivity the mutations also conferred other properties such as altered vacuolar morphology and secretion of the vacuolar enzymes carboxypeptidase Y, proteinase A, proteinase B and alpha-mannosidase. The mutants fall into at least 17 complementation groups, termed ssv for salt-sensitive vacuolar mutants, of which two are identical to complementation groups isolated by others. We conclude that in Saccharomyces cerevisiae correct vacuolar biogenesis and protein targeting is required for osmotolerance as well as other important cellular processes.

Ethyl Methanesulfonate↗
PubMed1988

Isolation and expression of a pea vicilin cDNA in the yeast Saccharomyces cerevisiae.

A cDNA clone containing the complete coding sequence for vicilin from pea (Pisum sativum L.) was isolated. It specifies a 50,000-Mr protein that in pea is neither post-translationally processed nor glycosylated. The cDNA clone was expressed in yeast from a 2 micron plasmid by using the yeast phosphoglycerate kinase promoter and initiator codon. The resultant fusion protein, which contains the first 16 amino acid residues of phosphoglycerate kinase in addition to the vicilin sequence, was purified and subsequently characterized. It has slightly slower mobility on SDS/polyacrylamide-gel electrophoresis than standard pea vicilin and forms a mixture of multimers, some of which resemble the native protein.

Amino Acid Sequence↗
PubMed1988

Ti plasmid-specified chemotaxis of Agrobacterium tumefaciens C58C1 toward vir-inducing phenolic compounds and soluble factors from monocotyledonous and dicotyledonous plants.

Twelve phenolic compounds with related structures were analyzed for their ability to act as chemoattractants for Agrobacterium tumefaciens C58C1 and as inducers of the Ti plasmid virulence operons. The results divided the phenolic compounds into three groups: compounds that act as strong vir inducers and are chemoattractants for A. tumefaciens C58C1 harboring the nopaline Ti plasmid pDUB1003 delta 31, but not the isogenic cured strain; compounds that are at best weak vir inducers and are weak chemoattractants for Ti plasmid-harboring and cured A. tumefaciens C58C1; and compounds that are vir noninducers and are also nonattractants. A strong correlation between vir-inducing ability and Ti plasmid requirement for chemotaxis is thus established. In addition, chemical structure rules for vir induction and chemotaxis are outlined. Positive chemotaxis toward root and shoot homogenates from monocotyledonous and dicotyledonous plants was observed. At low extract concentrations, chemotaxis was enhanced by the presence of Ti plasmid. The chemoattractants do not derive from intact cell walls. Lack of attraction is not responsible for the apparent block to monocot transformation by A. tumefaciens.

Chemical Phenomena↗
PubMed1984

A functional map of the nopaline synthase promoter.

This paper describes the first functional map of a promoter expressed from the plant chromosome. We have constructed a series of overlapping deletion mutants within the region upstream of the Ti-plasmid encoded nopaline synthase (nos) gene. By monitoring nos expression in tumour tissue we have inferred a functional map of the nos promoter. The maximum length of sequence upstream of the transcription initiation point required to express wild type levels of nopaline synthase is 88 bp. Within this region, the "CAAT" box is essential for maximal activity; deletion of this sequence reduced apparent nos expression by over 80%. Presence of an intact or partial "TATA" box in the absence of the "CAAT" box supports a barely detectable level of nopaline synthase. Removal of all sequences upstream of the nos coding sequence results in no detectable activity.

Amino Acid Oxidoreductases↗
PubMed1984

The right hand copy of the nopaline Ti-plasmid 25 bp repeat is required for tumour formation.

At either end of the nopaline Ti-plasmid T-region resides a copy of a 25 bp repeated element. The normal T-DNA endpoint is 1 bp internal of the right copy, with the transcription initiation site of the nopaline synthase (nos) gene being approximately 300 bp away in the same direction. Here we describe results which demonstrate that deletion of any combination of sequences between the nos initiation site and the right copy of the 25 bp repeat does not affect oncogenicity. Thus a mutant retaining the right copy and only 3 bp internal of it is indistinguishable from the wild type parent in its oncogenic properties. However deletion of a further 39 bp, including complete removal of the right copy abolishes crown gall tumour formation on Kalanchöe and tobacco. From these results we infer that unlike the left border, the right copy of the 25 bp repeat is required for T-DNA transfer and/or integration. This is the first conclusive demonstration of the involvement of a copy of the repeats in this process.

Chromosome Deletion↗
PubMed1979

Positive control of ilvC expression in Escherichia coli K-12; identification and mapping of regulatory gene ilvY.

The construction of a plasmid carrying the ilvC::lacZ fusion is described. This plasmid provides a convenient source of template deoxyribonucleic acid for use in an in vitro protein-synthesizing system. We screened strains deleted in regions of the ilv cluster for their ability to support ilvC-dependent beta-galactosidase synthesis. The fact that two deletions prevented beta-galactosidase production indicated that ilv-C expression is under positive control. By use of plasmids carrying the positive-control factor structural gene ilvY, we were able to restore protein-synthesizing ability to these strains. These plasmids also enabled us to map ilvY between ilvA and ilvC.

Chromosome Mapping↗
PubMedDate not supplied

Natural cytotoxicity towards allogeneic tumour targets in Xenopus mediated by diverse splenocyte populations.

We have recently demonstrated NK-like activity in the spleen of the clawed frog, Xenopus laevis. This paper investigates the cellular basis of this natural cytotoxicity. Significant levels of cytotoxicity towards B3B7 allogeneic thymus tumour targets, that express neither class Ia nor class II MHC proteins, occurred after splenocytes from either control or early-thymectomized (Tx) year-old Xenopus were cultured for 48 hours. Killing by Tx cells required their culture in growth factor-rich medium (GFM) obtained from concanavalin A-stimulated cells. Immunomagnetic cell sorting revealed that cytotoxic effectors in both control and Tx frogs were found in the B cell-depleted population, but never in the B cell-enriched fraction. Splenocytes from control Xenopus, depleted of T cells by magnetic sorting and following culture in GFM, also developed natural cytotoxicity towards allotumour cells. Magnetic cell sorting also revealed that purified (CD5+) T cells cultured for 48 hours in GFM also became able to lyse the allogeneic tumour targets. Cytotoxicity mediated by T cells resided not only in the CD5+, CD8+ population, but also in the CD5+, CD8- (putative CD4+) T cell subset. Ontogenetic studies revealed that splenocytes from 6-7 week-old (stage 56-57) control larvae, even after 48 hr culture in GFM, were unable to spontaneously lyse the allotumour targets, whereas cultured splenocytes from 6 month old froglets were effective killers. Thymocytes from larvae or adults routinely failed to kill tumour cells. The work highlights the need to use Tx Xenopus to further explore non-T-cell-mediated, NK-like cytotoxicity at the amphibian level of evolution.

Animals↗
PubMedDate not supplied

Incidence of injuries in high school track and field athletes and its relation to performance ability.

Two hundred fifty-seven high school track athletes from 17 teams were observed prospectively for one complete season (77 days) to study the incidence and types of injuries and to establish the relationship among injuries, duration of training, and individual performance ability. One hundred seventy-four (68%) of the athletes were male and 83 (33%) were female. A total of 41 injuries was observed over this period of time. One injury occurred for every 5.8 males and every 7.5 females. On the average, an injury resulted in 8.1 days of missed practice, 8.7 days for males and 6.6 days for females. Sprinting events were responsible for 46% of all injuries. The majority (83%) of injuries involved the lower extremities. Management of these injuries varied greatly. A direct correlation was noted between performance level of the athlete and incidence of injuries. The average noninjured athlete ranked at the 57.4 percentile based on best seasonal performance while the average injured athlete ranked at the 75.4 percentile. This direct relationship was present for both sexes and within all events, although some variation was noted within these separate groups.

Adolescent↗