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D A Mills

Publications and source records attributed to D A Mills.

34 records · Page 2Linked to original sources
PubMed1994

Genetic analysis of regions of the Lactococcus lactis subsp. lactis plasmid pRS01 involved in conjugative transfer.

The genes responsible for conjugative transfer of the 48.4-kb Lactococcus lactis subsp. lactis ML3 plasmid pRS01 were localized by insertional mutagenesis. Integration of the IS946-containing plasmid pTRK28 into pRS01 generated a pool of stable cointegrates, including a number of plasmids altered in conjugative proficiency. Mapping of pTRK28 insertions and phenotypic analysis of cointegrate plasmids identified four distinct regions (Tra1, Tra2, Tra3, and Tra4) involved in pRS01 conjugative transfer. Tra3 corresponds closely to a region previously identified (D. G. Anderson and L. L. McKay, J. Bacteriol. 158:954-962, 1984). Another region (Tra4) was localized within an inversion sequence shown to correlate with a cell aggregation phenotype. Tra1 and Tra2, two previously unidentified regions, were located at a distance of 9 kb from Tra3. When provided in trans, a cloned portion of the Tra3 region complemented Tra3 mutants.

Chromosome Mapping↗
PubMed1993

Inhibition by tentoxin of cooperativity among nucleotide binding sites on chloroplast coupling factor 1.

Tentoxin, a cyclic tetrapeptide produced by the fungus Alternaria tenuis, is a potent inhibitor of the chloroplast coupling factor 1 from certain sensitive species of plants. We have shown that the beta subunit is at least partly responsible for conferring sensitivity to the toxin. This was confirmed by Avni et al. (Avni, A., Anderson, J.D., Holland, N., Rochaix, J-D., Gromet-Elhanan, Z., and Edelman, M. (1992) Science 257, 1245-1247) who demonstrated the importance for tentoxin sensitivity of an acidic amino acid residue at position 83 in the beta subunit sequence. In this paper we show that the Ca(2+)-ATPase and Mg(2+)-ATPase activities of CF1 lacking the delta and epsilon subunits, CF1(-delta epsilon), were fully sensitive to tentoxin, even after the gamma subunit is cleaved by trypsin into several smaller fragments. We also show that the isolated reconstitutively active beta subunit of CF1 does not effectively compete with CF1(-delta epsilon) for tentoxin binding. The results suggest that tight tentoxin binding requires the presence of at least the alpha and beta subunits but is independent of the delta and epsilon subunits. Tentoxin inhibited the release of a tightly bound molecule of ADP from CF1, which was induced by the binding of the ATP analogue adenylyl-beta,gamma-imidodiphosphate (AMP-PNP). AMP-PNP was shown previously (Shapiro, A.B., and McCarty, R.E. (1990) J. Biol. Chem. 265, 4340-4347) to cause two adenine nucleotide binding sites on CF1, sites 1 and 3, to switch their properties, possibly as part of an alternating site catalytic cooperativity mechanism (Boyer, P.D. (1989) FASEB J. 3, 2164-2178). It is proposed that the effect of tentoxin on catalytic cooperativity in CF1 results from tentoxin binding at an interface between alpha and beta subunits, preventing transfer of information between different nucleotide binding sites on the enzyme.

Adenosine Diphosphate↗
PubMed1993

Cloning and sequence analysis of the meso-diaminopimelate decarboxylase gene from Bacillus methanolicus MGA3 and comparison to other decarboxylase genes.

The lysA gene of Bacillus methanolicus MGA3 was cloned by complementation of an auxotrophic Escherichia coli lysA22 mutant with a genomic library of B. methanolicus MGA3 chromosomal DNA. Subcloning localized the B. methanolicus MGA3 lysA gene into a 2.3-kb SmaI-SstI fragment. Sequence analysis of the 2.3-kb fragment indicated an open reading frame encoding a protein of 48,223 Da, which was similar to the meso-diaminopimelate (DAP) decarboxylase amino acid sequences of Bacillus subtilis (62%) and Corynebacterium glutamicum (40%). Amino acid sequence analysis indicated several regions of conservation among bacterial DAP decarboxylases, eukaryotic ornithine decarboxylases, and arginine decarboxylases, suggesting a common structural arrangement for positioning of substrate and the cofactor pyridoxal 5'-phosphate. The B. methanolicus MGA3 DAP decarboxylase was shown to be a dimer (M(r) 86,000) with a subunit molecular mass of approximately 50,000 Da. This decarboxylase is inhibited by lysine (Ki = 0.93 mM) with a Km of 0.8 mM for DAP. The inhibition pattern suggests that the activity of this enzyme in lysine-overproducing strains of B. methanolicus MGA3 may limit lysine synthesis.

Amino Acid Sequence↗
PubMed1992

Structural mapping of cysteine-63 of the chloroplast ATP synthase beta subunit.

The single sulfhydryl residue (cysteine-63) of the beta subunit of the chloroplast ATP synthase F1 (CF1) was accessible to labeling reagents only after removal of the beta subunit from the enzyme complex. This suggests that cysteine-63 may be located at an interface between the beta and the alpha subunits of CF1, although alternative explanations such as a conformational change in beta brought about by its release from CF1 cannot be ruled out. Cysteine-63 was specifically labeled with [(diethylamino)methylcoumarinyl]-maleimide, and the distance between this site and trinitrophenyl-ADP at the nucleotide binding site on beta was mapped using fluorescence resonance energy transfer. Cysteine-63 is located in a hydrophobic pocket, 42 A away from the nucleotide binding site on beta.

Amino Acid Sequence↗
PubMed1991

Nucleotide binding to the isolated beta subunit of the chloroplast ATP synthase.

The beta subunit isolated from the chloroplast ATP synthase F1 (CF1) has a single dissociable nucleotide binding site, consistent with the proposed function of this subunit in nucleotide binding and catalysis. The beta subunit bound the nucleotide analogs trinitrophenyl-ATP (TNP-ATP) or trinitrophenyl-ADP (TNP-ADP) with nearly equal affinities (Kd = 1-2 microM) but did not bind trinitrophenyl-AMP. Both ATP and ADP effectively competed with TNP-ATP for binding. Other nucleoside triphosphates were also able to compete with TNP-ATP for binding to beta; their order of effectiveness (ATP greater than GTP, ITP greater than CTP) mimicked the normal substrate specificity of CF1. The single nucleotide binding site on the isolated beta subunit very closely resembles the low affinity catalytic site (site 3) of CF1 (Bruist, M.F., and Hammes, G. G. (1981) Biochemistry 20, 6298-6305), suggesting that tight nucleotide binding by other sites on the enzyme involves other CF1 subunits in addition to the beta subunit. The results are inconsistent with an earlier report (Frasch, W.D., Green, J., Caguial, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069), which suggested more than one nucleotide binding site per beta subunit. Binding of nucleotides to the isolated beta subunit was eliminated by a brief heat treatment (40 degrees C for 10 min) of the protein. A small change in the circular dichroism spectrum of beta accompanied the heat treatment indicating that a localized (rather than global) change in the folding of beta, involving at least part of the nucleotide binding domain, had occurred. Also accompanying the loss of nucleotide binding was a loss of the reconstitutive capacity of the beta subunit. ATP protected against the effects of the heat treatment.

Adenosine Diphosphate↗
PubMed1988

NMR assignments of the four histidines of staphylococcal nuclease in native and denatured states.

NMR signals from all four histidine ring C epsilon protons and three of the four histidine C delta protons in the protein staphylococcal nuclease have been assigned by comparing spectra of the wild-type (Foggi strain) protein to spectra of three variants that each lack a different histidine residue. All proteins studied were cloned and overproduced in Escherichia coli. The NMR spectra of the three mutant proteins (H8R, H46Y, and H124L) used to make these assignments were similar to one another and to those of the wild type, except for signals from the mutated residues. The pKa values of those histidines conserved between the wild type and the mutants remained essentially unchanged. Multiple histidine C epsilon proton resonances due to non-native forms of nuclease were observed in both thermally induced and acid-induced unfolding. Residue-specific assignments of H epsilon protons in the thermally denatured forms of the mutant H46Y were obtained from connectivities to the native state by saturation transfer.

Escherichia coli↗
PubMed1984

How many well-baby visits are necessary in the first 2 years of life?

Since we could find no scientific basis for the 10 well-baby visits recommended in the first 2 years of life in Ontario, we carried out a randomized trial in 570 healthy, low-risk newborns to determine the efficacy and safety of decreasing the number of scheduled well-baby visits from 10 to 5. Among the 466 babies still in the trial at the end of the study period those in the 10-visit group had had a mean of 7.63 scheduled and 0.26 unscheduled well-baby visits and those in the 5-visit group a mean of 4.77 scheduled and 1.42 unscheduled well-baby visits. Reducing the recommended number of visits did not result in an increased incidence of illness or an increased prevalence of undetected abnormality, and the physical development of the babies in the two groups was almost identical. The mean scores of the two groups in assessments of mental development, maternal-child relations, maternal anxiety and parental satisfaction with health care were close. The results suggest that the recommended number of well-baby visits for healthy, low-risk newborns can be reduced to five for children of multiparas and six for children of primiparas. Any additional well-baby visits should be scheduled according to the needs and experience of the parents.

Child Development↗
PubMed1980

Rubella antibody titres and immunization status in a family practice.

Rubella vaccination status and immunity to rubella were studied in 230 "active patients" aged 8 to 22 years in a teaching family practice by means of a chart review and measurement of the rubella antibody titre in a blood sample. Of the 200 patients who submitted a blood sample 161 (80%) were found to be immune, having a rubella hemagglutination-inhibiting antibody titre of 1:16 or greater. Log linear analysis showed that immunity to rubella was independent of a history of rubella, and that 94% of the vaccinated patients versus 74% of the unvaccinated patients (a significant difference; P = 0.007) were immune. In retrospect we estimated that 80% of the study group were protected at the start of the study. After surveillance and follow-up, with vaccination of 27 of the 39 patients identified as susceptible to rubella, this estimated proportion increased to 90%. The study showed that there is nothing to be gained by asking about a history of rubella but that vaccination against this disease is increasing among children aged 5 to 9 years.

Adolescent↗