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D C Alexander

Publications and source records attributed to D C Alexander.

At least 37 records · Page 2Linked to original sources
PubMed1994

Cloning, sequencing and disruption of a gene from Streptomyces clavuligerus involved in clavulanic acid biosynthesis.

During the purification of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Streptomyces clavuligerus, a small protein (CLA) involved in clavulanic acid production co-purified with ACVS. A 24-mer mixed-DNA probe based on the N-terminal amino-acid sequence of CLA was used to isolate the corresponding gene (cla), located near one end of the known cluster of penicillin and cephamycin biosynthetic genes, 5.7 kb downstream from the pcbC gene which encodes isopenicillin-N synthase. The sequence of cla would encode a protein of 313 aa with a high degree of similarity to amidinohydrolase enzymes. The cla gene is located immediately upstream from the previously described clavaminate synthase 2-encoding gene (cs2), and cla homologs were only present in streptomycetes which produced clavam compounds. Replacement of cla with a disrupted copy of the gene blocked the production of clavulanic acid in starch asparagine medium (SA).

Amino Acid Sequence↗
PubMed1994

Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine.

We report that rfe mutants of wild-type strains of Escherichia coli O7, O18, O75, and O111 did not express O-specific polysaccharide unless the rfe mutation was complemented by a cloned rfe gene supplied in a plasmid. The O polysaccharides in these strains are known to have N-acetylglucosamine (GlcNAc) in their O repeats. In addition, in vitro transferase assays with bacterial membranes from either the O7 wild-type strain or its isogenic rfe mutant showed that GlcNAc is the first carbohydrate added onto the lipid acceptor in the assembly of the O7 repeat and that this function is inhibited by tunicamycin. Our results indicate that the rfe gene product is a general requirement for the synthesis of O polysaccharides containing GlcNAc.

Acetylglucosamine↗
PubMed1993

Isolation of a tomato alcohol dehydrogenase 2-encoding cDNA using phage-promoted antibody screening of a plasmid cDNA library.

We describe the cloning of a cDNA encoding tomato alcohol dehydrogenase 2 (Adh2) by screening plasmid cDNA clones in phage plaques. A cDNA library constructed in a plasmid vector containing a unique SstI site at the 5' end of the cDNA insert was transferred into the SstI site of the lacZ gene of phage lambda Charon16, and screened by anti-Adh2 antibody to identify reactive plaques. Plasmid cDNA clones were recovered by SstI digestion, ligation, and transformation from phage minipreps for subsequent characterization. This system preserves the original plasmid library for subsequent screening with nucleic acid probes to identify full-length, multiple independent, or related cDNA clones not subject to the selection pressure of phage growth or lysogeny, or negative antibody reactivity. Thirty-two cDNA clones were identified with polyclonal antiserum to Adh2. Three of these reacted with monoclonal anti-Adh2 and only those three hybridized to maize adh1 sequence. One of these cDNAs, Adh31, was further characterized as encoding Adh2 by hybrid-selected translation and high sequence homology with the maize adh1 gene.

Alcohol Dehydrogenase↗
PubMed1992

U.S. system for organ procurement and transplantation.

The establishment and responsibilities of the organ procurement and transplantation network in the United States are discussed, and the process of receiving an organ transplant through the system is described. The National Organ Transplant Act of 1984 provided for a federally funded network for organ procurement and transplantation, which would function as a private, non-profit organization. This organization is the United Network for Organ Sharing (UNOS). UNOS monitors the activities of and provides service to transplant centers and organ procurement organizations (OPOs). The names of all candidates awaiting cadaveric organ transplants are placed on a central waiting list maintained by UNOS; UNOS also is responsible for maintaining a scientific registry to collect relevant data from transplant centers on the recipients of organ transplants. Although there is a national list of more than 25,000 persons waiting at any given time for a cadaveric organ, there are far fewer actual organ donors (less than 5000 in 1991). The hospital's best resource with respect to the donation process is the local OPO, which provides services related to organ donor referral, evaluation, and surgical recovery. The organ donation process consists of eight components: donor identification, referral, evaluation, consent, management, recovery of organs, allocation, and follow-up. An organ recovery coordinator from the local OPO helps the hospital staff in determining donation potential, seeking consent from the next of kin, and managing the donor after consent has been obtained. The OPO--never the donor's family or their insurer--is billed for charges relating to the donation. The OPO then bills the costs associated with the donation to the transplant centers receiving each organ for implantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed1989

Changing trends in dental restorative treatment needs of naval recruits 1987-89--disease or diagnosis?

The restorative treatment needs of 9960 young men and women who entered the Naval Service at HMS Raleigh between January 1987 and April 1989 are summarised. A marked increase in the numbers of fillings required and a decrease in the proportion considered to be dentally fit occurred in May 1988 and has continued as a trend thereafter. Weighted non-orthogonal analysis of variances have been conducted to assess the significance of clinical and period variables upon the changes.

Dental Caries↗
PubMed1987

cDNA cloning and in vitro synthesis of the Dolichos biflorus seed lectin.

The Dolichos biflorus seed lectin contains two structurally related subunits. A cDNA library was constructed using RNA isolated from D. biflorus seeds actively synthesizing the seed lectin. The library was expressed in Escherichia coli using a lambda Charon 16 vector, and lectin-specific antiserum was used to isolate a seed lectin cDNA. Hybridization of the D. biflorus seed lectin cDNA to RNA isolated from seeds actively producing both lectin subunits identifies a single-size RNA of 1100 bases. An oligodeoxyribonucleotide probe, constructed from an amino acid sequence common to both lectin subunits, detects the same size RNA. Translation of seed mRNA in vitro and immunoprecipitation of translation products using a lectin-specific antiserum yields a single polypeptide of slightly higher molecular mass than the largest seed lectin subunit. This seed lectin precursor is indistinguishable from a polypeptide synthesized from mRNA hybrid selected by the seed lectin cDNA. These data support the existence of a single polypeptide precursor for both subunit types of the D. biflorus seed lectin and suggest that differences between the subunit types arise by posttranslational processing.

Amino Acid Sequence↗
PubMed1987

A nutritional analysis of food provided to Royal Naval personnel at sea.

The aim of this study was to determine the nutritional content of the foods provided to, and the confectionary purchased by, personnel serving at sea in British warships. Data were collected from the stores accounts of six ships over a period of 32,354 man-victualling-days. Analysis was carried out at the Nuffield Laboratories of Comparative Medicine. The food from the ships' gallies provided mean daily intakes of 3750 kcal, of which 42 per cent were derived from fats (17 per cent from saturated fats) and 9 per cent from added sugars. In taking account of confectionary purchased from NAAFI sources the total energy intake was raised to 4200 kcal of which 40 per cent were derived from fats (15 per cent saturated fats) and 12 per cent from added sugars. The results do not compare favourably with the recommendations of the reports of expert committees.

Adult↗
PubMed1986

Nucleotide sequence and molecular evolution of two tomato genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase.

We have isolated and sequenced two cDNA clones (LESS5 and LESS17) encoding the small subunit of ribulose-1,5-bisphosphate carboxylase of tomato (Lycopersicon esculentum). At the nucleotide level, the protein-coding regions of these genes are 85% conserved, while the untranslated 3' regions are only 55% conserved. Comparison with rbcS genes from other species of Solanaceae suggests that the tomato LESS5 gene, the Nicotiana tabacum NTSS23 gene and the Petunia hybrida SSU8 gene are orthologous members of the rbcS gene family. In addition, the tomato gene LESS17, and the Petunia hybrida gene SSU611, may also be orthologous, since their untranslated 3' regions are related. There is a large difference between the two tomato rbcS genes in the frequency of the CG dinucleotide. This difference may reflect different levels of methylation, and therefore expression, of the tomato genes. Many of the differences involving the CG dinucleotide can be represented as transitions between C and T on the noncoding strand. Such changes are consistent with observations that methylated cytosines are hot-spots for transitions.

Base Sequence↗
PubMed1986

Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening.

The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-alpha-D-galacturonide) glucanohydrolase, EC 3.2.1.15] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting post-translational processing. A plasmid cDNA library was prepared from poly(A)(+) RNA isolated from ripe tomato fruit. The cDNA library was inserted into a lambda-based expression vector, and polygalacturonase cDNA clones were identified by immunological screening. Hybrid-select translation experiments indicated that the cDNAs encode a 54-kDa in vitro translation product that is specifically immunoprecipitated with polygalacturonase antiserum. RNA-blot analysis indicated that the 1.9-kilobase polygalacturonase mRNA was virtually absent from immature-green fruit, accumulated steadily during the ripening process, and was at its highest level in red-ripe fruit. There was at least a 2000-fold increase in the level of polygalacturonase mRNA between immature-green and red-ripe tomato fruit. These studies show that the levels of polygalacturonase mRNA are developmentally regulated during tomato fruit ripening.

Journal Article↗
PubMed1984

A simplified and efficient vector-primer cDNA cloning system.

A simplified, efficient, and versatile vector-primer cDNA cloning system is presented. The dimer-primer system is a modification of the method of Okayama and Berg (1982) with the following features: (i) the vector-primer molecules are more rapidly and reliably prepared by virtue of the elimination of an endonuclease digestion and the agarose gel purification step from the original method, and (ii) the final cDNA products contain polylinkers at both cDNA-vector junctions, simplifying the size analysis, subcloning, and sequencing of inserts. The system is highly efficient, yielding greater than 10(5) transformants using 1 microgram mRNA and 1 pmol of vector-primer ends, with 75% or more of the transformants having inserts. The ability of the system to produce clones of full-length or near full-length is demonstrated by the analysis of 32 ribulose-1,5-bisphosphate carboxylase small subunit cDNA clones from tomato.

Animals↗
PubMed1983

Regulation of beta-chain mRNA of ovine follicle-stimulating hormone by 17 beta-estradiol.

17 beta-Estradiol (E2) rapidly and reversibly decreases the synthesis of follicle-stimulating hormone (FSH) in primary dispersed cell cultures of ovine pituitaries. Similarly, E2 also causes a decrease in the messenger RNA for the beta subunit of ovine FSH (FSH beta-mRNA) as measured in an in vitro translation assay. These results are consistent with the concept that E2 directly regulates either transcription of the FSH beta gene or processing of FSH beta-mRNA in sheep. Of additional interest is the observation that pituitary cultures from various species respond differently to E2 in terms of FSH synthesis. For example, E2 stimulates FSH synthesis in rat pituitary cultures, has no effect in similar rabbit cultures, and inhibits FSH synthesis in ovine cultures. Thus, a set of eukaryotic 'mutants' may exist to aid studies of the effect of E2 on FSH synthesis and secretion.

Animals↗
PubMed1981

mRNA for ovine follicle-stimulating hormone beta-chain. An in vitro translational assay.

Polyadenylated mRNA from pituitary glands of castrate male sheep was used to demonstrate the presence of an mRNA coding for the beta-subunit of follicle-stimulating hormone (FSH). The mRNA fraction was translated in a wheat germ membrane-free system in the presence of [35S]cysteine. Affinity-purified antibodies, raised against the reduced and carbamylmethylated beta-subunit of ovine FSH (RCM-oFSH beta), were used to extract an in vitro translation product of Mr approximately equal to 15,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody binding of the product was readily blocked by unlabeled RCM-oFSH beta, but not by the RCM derivatives of the alpha- or beta-subunits of ovine luteinizing hormone. Tryptic peptide analysis using reversed phase high performance liquid chromatography yielded very similar patterns for the immunospecific translation product and authentic oFSH beta. FSH beta translation product was found to account for approximately 0.1-0.4% of the total isotope incorporation when mRNA preparations from castrate male sheep were used. Backgrounds for the immunoextraction procedure were kept at approximately 0.02% by using a unique two-cycle specific extraction method.

Animals↗