Biomedical subjects

D L Robertson

Publications and source records attributed to D L Robertson.

11 recordsLinked to original sources

PubMed1992

Stereochemical and positional specificity of the lipase/acyltransferase produced by Aeromonas hydrophila.

Aeromonas species secrete a glycerophospholipid-cholesterol acyltransferase (GCAT) which shares many properties with mammalian plasma lecithin-cholesterol acetyltransferase (LCAT). We have studied the stereochemical and positional specificity of GCAT against a variety of lipid substrates using NMR spectroscopy as well as other assay methods. The results show that both the primary and secondary acyl ester bonds of L-phosphatidylcholine can be hydrolyzed but only the sn-2 fatty acid can be transferred to cholesterol. The enzyme has an absolute requirement for the L configuration at the sn-2 position of phosphatidylcholine. The secondary ester bond of D-phosphatidylcholine cannot be hydrolyzed, and this lipid is not a substrate for acyl transfer. In contrast to the phospholipases, but similar to LCAT, the enzyme does not interact stereochemically with the phosphorus of phosphatidylcholine. In fact, the phosphorus is not required for enzyme activity, as GCAT will also hydrolyze monolayers of diglyceride, although at much lower rates.

Acyltransferases↗

PubMed1992

Multiple evolutionary origins of prochlorophytes within the cyanobacterial radiation.

The taxonomic group Prochlorales (Lewin 1977) Burger-Wiersma, Stal and Mur 1989 was established to accommodate a set of prokaryotic oxygenic phototrophs which, like plant, green algal and euglenoid chloroplasts, contain chlorophyll b instead of phycobiliproteins. Prochlorophytes were originally proposed (with concomitant scepticism) to be a monophyletic group sharing a common ancestry with these 'green' chloroplasts. Results from molecular sequence phylogenies, however, have suggested that Prochlorothrix hollandica is not on a lineage that leads to plastids. Our results from 16S ribosomal RNA sequence comparisons, which include new sequences from the marine picoplankter Prochlorococcus marinus and the Lissoclinum patella symbiont Prochloron sp., indicate that prochlorophytes are polyphyletic within the cyanobacterial radiation, and suggest that none of the known species is specifically related to chloroplasts. This implies that the three prochlorophytes and the green chloroplast ancestor acquired chlorophyll b and its associated structural proteins in convergent evolutionary events. We report further that the 16S rRNA gene sequence from Prochlorococcus is very similar to those of open ocean Synechococcus strains (marine cluster A), and to a family of 16S rRNA genes shotgun-cloned from plankton in the north Atlantic and Pacific Oceans.

Base Sequence↗

PubMed1992

Detection of spores of Bacillus anthracis using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to identify spores of Bacillus anthracis. By using an assay capable of amplifying a 1247-bp fragment from the gene that encodes the edema factor of B. anthracis, as few as 10(3) copies of a plasmid containing the edema factor gene and as few as 2 x 10(4) spores were detected. Subjecting the product of this PCR to a second PCR designed to amplify a 208-bp fragment nested within the 1247-bp product improved detection to a single plasmid copy per PCR and to two spores of B. anthracis per PCR.

Bacillus anthracis↗

PubMed1991

Complete cloning of the chloroplast genome of safflower in lambda EMBL3 and mapping of 23S and 16S rRNA genes.

A recombinant DNA library was constructed from partial BamHI or MboI digests of safflower (Carthamus tinctorius L.) chloroplast DNA, in the BamHI site of lambda EMBL3. Seventeen lambda recombinants, selected by chromosome walking, were found to contain overlapping fragments of the entire chloroplast genome. These clones were mapped using single and double digests of BamHI, EcoRI and HindIII. cDNAs synthesized from isolated 16S and 23S chloroplast rRNAs were used to map the ribosomal RNA genes relative to physical maps of the above restriction enzymes. The mapped positions of the rRNA genes for the safflower chloroplast DNA are in good agreement with previously published data for tobacco, spinach and several other higher plants.

Bacteriophage lambda↗

PubMed1990

Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA.

The discovery of RNA enzymes has, for the first time, provided a single molecule that has both genetic and catalytic properties. We have devised techniques for the mutation, selection and amplification of catalytic RNA, all of which can be performed rapidly in vitro. Here we describe how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution experiments to be carried out in response to artificially imposed selection constraints. We worked with the Tetrahymena ribozyme, a self-splicing group I intron derived from the large ribosomal RNA precursor of Tetrahymena thermophila that catalyses sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. We selected for variant forms of the enzyme that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected molecule represents the discovery of the first RNA enzyme known to cleave single-stranded DNA specifically.

Animals↗

PubMed1979

Stability-indicating high-performance liquid chromatographic determination of chlorpropamide, tolbutamide, and their respective sulfonamide degradates.

A quantitative high-performance liquid chromatographic method for the determination of chlorpropamide, tolbutamide, and their respective hydrolysis products, p-chlorobenzenesulfonamide and p-toluenesulfonamide, in solid dosage forms was developed. The method is stability indicating and can be used to determine the sulfonamide hydrolysis product and the intact drug in the presence of minor degradates. Method reproducibility, demonstrated by repeated injections of a calibration standard, was 1.21%. The lower limit of quantitation of the hydrolysis products, p-chlorobenzenesulfonamide and p-toluenesulfonamide, was 0.2 microgram/5-microliter injection. The accuracy of the method for intact drugs was determined by comparison of the HPLC results to those obtained by the appropriate USP or BP assays. The mean of the results obtained by the two methods differed by 0.7% for chlorpropamide and 0.3% for tolbutamide. Pure drug samples were spiked with amounts of the hydrolysis products ranging from 20 to 120% of the intact content. The mean percent recovery for p-chlorobenzenesulfonamide was 98.6%; for p-toluenesulfonamide, it was 100.6%. A qualitative TLC procedure for the detection of chlorpropamide, p-chlorobenzenesulfonamide, dipropylurea, propylurea, n-propylamine, tolbutamide, p-toluenesulfonamide, dibutylurea, butylurea, and n-butylamine is also described.

Chlorpropamide↗

PubMed1979

Structural analysis of the intracellular RNAs of murine mammary tumor virus.

We have characterized murine mammary tumor virus (MuMTV)-specific RNA in several types of cells in which viral DNA is transcribed into RNA: cultured GR mouse mammary tumor cells, S49 lymphoma cells from BALB/c mice, lactating mammary glands from C57BL/6 mice, and mink lung cells infected in vitro with MuMTV. In all cell types studied, there are three distinct species of intracellular viral RNA, with sedimentation coefficients of 35S, 24S, and 13S (or molecular weights of 3.1 X 10(6), 1.5 X 10(6), and 0.37 X 10(6), as determined by rate-zonal sedimentation in sucrose gradients and by electrophoresis in agarose gels under denaturing conditions. These three viral RNA species appear to be present regardless of viral RNA concentration, responsiveness to glucocorticoid hormones, production of extracellular virus, and use of either endogenous or acquired MuMTV proviral DNA as template. The three viral RNAs display characteristics of mRNAs in that they are polyadenylated, associated with polyribosomes, and released from polyribosomes by treatment with EDTA; hence all three species presumably direct the synthesis of virus-coded proteins. The two larger species of viral RNA are probably responsible for synthesis of the structural proteins of the virion, but the function of the 13S RNA is not known. Both of the subgenomic RNAs contain sequences found at the 3' terminus of 35S (or genomic) RNA. However, only the 24S RNA (not the 13S RNA) contains sequences which are located at the 5' terminus of 35S RNA and are apparently transposed during RNA synthesis of maturation, as described for subgenomic mRNA's of other retroviruses.

Base Sequence↗

PubMed1979

Characterization of the proteins of intracisternal type A and extracellular oncornavirus-like particles produced by MOPC-460 myeloma cells.

The mouse plasmacytoma cell line, MOPC-460, produces both intracisternal and intracytoplasmic A-type particles when grown as a solid tumor. When these cells are grown either as an ascites tumor or in tissue culture, a third type of particle is produced extracellularly. This particle, the "myeloma-associated virus," is closely related to, and probably an alternate form of, the intracisternal A-type particle. The proteins present in these two types of particles were compared by tryptic peptide mapping. Both types of particles were found to contain essentially the same major proteins of 76,000 (p76), 68,000 to 70,000 (p68-70), and 45,000 (p45) daltons, in addition to varying amounts of smaller proteins. The relative proportions of all these proteins varied from preparation to preparation in an unpredictable way. The p45, p68, and p70 proteins all contained sequences found in p76, suggesting precursor-product relationships of p76 leads to p70 leads to p45 for solid tumor A-type particles and p76 leads to p68 leads to p45 for extracellular myeloma-associated virus. In addition, immune precipitation experiments have established that p76 contains at least some of the antigenic determinants characteristic of murine leukemia virus p30. This confirms earlier nucleic acid hybridization studies which indicated a moderate degree of relatedness between MOPC-460 A-type particles and several standard murine leukemia and sarcoma viruses. Taken together, our results provide evidence supporting the concept that MOPC-460 A-type particles may represent aberrant forms of C-type murine viruses.

Animals↗

PubMed1976

Relationships between intracisternal type A and extracellular oncornavirus-like particles produced in murine MOPC-460 myeloma cells.

Oncornavirus-like particles of the "A" (both intracisternal and intracytoplasmic) and "B" or "C" (extracellular) types are produced by murine MOPC-460 myeloma cells. This communication describes a comparative study on tracisternal A and extracellular particles. Both types of particles contain an RNA-dependent DNA polymerase activity, traces of 35S and 70 S RNA in addition to larger amounts of degraded RNA, and proteins of approximately 76,000 and 45, 000 daltons. The 76,000-dalton proteins from intracisternal A and extracellular particles have the same cyanogen bromide peptides. Hybridization kinetic analysis indicates that the RNAs in the two particles are identical or very closely related and share partial homology with Moloney leukemia virus RNA. In contrast, the particles appear to have little or no relationship to murine mammary tumor virus as judged by several different criteria. Electron microscope studies indicate that the extracellular particles arise from the budding of core components through the plasma membrane. These results suggest that the intracisternal A and extracellular oncornavirus-like particles produced by MOPC-460 cells are closely related.

Animals↗

PubMed1975

Reverse transcriptase associated with A-type particles from murine myeloma cells.

Our findings may be summarized as follows: (a) a DNA polymerase is associated with murine type A particles, which is very similar to the reverse transcriptases found in true RNA tumor viruses; (b) the majority of the RNA isolated from A particles is of low molecular weight (probably due to degradation), byt appears to contain an amount of unique sequence information comparable to that found in other oncornavirus particles. These results suggest that A-type particles may be very similar, and perhaps related, to the two other classes (B and C) of oncornaviruses.

Base Sequence↗

PubMed1975

Characterization of DNA polymerase and RNA associated with A-type particles from murine myeloma cells.

The RNA-dependent DNA polymerase present in intracisternal A-type particles from mouse myeloma tumor cells has been studied. This polymerase can use either endogenous A particle RNA or an exogenous synthetic polynucleotide [poly (rA)] as a template. The DNA reaction product is small (4S-10S) and over 90% of it hybridizes to A particle RNA, whereas up to 50% of it hybridizes to murine sarcoma-leukemia virus RNAs. The RNA isolated from purified A particles is generally of low molecular weight (5S-15S) but contains small amount of 70S and 35S components. These results suggest that A-type particles may be related to C-type oncornaviruses.

Animals↗