Purification of DNA by affinity chromatography removal of polysaccharide contaminants.
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Biomedical subjects
Publications and source records attributed to M Edelman.
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Cucumber hypocotyl DNA in neutral CsCl distributes into a mainband comprising 59% of the total, and two large satellite bands which contribute 41% to the DNA pattern. Organelle enrichment studies show that the densities of mitochondrial and chloroplast DNA coincide with those of the satellite bands. At least 12-19% of total cucumber DNA is associated with the cytoplasmic organelles. These values, which are several times larger than those usually quoted for higher plants, are correlated with an unusually low amount of DNA per haploid nucleus in cucumber. Synthesis of the satellite DNAs, as well as mainband DNA, is appreciably stimulated in vivo by application of the plant hormone, gibberellin. Endogenous and hormone-enhanced synthesis of the satellite DNAs is proportionately greater in target tissue showing a high rate of organelle synthetic activity.
Ribulose 1,5-diphosphate carboxylase (RUDPcase) from autotrophically grown Euglena gracilis was purified to homogeneity as measured by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and immunoprecipitation reactions. The enzyme represented about 9% of total protein and 24% of soluble protein in the autotrophic cell. Light-grown, heterotrophic cells seemed to contain considerably less RUDPcase. Native carboxylase from autotrophic Euglena showed an s(20, w) at low protein concentrations of 17 to 17.5, suggesting a molecular weight of >500,000 daltons. Upon denaturation, the enzyme dissociated into two subunits having different amino acid compositions and molecular weights of 59,000 and 12,000 daltons. Based upon the amino acid mass ratios, a quaternary organization of 7 to 8 large and 8 to 10 small subunits per native enzyme molecule was indicated.The phylogenetic relationship of carboxylase from Euglena and from three higher plants was investigated. In general, the size, subunit formation, and quaternary structure of RUDPcase from the various sources seemed to be similar. A partial immunochemical reaction between anti-RUDPcase serum from Euglena and the enzymes from lettuce, cucumber, and New Zealand spinach suggested that the algal and higher plant carboxylases were related but not identical. This was borne out by amino acid analyses which showed a close correspondence between the large, but not the small, subunits of Euglena and lettuce.
Polyribonucleotide segments, about 60 nucleotides long and consisting of about 95% adenylic acid residues, were isolated from whole cell ribonucleic acid of the deuteromyceteous fungus Trichoderma viride. Similar findings in two other groups of the true fungi raise the possibility that short polyadenylate sequences may be a feature of these relatively simple organisms.
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The RNA associated with poly(A) sequences from Euglena gracilis and Vicia faba has been isolated by binding to millipore filters and characterized by sedimentation velocity centrifugation and electrophoretic mobility. Poly(A)-associated RNA as isolated in solution was highly aggregated. When denatured, it sedimented as a broad peak with a mean value of 16-18 S. This RNA was shown to be covalently linked to poly(A) sequences which are 150-250 nucleotides long. Our size estimates for plant poly(A) and poly(A)-associated RNA are similar to those obtained for animal cells.
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