Quick and high-efficiency electroelution of nucleic acid fragments.

We describe a quick, simple, and inexpensive technique for the electroelution of nucleic acid fragments that provides a high yield of DNA by using common laboratory components. The quantity of buffer used for the recovery of nucleic acid fragments is relatively small, the quality of the DNA recovered is relatively high, and more than one electroelution can be carried out at the same time.     

Changes in nucleic acid and protein synthesis during starvation and spherule formation inPhysarum polycephalum

Summary The synthesis of protein and nucleic acids was studied by isotope incorporation and dilution in the plasmodia ofPhysarum polycephalum during periods of growth and differentiation (spherule formation). The total protein content decreased during starvation, but protein synthesis still occurred, probably at the expense of proteins previously synthesized during growth. Studies on leucine incorporation showed that protein synthesized during growth had a greater turnover than did protein formed by starving cultures, when both types of cultures were transferred to starvation conditions. Protein synthesis after prolonged starvation was rapidly and markedly decreased following the inhibition of RNA synthesis, whereas no such direct dependence on RNA synthesis was observed in growing cultures or during early starvation.The kinetics of RNA synthesis and the types of RNA formed were also shown to differ in growth and starvation. RNA turnover was low in growing cultures but substantial in starving cultures that were returned to growth medium. Qualitative differences in pulse-labeled RNA extracted from growing or starving cultures were revealed by methylated-albumin-kieselguhr column chromatography and sucrose gradient centrifugation. In starving cultures proportionately more labeled RNA was found in the lighter, non-ribosomal region of the gradient, and RNA from this region hybridized with denatured DNA to a greater extent than did other RNA fractions.This work was supported in part by Grant CA-07175 from the National Cancer Institute and by a grant from the Alexander and Margaret Stewart Trust Fund. The authors express their appreciation to Dr. H. Kubinski for helpful suggestions.One of us (H.W.S.) was in part supported by the Deutsche Forschungsgemeinschaft.     

Effect of saffron on cell colony formation and cellular nucleic acid and protein synthesis.

A concentrated extract of saffron was prepared from the flowers of Crocus sativis. The effect of this extract on the ability of HeLa cells to form colonies, and on cellular DNA, RNA and protein synthesis was examined. Incubation of cells with extract for 3 h resulted in significant inhibition of colony formation and cellular nucleic acid synthesis with 50% inhibition at concentrations of approximately 100-150 micrograms/ml. In contrast there was no inhibition of cellular protein synthesis at concentrations of extract as high as 400 micrograms/ml.     

Differences in actin localization during bud and hypha formation in the yeast Candida albicans

Stationary phase cells of Candida albicans can form either a bud or a hypha, depending upon the pH of the medium into which they are released. At low pH, cells form an ellipsoidal bud and at high pH, cells form an elongated hypha. By staining cells with rhodamine-conjugated phalloidin, we have compared the dynamics of actin localization during the formation of buds and hyphae. Before evagination, actin granules were distributed throughout the cytoplasmic cortex in both budding and hypha-forming cells. Just before evagination, actin granules clustered at the site of evagination, then filled the early evagination in both budding and hypha-forming cells. With continued bud growth, the actin granules then redistributed throughout the cytoplasmic cortex. In marked contrast, with continued hyphal growth, the majority of actin granules clustered at the hyphal apex. This distinct difference in actin granule localization may be related to the distinct differences in the expansion zones of the cell wall recently demonstrated between growing buds and hyphae. The spatial and temporal dynamics of the large neck actin granules and of actin fibres are also described.     

Bud formation in leaves,leaf fragments and midrib pieces of Heloniopsis orientalis (Liliaceae)

Summary Isolated leaves, leaf fragments and pieces of the midrib portion devoid of lamina, of Heloniopsis orientalis were grown on an inorganic nutrient medium without organic nutrients and growth regulators in order to investigate their regenerative ability. Bud formation in intact, attached leaves occurs only at the tip, in isolated leaves at the tip and the base, whereas leaf fragments cut transversely at a distance from the tip and isolated midrib pieces form numerous shoot buds in a random distribution. Lamina fragments lacking midrib frequently fail to regenerate even after a long time of culture. It is suggested that endogeneous growth regulators in the leaf, especially the vascular tissues, play an important role in bud initiation. Very young leaves of Heloniopsis are capable forming buds and roots when isolated from the mother plants.     

Onset of nucleic acid synthesis during germination of Pisum sativum L.

Summary Measurments of total nucleic acid content of the embryonic axis indicated that massive net synthesis of both DNA and RNA was initiated at approximately 30 h after the onset of germination. The onset of net nucleic acid synthesis was marked by an increase in the rate of incorporation of [³H]thymidine into DNA, and of [³H]orotic acid and [³H]uridine into both DNA and RNA. rRNA was usually more heavily labelled than tRNA, but was not preferentially accumulated, suggesting a grater rate of turnover of rRNA than tRNA. Some incorporation of precursors occurred prior to the onset of net nucleic acid synthesis, particularly into RNA. This was taken to represent nucleic acid turnover. There was no evidence that the scavenging pathways for nucleotide biosynthesis were more important than the normal pathways in contributing precursors for net nucleic acid synthesis.     

Effect of picroliv on protein and nucleic acid synthesis.

Oral administration of picroliv, a standardised fraction of roots and rhizomes of Picrorhiza kurroa, showed stimulation of nucleic acid and protein synthesis in rat liver. Results are comparable with a standard hepatoprotective agent, silymarin.     

Bud formation on excised Heloniopsis leaf fragments: Effects of leaf age and the midrib

Detached young leaves and their segments of Heloniopsis orientalisproduced adventitious buds more readily than did detached matureleaves or their segments. Media composition had a greater influenceon bud regeneration in segments of mature leaves than in segmentsof young ones. Detached young leaves and their fragments couldform buds when placed on filter paper moistened with deionizedwater only or on an agar medium without the aid of exogenousinorganic salts and growth regulators. The presence of the midribenhanced bud formation especially in segments of mature leaves.The number of buds in midrib-less segments treated with cytokininwas greater than in untreated segments with the midrib. Theseand other results suggest that regeneration is controlled byendogenous growth regulating substances supplied via vasculartissue. (Received September 4, 1973; )     

Protein and nucleic acid synthesis during imbibition of dormant and after-ripened Vaccaria pyramidata seeds.

The regulation of nucleic acid and protein synthesis in dormant, thermodormant, and after-ripened embryos of Vaccaria pyramidata (Caryophyllaceae) has been studied. Germination of after-ripened V. pyramidata seeds is prevented by inhibitors of protein, RNA, and DNA synthesis. The synthesis of both protein and RNA is activated at the beginning of imbibition, whereas [³H]thymidine incorporation does not start until the second period of the imbibition phase. [³H]Thymidine incorporation is greatly reduced in embryos treated with cycloheximide or 6-methylpurine. There is no correlation between the level of [³H]uracil and l-[¹⁴C]leucine incorporation into macromolecules and the physiological state of the seeds: tRNA, ribosomal RNA, and poly(A)-containing RNA (probably mRNA) as well as proteins are synthesized at the same rate in both dormant and thermodormant embryos as in after-ripened embryos. The protein patterns of dormant and after-ripened embryos are similar, as shown by electrophoresis and electrofocusing of double-labeled proteins. The level of DNA synthesis, measured as [³H]thymidine incorporation, may, on the other hand, indicate the physiological activity of the seeds: [³H]Thymidine is incorporated at a high rate in after-ripened embryos only and remains at a low level in dormant or thermodormant embryos. This correlation is, however, observed only in the axes. DNA synthesis in the cotyledons does not show any relation to the developmental stage of the seeds. These results are discussed in relation to the regulation of dormancy and after-ripening of seeds.     

Development of the metanephric kidney. Protein and nucleic acid synthesis

The metanephric kidney was studied in fetal and older mice beginning at 16 days after mating of the parents. Polyribosomes from fetal kidneys labeled in vitro with 14C-labeled amino acids had 10-20 times more acid-precipitable radioactivity associated with them than polysomes from adult kidneys similarly labeled. Between 3 and 6 days after birth the rate incorporation of labeled amino acids by polyribosomes from neonatal kidneys declined sharply to only twice the value found for adult kidneys. There was no change in the shape of the polyribosome profile with increasing age, but before birth few, if any, ribosomes were bound to membranes compared with 20% 2 days after birth and between 20 and 30% in the adult. Total protein represented less than 10% of the wet weight in the fetal kidney but increased to 17% of the wet weight in the adult kidney. There was a steady decline in the concentration of RNA and DNA with respect to dry weight throughout kidney development. DNA concentration declined more rapidly than RNA concentration, so that the milligram to milligram ratio of RNA to DNA increased. In males the RNA/DNA ratio was stable at 1.3 at 40 days after birth; but in females the decline in DNA concentration was more protracted, and at 200 days after birth the RNA/DNA ratio was only 0.99. Thus, total nucleic acids show only gradual changes in concentration throughout development of the kidney, but a sharp change in the synthetic activity of the ribosomes and in their binding to membranes occurs in kidneys soon after birth.     

Short-chain fatty acid synthesis in brain. Subcellular localization and changes during development.

Acetyl-CoA synthase (EC 6.2.1.1), Propionyl-CoA synthase (EC 6.2.1.-) and butyryl-CoA synthase (EC 6.2.1.2) were measured in subcellular fractions prepared by primary and density-gradient fractionation from adult rat brain by a method resulting in recoveries close to 100%. Most of the activity of the three enzymes was recovered in the crude mitochondrial fraction. On subfractionation of this crude mitochondrial fraction with continuous sucrose density gradients, most of the activity of the three enzymes was found at a higher density than NAD+-isocitrate dehydrogenase and at about the same density as glutamate dehydrogenase, confirming earlier reported data for acetyl-CoA synthase. The finding that propionyl-CoA synthase and butyryl-CoA synthase had about the same distribution in the gradients as acetyl-CoA synthase adds support to the hypothesis that mitochondria involved in the metabolism of these short-chain fatty acids (all three of which have been shown to result in a rapid and high labelling of glutamine in vivo) form a distinct subpopulation of the total mitochondrial population. The three synthase activities were found to differ from each other in their rate of change and their subcellular localization during rat brain development. This, in combination with the observation that in gradients of adult brain preparations the three activities did not completely overlap, suggests that the three synthase activities are not present in the same proportion to each other in the same subpopulation (s) of mitochondria in the brain.