Changes in the transcription pattern of flax cotyledons after inoculation with flax rust

The rate of ³²P incorporation into RNA fractions of flax cotyledons (Linum usitatissimum L. var. Bison) was found to increase two- to three-fold by 48h after inoculation with flax rust [Melampsora lini (Pers.) Lev., race no. 3]. This was accompanied by a change in the nucleotide composition of the newly transcribed sodium chloride-soluble RNA fraction. A comparison of the nucleotide composition of the RNA synthesized in the host–parasite complex at different stages of development indicated the preferential synthesis of one or more molecular species of RNA with a high A+U/G+C ratio at a relatively early stage of infection. Treatment of healthy plants with indol-3-ylacetic acid also resulted in a substantial stimulation in the rate of ³²P incorporation into RNA but this was not accompanied by a detectable change in the nucleotide ratios of the newly synthesized RNA. These results suggest that the synthesis of one of more additional RNA species or the augmented synthesis of certain species of RNA may be a specific phenomenon elicited by host–pathogen interaction.     

Nucleic acid metabolism in cold-treated wheat embryos

The incorporation of ₃₂P into nucleic acid fractions separatedon a MAK column was compared for normally germinated and cold-treatedwheat embryos. ₃₂P accumulation in DNA fraction was decreasedby cold treatment, although that in the RNA fractions was slightlypromoted. The synthesis of the fraction, probably mRNA, elutedafter the peak of heavy rRNA was enhanced in cold-treated embryosand suppressed when the embryos were cold-treated in the presenceof 8-azaguanine, an inhibitor of vernalization. (Received May 2, 1975; )     

Catecholamine-induced phosphorylation of cardiac muscle proteins

The catecholamine-induced phosphorylation of cardiac muscle protein was investigated using a rat ventricular muscle slice preparation. Slices 0.5 mm thick and weighing 40–50 mg were incubated for 40 min in oxygenated bathing medium containing ³²P to partially label intracellular ATP. Subsequent addition of 10^?5 M isoproterenol for 10 min resulted in a 44–63% (based on protein) or a 63–70% (based on inorganic phosphate) increase in ³²P incorporation into 100 000 × g particulate and 100 000 × g supernatant (soluble) fractions without an increase into homogenates, 1000 and 29 000 × g particulate fractions prepared from the slices. The catecholamines also produced a 93% increase in ³²P incorporation ans a 27% increase in inorganic phosphate in trichloroacetic acid-insoluble protein that was obtained from ventricular slice homogenates. A significant increase in the incorporation of ³²P occurred in the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein within 2 and 1 min, respectively. While the β-adrenergic blocking agent propanolol had no effect by itself on ³²P incorporation, it prevented the isoproterenol-induced incorporation of ³²P into the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein. Removal of isoproterenol from the bathing medium eliminated the differences in ³²P incorporation, indicating that the effects of the catecholamine were reversible. Norepinephrine and ipinephrine at 10^?5 M caused phosphorylation effects similar to that of isoproterenol. When the slices were bathed under anoxic conditions isoproterenol failed to enhance the incorporation of ³²P into proteins of the 100 000 ×g particulate and supernatant fractions or acid-insoluble protein. SDS gel eloectrophoresis of ventricular slice homogenates revealed that isoproterenol enhanced the ³²P incorporation into several myocardial proteins having molecular weights of 155, 94 (glycogen phosphorylase), 79, 68–77, and 54–59 · 10³ and decreased the incorporation into a 30 · 10³ dalton protein(s). These results are consistent with the notion that catecholamines may increase the phosphorylation of myocardial proteins in the intact myocardium which in turn may play a role in catecholamine-induced glycogenolysis and augmentation of contractility.     

Evidence for Two Metabolically Distinct Types of Ribonucleic Acid in Chromatin and Nucleoli

Patterns of radioisotope incorporation are useful characteristics in describing cellular RNA fractions, and have indicated a distinctive "nuclear" RNA. In order to characterize the RNA fractions of the two nuclear components, nucleoli and chromatin, and to determine thereby the precise localization of the RNA typical of isolated nuclei, time-courses of P³² incorporation into nucleolar, chromosomal, and cytoplasmic RNA of Drosophila salivary glands have been determined from autoradiograms. Two experiments are reported which cover 12 and 18 hour periods, including an initial 2 hour feeding on P³². Concentrations of RNA-P³² (identified by ribonuclease digestion) were determined by grain counts. After 1 hour only the nucleolar RNA is labelled. Activity is detectible in chromosomal and cytoplasmic RNA after the 2nd hour. The nucleolar fraction reaches its maximum activity shortly after transfer of the larvae to non-radioactive food, the other fractions several hours later. Maximum activities persist in the chromosomal and cytoplasmic fractions; nucleolar activity decreases after the 9th hour. The observed differences in times at which incorporation begins and maximum activities are reached, and in maintenance of maximum activities indicate that chromosomal and nucleolar RNA are distinct fractions. The metabolic characteristics which have been ascribed to "nuclear" RNA apply only to the nucleolar fraction.     

A comparative study of RNA fractions mediating delayed sensitivity to a chemically defined antigen in vitro.

Hot-cold phenol extracts of RNA prepared from guinea pigs sensitized to mono (p-azobenzene-arsonate)-N-chloroacetyl-l-tyrosine (ARSNAT) contains limited but distinct fractions able to transfer ARSNAT or KLH sensitivity to guinea pig peritoneal exudate cells in vitro. Each of these fractions have been compared by oligo(dT) affinity chromatography and sucrose density gradient analysis. One RNA fraction initially obtained from a sucrose density gradient (designated as B fraction) possessed two separate peaks and contained polyadenylic acid sequences as evidenced by its ability to bind to an oligo (dT) column. Another fraction (Fraction II) initially isolated by oligo (dT) affinity chromatography possessed two peaks after sucrose density gradient analysis, contained poly-A sequences, and had an S-value range approximating the B fraction. RNA fractions prepared from the liver or skeletal muscle of sensitized guinea pigs fails to transfer ARSNAT sensitivity and all fractions are completely inactivated by bovine pancreatic RNase. The results suggest that portions of density gradient prepared B fraction and Fraction II binding to oligo (dT) cellulose may represent the same and/or similar moieties of immunobiologically active RNA.     

Some properties of cortisol-receptor complex isolated from cytoplasm of rat liver and its effect on biosynthesis of nuclear RNA

The cortisol binding compound, from the cytoplasm of rat liver, was purified by gel filtration and precipitation with ammonium sulphate. The binding compound from rat liver cytoplasm, has been found to consist of two distinct protein fractions. The proteins eluted from DEAE-cellulose column chromatography at 0.04 M and 0.18 M concentrations of KCl, have two different isoelectric points, one at pH 4.85–5.00, and another at pH 5.85–6,10, but only the fraction eluted at 0.04 M KCl was found to be able to stimulate the incorporation of ³²P into RNA of incubated rat liver nuclei. The highest values of ³²P incorporation in the nucleic acids of incubated nuclei were obtained with partially purified hormone protein (s) complexes wich were precipitated with ammonium sulphate saturation between 20–25% and 25–30%.     

Metabolism of nucleic acids in wheat roots in dependence on nutritive conditions

Influence on individual types (fractions) of nucleic acids (NA) was studied in roots of wheat plants grown in various cultivation media, namely in distilled water, in sodium humate and in a nutrient solution. NA’s were prepared by means of the phenol technique. Using separation on a methylalbumine column (MAK) five fractions were obtained, namely: fraction I.— low molecular weight substances, fraction II.—soluble RNA, fraction III.—DNA-RNA, and the ribosomal RNA in two fractions, IV.—(l r-RNA) and V.—(h r-RNA). Of the NA fractions investigated, the r-RNA fraction was noticeably influenced by the kind of nutrition, its amount varying in a certain proportion to the growth intensity affected by the cultivation medium. The other NA fractions were not apparently affected. The metabolical turnover of the individual NA types (as observed from the specific³²P activity) was considerably influenced by the kind of nutrition as well. The specific³²P activity in all NA fractions of wheat roots cultivated in a nutrient solution was approximately double that in roots of wheat plants cultivated in distilled water and Na-humate. Changes in the specific³²P activity of r-RNA were again considerably evident. With regard to root growth their relative values appeared in an inverse proportion to the changes in the r-RNA content. The specific³²P activity decreased with increasing growth intensity. Besides changes in the r-RNA fraction, changes in fraction I. were apparent. An especially striking decrease in the specific³²P activity was found in roots of plants grown in H₂O, namely by about one order in comparison with its specific activity in fraction I. from roots of plants grown in Na-humate and in a nutrient solution.     

Effect of light on ribonucleic Acid metabolism in greening maize leaves

The effect of illumination on the incorporation of labeled precursors into RNA of dark-grown maize (Zea mays) leaves was studied using either ³²P-phosphate or double labeling with ¹⁴C- and ³H-uridine. In the dark, label was preferentially incorporated into etioplast ribosomal RNAs. Incorporation into this fraction and into lower molecular weight fractions was strongly and preferentially stimulated by light during the first 2 hours of illumination. The effect persisted after illumination was terminated. The possibility that light-induced alterations in plastid ribosomal RNA metabolism may not be required for chlorophyll accumulation in maize is discussed.     

Biochemical changes in mouse liver after palmitoyl-ethanolamide (PEA) administration

PEA (palmitoylethanolamide; N-(2-hydroxyethyl)palmitamide) daily and orally administered to male mice caused: (1) increased incorporation of labelled orotic acid into DNA and RNA, (2) an increase in the activity of uridine kinase and decrease of tryptophan pyrrolase, (3) decreased ribonuclease activity of isolated liver ribosomes, (4) raising of specific radioactivity after injection of labelled amino acids in both mitochondrial and microsomal fractions of liver homogenate, (5) increased incorporation of [¹⁴C]-palmitic acid and ³²P into liver phospholipids.     

Effect of 5-fluoroorotic acid administration of the 32 P base composition, DNA-RNA hybridization properties, and labeling of polyadenylate-rich RNA in the cytoplasm of rat liver cells

5-Fluoroorotic acid treatment lowered the (Guanine + Cytosine)/(Adenine + Uracil) base ratio of ³²P-labeled microsomal RNA from a control value of 1.36 to 1.00. Low doses of actinomycin D, which are effective in inhibiting ribosomal RNA synthesis without significantly affecting messenger RNA synthesis, caused a similar decrease in the base ratio. Microsomal RNA labeled by [³H]orotate in the presence of 5-fluoroorotic acid had approximately $\text{1}\text{2}$ the specific radioactivity but twice the hybridization efficiency of RNA labeled in its absence. Evidence is presented that this RNA (1) has a different structure from that of ribosomal RNA, (2) hybridizes to DNA with an efficiency consistent with that of other published studies of polysome-associated messenger RNA, and (3) possesses sequences which are present in other samples of liver microsomal RNA but not in kidney microsomal RNA. These properties differ from those known to be exhibited by 18 S and 28 S ribosomal RNA. Electrophoretic analysis of this [³H]orotate-labeled microsomal RNA indicated that the analogue greatly inhibited precursor incorporation into ribosomal RNA but had little or no effect on incorporation into messenger RNA. Ribosomal RNA and polyadenylate-rich nonribosomal RNA were prepared from total polyribosomes by phenol extraction at pH 7.6 and pH 9.0, respectively. 5-Fluoroorotic acid inhibited [³H]orotate or ³²P_i incorporation into the pH 7.6 fraction much more effectively than incorporation into the pH 9.0 fraction. A subfraction of the pH 9.0 RNA which was retained by a polythymidylate-cellulose column had a greatly increased adenylate content.     

Plasmodium berghei: I. Immunochemical properties of fractions of a soluble extract

Soluble extracts of Plasmodium berghei were separated into 12 fractions following preparative disc electrophoresis in polyacrylamide gel. One or two protein bands were detected in each fraction by analytical disc electrophoresis. Similarly, one or two precipitinogens were generally detected in each of Fractions 1 through 11 by immunoelectrophoresis and by double immunodiffusion in agar gel, while the unfractionated extract contained 10 precipitinogens. Antisera produced in rabbits against each fraction each contained two or three (sometimes five) antiplasmodial precipitins demonstrable by immunoelectrophoresis. Serial fractions obtained in separate runs were closely similar to each other, although some degree of overlapping sometimes occurred between neighboring fractions. Glycoproteins were detected in all the fractions, but chiefly in Fractions 4 and 12. The bulk of the RNA in the extract was located in Fraction 4, while hemoglobin was usually confined to Fraction 6. The molecular weights of the soluble components of P. berghei range between 8000 and 130,000.     

Changes in Nucleic Acid Metabolism of Excised Barley Leaves During Senescence and the Effect of Kinetin on these Changes

The changes in the amount, rale of synthesis and the nucleotide composition of different RNA fractions in excised barley leaves floated on water or kinetin (10 mg/l) in the dark were examined. In excised leaves floated on water all nucleic acid components declined and these declines were retarded by kinetin. Barley leaves floated on water showed a stimulation of ³²P incorporation into various RNA fractions within 48 hours followed by a decline after 96–144 hours. The leaves floated on kinetin, however, showed an even higher incorporation of ³²P into UNA by 48 hours which remained at a comparatively higher level throughout the experiment. In spite of the above changes in RNA synthesis significant differences in the ³²P sucrose gradient profiles or in the ³²P nucleotide composition of UNA from water and kinetin floated leaves were not noted. The results of this study show that important changes in nucleic acid metabolism occur during the early stages of leaf senescence and that alterations in nucleic acid metabolism during senescence and during kinetin treatment may involve quantitative and only subtle qualitative changes.     

Phospholipide Turnover in Microsomal Membranes of the Pancreas during Enzyme Secretion

After incubation of pigeon pancreas slices with P³² and isolation of various fractions by differential centrifugation the deoxycholate extract of the microsome fraction was found to account for over half of the phospholipide P and over half of the P³² incorporated into the phospholipides. The remaining phospholipide P and P³² were fairly evenly distributed in the nuclei, zymogen granules, mitochondria, microsomal ribonucleoprotein particles, and the soluble fraction. When enzyme secretion was stimulated with acetylcholine about two-thirds of the increment in radioactivity in the total phospholipides was found in deoxycholate soluble components of the microsome fraction. The remainder of the increment was distributed in the other fractions. This indicates that the cellular component in which the increase in phospholipide turnover occurs on stimulation of secretion is a membranous structure. Evidence is presented which indicates that the increment in radioactivity in the non-microsomal fractions on stimulation of secretion is due to contamination of these fractions with fragments of the stimulated membranous structure. The distribution of P³² radioactivity in each of the chromatographically separated phospholipides in the various fractions from unstimulated tissue paralleled the distribution of radioactivity in the total phospholipide fraction, indicating that individual phospholipides are not concentrated in different fractions but are associated together in the membranous structures of the microsome fraction. The major proportion of the stimulation of the turnover of the individual phospholipides also occurred in the microsome fraction. The distribution of radioactivity from glycerol-1-C¹⁴ in the total phospholipides and in the individual phospholipides in the various fractions was similar to the distribution of P³². In the microsome fraction acetylcholine stimulated the incorporation of glycerol-1-C¹⁴ in each phospholipide which showed a stimulation of P³² incorporation. The significance of the turnover of phosphatides in microsomal membranes in relation to the mechanism of secretion is discussed.     

Auxin-stimulated RNA Synthesis in Oat Coleoptile Cells

Using oat coleoptile segments the following results were obtained. Ten mg/l auxin (indole-3-acetic acid) increased the incorporation of uracil-2-¹⁴C and orthophosphate-³²P into RNA fraction during a relatively short incubation period. Stimulation of ³²P incorporation due to auxin was found only in the region heavier than ribosomal RNA, probably in the messenger RNA region. The stimulation of uracil-2-¹⁴C incorporation into RNA caused by auxin was not influenced by the presence of 0.3 M mannitol which prevents osmotically the water absorption of cells. It is concluded that auxin primarily stimulates the biosynthesis of RNA, possibly messenger, in oat coleoptile cells.     

In situ phosphorylation of tyrosine hydroxylase in chromaffin cells: Localization to soluble compartments

The present studies investigated the subcellular distribution of acetylcholine's effects upon the phosphorylation of tyrosine hydroxylase in isolated purified bovine adrenal chromaffin cells. After labeling the intact chromaffin cells with ³²P_i, over 90% of the [³²P]tyrosine hydroxylase was found in soluble fractions. Stimulation of the cells with acetylcholine, the natural secretagogue of chromaffin cells, increased the phosphorylation of tyrosine hydroxylase and over 90% of the increase was associated with soluble tyrosine hydroxylase. Homogenates and subcellular fractions from chromaffin cells were also prepared and phosphorylated in vitro in an attempt to optimize detection of tyrosine hydroxylase phosphorylation. In chromaffin cell homogenates, both 8-bromo-cyclic AMP and calcium increased ³²P incorporation into tyrosine hydroxylase, and again over 90% of the increase was observed in soluble fractions. In the particulate fraction, phosphorylation of a band which comigrated with tyrosine hydroxylase in electrophoresis was occasionally detected but only with very long autoradiographic exposures.Tyrosine hydroxylase enzymatic activity in the isolated purified chromaffin cells was also found to be associated predominantly (approx 90%) with soluble fractions. In contrast, a large portion (40–50%) of the tyrosine hydroxylase activity from crude bovine adrenal medullae was associated with the particulate fraction.The data indicate that although tyrosine hydroxylase (and possibly kinases) can associate with particulate fractions when isolated from crude bovine adrenal medullae, the enzyme is predominantly soluble when isolated from the isolated cells. Further, the effects of acetylcholine on the isolated chromaffin cells are predominantly associated with this soluble tyrosine hydroxylase and its attendant kinases.     

Bean abscission cellulase : characterization of a cDNA clone and regulation of gene expression by ethylene and auxin

The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACl). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACl codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)⁺ RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACl decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACl or expression of cellulase activity.     

RNA extracts with polyadenylic acid sequences transfer specific sensitivity for a low molecular weight antigen (MW 486).

RNA prepared from the lymphoid tissues of guinea pigs specifically sensitized to mono(p-azobenzene-arsonate)-N-chloroacetyl-l-tyrosine (ARSNAT) (MW = 486) was fractionated by oligo(dT)-cellulose affinity chromatography. Two fractions designated as I and II were eluted from the column. Fraction II, binding to the column and containing polyadenylic acid sequences, transferred ARSNAT or keyhole-limpet-hemocyanin (KLH) sensitivity to nonsensitized guinea pig peritoneal exudate cells (GP-PEC) in 14 experiments. Fraction I was unable to transfer KLH or ARSNAT sensitivity to GP-PEC. The amount of Fraction II needed to transfer ARSNAT sensitivity was 10 times less than previously reported. Synthetic nonlymphoid cell poly(A) tested in this system failed to transfer ARSNAT or KLH sensitivity to GP-PEC. Both Fractions I and II were inactivated by ribonuclease. The results suggest a possible messenger function for the poly (A)-containing RNA fractions.     

Synthesis of mitochondrial RNA in disaggregated embryos of Xenopus laevis

Mitochondrial RNA synthesis was studied during early Xenopus laevis development using disaggregated embryos. By gel electrophoresis, the labeled mitochondrial RNA consisted of three discrete species. Transfer RNA was the only mitochondrial RNA species which was methylated. Incorporation of ³²P into mitochondrial RNA was detectable as early as the blastula stages. The level of incorporation was found to increase with increasing developmental age. All species of mitochondrial RNA appeared to be labeled at a constant rate relative to one another. Partial analysis of the acid-soluble nucleotide pool indicated that the observed incorporation probably represents a net increase in the synthetic rate for mitochondrial RNA as development proceeds.     

Properties of Single- and Double-stranded Ribonucleic Acid from Barley Plants Infected with Bromegrass Mosaic Virus

Incorporation of ³²P into nucleic acids in barley plants infected with bromegrass mosaic virus (BMV) was analyzed by chromatography on methylated albumin kieselguhr (MAK) columns. Treatment with actinomycin D reduced the synthesis of ribosomal ribonucleic acid (RNA) to low levels and allowed the detection of the three components of BMV-RNA in vivo. The kinetic study on ³²P incorporation into these BMV-RNA components suggested that a single cleavage occurred in some of the intact RNA shortly after completion of its synthesis, giving rise to the small and medium components. Chromatographic analyses also revealed a double-stranded, ribonuclease-resistant RNA which has been purified by differential extraction, sucrose-density gradient centrifugation, and MAK column chromatography. This RNA sediments at approximately 14S, is alkali-labile, and has a sharp thermal transition with a T_m of 96 C in 0.1 × standard saline citrate buffer, as determined by susceptibility to ribonuclease. The RNA is absent in uninfected barley plants.