TRANSCRIPTIONAL CONTROL OF PROTEIN SYNTHISIS IN THE DEVELOPING OVARIES OF BOMBYX MORI*

Amino acid incorporation was studied with cell-free extracts and ribosomes prepared from pupal ovaries at different ages of Bombyx mori. Poly(U)-directed ³H-phenylalanine incorporation attained a maximum rate at a certain stage of development, but soon dropped to a low level and was replaced by ³H-leucine incorporation, which was due to endogenous mRNA. The latter incorporation occurred at the stage when actual protein synthesis takes place in the ovaries. “Run-off” of the ribosomes which had a high endogenous activity resulted in an enhancement of the poly(U)-dependent activity. The results indicate that the protein synthesis in the ovary is mainly controlled at the level of mRNA. This was further supported by the fact that the relative amount of an ovarian poly(A)-containing “mRNA” fraction increased in parallel with the endogenous activity.     

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.     

Untersuchungen zur Entwicklung des Phycomyceten Allomyces arbuscula

Polysomes were isolated both from growing gametophytes of Allomyces arbuscula and from gametangia prepared from mycelia at different periods during gametogenesis. Analysis of polysomes by sucrose gradients showed that ribosomes present in the gametangia monosome pool were shifted into polysomes. This shift was found to be correlated with gametangia differentiation. The ribosome distribution remained virtually unchanged during the early stage of gamete formation. In mature gametes and swarming zygotes a low level of polysomes was detected. Labeling of rRNA by ³²PO₄ demonstrated a de novo synthesis of monosomes throughout the period of gametangia differentiation. No incorporation of ³²PO₄ was found to be present in ribosomes prepared from gametangia after onset of gamete formation. On the basis of these labeling experiments it is concluded that radioactivity in polysomes extracted from mature gametes and swarming zygotes can be attributed in part to conserved mRNA.Synchronous formation of gametangia was induced by transferring the vegetative mycelia from growth medium into a low salt buffer. Under these conditions the incorporation of either ³²PO₄ or ³H-uridine into RNA, particularly into rRNA, was found to be markedly decreased. This obviously indicates a shutdown of RNA synthesis. rRNA from induced mycelia examined by polyacrylamide gel electrophoresis was found to be severely degraded. In contrast to this, rRNA isolated from ribosomes of developing gametangia and from gametes exhibited no degradation products. It is suggested that endonucleases cause rRNA hydrolysis in the hyphal cytoplasm during gametangia differentiation. Ribosomes compartmentalized in gametangia seem to be inaccessible to nucleases during the later process of gametogenesis.

---

Abkürzungen MAK Methyl-Albumin-Kieselgur - PAA Polyacrylamid - stains all 4,5,4,5-Dibenzo-3,3-diäthyl-9-methylthiacarbocyaninbromid (Serva, Heidelberg)     

Transfer Ribonucleic Acid in KB Cells Infected with Adenovirus Type 2

Populations of transfer ribonucleic acid (tRNA) extracted from control and type 2 adenovirus (Ad2)-infected KB cells were compared. No consistent differences in acceptor activity for 11 amino acids were observed. Comparison of methylated albumin-kieselguhr (MAK) elution profiles of arginyl-tRNA from control and infected cells revealed a minor modification in that the proportion of arginyl-tRNA eluting at high salt concentration was somewhat greater in infected cells. No similar differences were observed in MAK elution profiles of aspartyl-, isoleucyl-, leucyl-, phenylalanyl-, seryl-, tyrosyl-, and valyl-tRNA. Hybridization of 4S RNA from infected cells labeled by incorporation of ³H-uridine with Ad2 deoxyribonucleic acid revealed the presence of a complementary species of RNA in this preparation. Hybridization of ³H-arginyl-tRNA and of ³H-aminoacyl-tRNA labeled by charging with ³H-arginine or a ³H-mixture of amino acids, respectively, failed to detect the presence of virus-specific tRNA in Ad2-infected cells.     

Biochemical properties of nucleic acids of chloroplasts in Spirogyra1

In chloroplasts and nuclei of Spirogyra separated by discontinuoussucrose dnesity gradient centrifugation, five types of nucleicacids were found by MAK column chromatography; tRNA, low molecularweight rRNA, DNA, light and heavy rRNAs. Chloroplast rRNA was found to differ from cytoplasmic rRNA,by comparative studies on its base composition, its elutionpattern by MAK column chromatography and its sedimentation profilewith sucrose density gradient centrifugation. Molecular species of chloroplast DNA differed from those ofnuclear DNA in their thermal denaturation points (Tm), theirbehaviour on an IRC-50 column, their buoyant density in CsCland their base analyses. (Received September 13, 1972; )     

Biosynthetic Incorporation of CIS-Parinaric Acid Into Radioactive SN-3-Phosphatidic Acid

Isolated guinea pig liver microsomal membranes catalyzed the incorporation of naturally occurring cis-parinaric acid into sn-3-[U-¹⁴ C]glycerophosphate. This resulted in the formation of sn-3-[¹⁴C](parinaroyl)phosphatidic acid, which was isolated by Chelex-100 and DEAE-cellulose column chromatography and further purified by Sephadex-G 25. The sn-3-[¹⁴C](parinaroyl)phosphatidic acid thus obtained exhibited absorption and fluorescence spectra substantially different from the cis-parinaric acid. Distribution of the incorporated cis-parinaric acid between the hydroxyl groups of biosynthesized sn-3-[¹⁴C]phosphatidic acid was determined by dearadation with Crotalus adamateus venom. This established that the major portion of the incorporated cis-panaric acid esterified the secondary hydroxyl group in the sn-3-[¹⁴C] phosphatidic acid, while the primary hydroxyl group was esterified to a significantly lesser degree. The similarity between the biochemical incorporation of isomeric doxyl stearic acids into lipids of biological membranes and that of cis-parinaric acid into sn-3-phosphatidic acid described 1n this communication are discussed in relation to the possible use of these probes in studies of intact biological membranes.     

TRANSFER RNA AND THE REGULATION OF PROTEIN SYNTHESIS IN RAT CEREBRAL CORTEX DURING NEURAL DEVELOPMENT

Protein synthesis was measured in ribosomal systems derived from the cerebral cortex of 5-and 35-day-old rats. Under optimal conditions incorporation of radioactive leucine per mg ribosomal protein was four times higher with ribosomes from the younger animals than with ribosomes from the 35-day-old rats. This suggests that a decrease in the rate of protein synthesis occurs during neural development. Both ribosomes and the pH enzyme fraction from the cerebral cortex of 35-day-old rats had lower activities than preparations from the younger rats. Cerebral cortical ribosomes from 35-day-old animals had a lower polyribosome content than similar preparations from 5-day-old rats. A three-fold higher requirement for the pH 5 enzyme fraction was observed with the ribosomal system from 5-day-old rats, an observation which correlated with the yields of pH 5 enzyme and ribosomal protein from the younger tissue. The nature of the changes in the composition of the pH 5 enzyme fraction was investigated. Methylated albumin kiesselguhr (MAK) and Sephadex G-75 column chromatography showed that RNA from the pH 5 enzyme fraction was heterogeneous, containing tRNA, rRNA, and a small molecular weight RNA. This latter RNA, perhaps a degradation product of rRNA, comprised the greatest portion of RNA from the pH 5 enzyme fraction of cerebral cortex. The data obtained with MAK chromatography were used to estimate the total tRNA content of the cerebral cortex, with no age-related differences being observed. Since evidence of RNA degradation was seen, tRNA was also isolated by phenol extraction of whole cerebral cortex in the presence of bentonite. Purification of tRNA by NaCl and isopropanol fractionation gave preparations with no detectable rRNA or small molecular weight RNA. With this purification method, the tRNA yield was greater than estimated by the MAK method, demonstrating that losses of tRNA occurred during the cell fractionation steps. With the purification method 1.6 times more tRNA was obtained from the cerebral cortex of 5-day-old animals than from the older tissue. This higher level of tRNA in the younger, more active tissue appeared to involve all tRNA species, since in vitro aminoacyiation studies revealed nearly identical acceptance values for 18 individual amino acids. These results suggest that the rate of protein synthesis in cerebral cortex is regulated in part by the total amount of tRNA present to translate the higher level of polysome-bound mRNA.     

Adaptation of the 3H-Leucine Incorporation Technique to Measure Heterotrophic Activity Associated with Biofilm on the Blades of the Seaweed Sargassum spp.

The ecological interaction between microorganisms and seaweeds depends on the production of secondary compounds that can influence microbial diversity in the water column and the composition of reef environments. We adapted the ³H-leucine incorporation technique to measure bacterial activity in biofilms associated with the blades of the macroalgae Sargassum spp. We evaluated (1) if the epiphytic bacteria on the blades were more active in detritus or in the biofilm, (2) substrate saturation and linearity of ³H-leucine incorporation, (3) the influence of specific metabolic inhibitors during ³H-leucine incorporation under the presence or absence of natural and artificial light, and (4) the efficiency of radiolabeled protein extraction. Scanning electron microscopy showed heterogeneous distribution of bacteria, diatoms, and polymeric extracellular secretions. Active bacteria were present in both biofilm and detritus on the blades. The highest ³H-leucine incorporation was obtained when incubating blades not colonized by macroepibionts. Incubations done under field conditions reported higher ³H-leucine incorporation than in the laboratory. Light quality and sampling manipulation seemed to be the main factors behind this difference. The use of specific metabolic inhibitors confirmed that bacteria are the main group incorporating ³H-leucine but their association with primary production suggested a symbiotic relationship between bacteria, diatoms, and the seaweed.     

The Interrelationship of 2,4-Dichlorophenoxyacetic Acid with Molecular Components of the Cell during Callus Induction

This study investigates the fate of ¹⁴C labelled 2,4-D used as a trigger compound during callus induction (dedifferentiation). It also traces the special relationship of 2,4-D with some high molecular weight components in vivo, in which 2,4-D-2-¹⁴C was found combined with high molecular weight component-fractions. Nucleic acids showed very low radioactivities, based on MAK column chromatography and centrifugal profiles from sucrose density gradient treatments, but there was no specificity in this radioactive incorporation. Centrifugal profiles and Pronase treatment indicate that protein combines with 2, 4-D-2-¹⁴C. These results support the claim for a model of auxin-induced derepression, in which auxin interacts with its binding protein.     

Biosynthesis of nucleic acids in pea (Pisum sativum L.) buds released of dominance

Biosynthesis of nucleic acids in the pea buds released from apical dominance by decapitation has been studied. The rate of biosynthesis of all nucleic acid classes separated by means of methylated albuminkieselgur (MAK) column chromatography was found to be increased in released pea buds as judged by the extent of orotic-6-¹⁴C acid incorporation. This increase has been observed in all experimental points examined (15, 24, 36 and 48 h after decapitation), being the most pronounced 15 h after decapitation. After that a certain decrease of the specific radioactivities of nucleic acids has been registered, but even 48 h after decapitation specific activities of the main nucleic acid classes were higher in comparison with those found in dominant buds. Variations of the free pyrimidine nucleotide pools were about 14% suggesting that this parameter did not influence the extent of the uptake of labelled orotic acid.     

Incorporation of cytokinin N-benzyladenine into tobacco callus transfer ribonucleic Acid and ribosomal ribonucleic Acid preparations

The incorporation of the cytokinin N⁶-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N⁶-benzyladenine-8-¹⁴C or N⁶-benzyladenine-8-¹⁴C: benzene-³H(G) has been examined. N⁶-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N⁶-benzyladenosine recovered from the RNA preparations was associated with the rRNA. Purification of the crude rRNA by either MAK chromatography or Sephadex G-200 gel filtration had no effect on the N⁶-benzyladenosine content of the RNA preparation. The distribution of N⁶-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N⁶-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T₂ and phosphatase, or ribonuclease T₂ and a 3′-nucleotidase. N⁶-benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T₂ or phosphatase alone or by successive treatment with ribonuclease T₂ and a 5′-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T₁ and pancreatic ribonuclease converted the N⁶-benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N⁶-benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant.     

Seasonal pattern of glycosylation in frog liver

The circannual behaviour of glycosylation and protein synthesis in frog liver slices was studied following the incorporation of³H-galactose and¹⁴C-glucosamine into glycolipids and glycoproteins and³H-leucine into proteins. The activity of two enzymes the galactosyl-transferase and the N-acetyl-glucosaminyl-1-P-transferase was determined. The incorporations of both sugars into the soluble fraction and into the lipid extract present a maximum during the spring-summer period. The incorporation into the protein fraction displays a different pattern:¹⁴C-Glucosamine and³H-leucine incorporation increases from winter to a maximum in autumn; the incorporation of³H-Galactose has a sharp peak during spring. The pattern of glycosyltransferase activities is similar to the pattern of incorporation of the two saccharides into proteins, indicating these enzymes as important control points for glycosylation in Anurae.     

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; )     

RNA synthesis in germinating red bean seeds

Nucleic acids from ³²P-labelled germinating red bean seeds wereinvestigated by means of MAK column chromatography. 1) In cotyledons,synthesis of D-RNA occurred in the early stages of germination,3 to 24 hr after the onset of imbibition. ³²P was also incorporatedinto rRNA continuously at rather a moderate rate. DNA-RNA hybridizationexperiments revealed that the proportion of heterogeneous RNA(D-RNA) to rRNA decreased gradually. Nucleotide analysis suggestedthat tRNA was synthesized de novo, and that its CCA-end exchangewas remarkable at early stages of germination. 2) In embryos,however, the incorporation of ³²P into rRNA was very much greaterthan into D-RNA, and the exchange reaction at CCA-end of tRNAwas not detected. The role of D-RNA, found in cotyledons inthe initial stages of germination, was discussed. ¹Present address: Research Institute for Biochemical Regulation,Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya,Japan. (Received May 10, 1972; )     

The influence of pH on arsenazo III.

None of the methods already reported for elimination of pectins from rRNA extracts allowed the complete removal of methylated polysaccharides from methyl-labeled cytoplasmic 17 and 26 S rRNA preparations of sycamore (Acer pseudoplatanus L.) cells. An improved procedure for purifying large amounts of higher plant cytoplasmic rRNA labeled on the methyl groups was investigated. Bulk cellular RNA from sycamore cells incubated for 24 to 36 h with methyl-labeled methionine was extracted at 4°C by the phenol-extraction procedure. Most of the pectic compounds (that accounted for about 30% of the total label of RNA extracts) was selectively precipitated, before the 66% ethanol precipitation of nucleic acid, by bringing the deproteinized aqueous layer to 10% ethanol ?0.15 m sodium acetate. Cytoplasmic rRNA, 17 and 26 S, were isolated by repeated sucrose gradient sedimentations and further chromatographed on a methylated albumin kieselgurh (MAK) column. The old-fashioned MAK chromatography proved to be very useful for elimination of residual pectins, since these compounds eluted in the void volume of the column. This purification procedure gave in a reproducible way cytoplasmic 17 and 26 rRNA virtually free of any labeled DNA, mRNA, plastid rRNA, and pectic compounds.     

Protein synthesis during the cell cycle of L5178Y cells

There are two peaks of ³H-leucine incorporation in the cell cycle of L5178Y cells. The first, during S stage, corresponds to a peak of ³H-leucine incorporation into the nuclear fraction. The second, during S or early G2, corresponds to a peak of ³H-leucine incorporation into the mitochondrial fraction. The rate of protein synthesis is unique for the proteins from each of the four fractions, nuclear, mitochondrial, microsomal, and soluble.The SDS polyacrylamide-gel electrophoretic patterns of ³H-leucine incorporation were different among three subcellular fractions: nuclear, mitochondrial, and microsomal + soluble. However, the incorporation pattern for each fraction remains qualitatively the same throughout the cell cycle.     

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of ovariectomy and administration of oestradiol-17beta on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomal preparation

1. Three procedures for isolating ribonucleoprotein particles from the cytoplasmic fraction of rat-uterus homogenates are described. By procedure 1, ribonucleoprotein particles were isolated in the presence of 5mm-Mg²⁺ and 25mm-K⁺, and the postmitochondrial supernatant fraction was made to 1·3% (w/v) in potassium deoxycholate. About 50% of the RNA and protein of the microsomal fraction was recovered in the monomeric ribosomes isolated. By procedure 2, ribonucleoprotein particles were isolated in the presence of 10mm-Mg²⁺ and 0·1m-K⁺, and in the absence of detergent. The ribosomes obtained were primarily polymeric, but recovery of microsomal RNA and protein was only 32%. By procedure 3, ribonucleoprotein particles were isolated according to procedure 1 but without the use of detergent. A mixture of polymeric and monomeric ribosomes was obtained, and the recovery of microsomal RNA and protein was about 60%. 2. Uterine polymeric and monomeric ribosomes, isolated by procedure 3 and designated `polyribosomal preparation', were examined for protein-synthesizing capabilities. The principal properties of the cell-free protein-synthesizing system containing the polyribosomal preparation are described. The efficiency of amino acid incorporation in the complete system incubated for 30min. and containing the polyribosomal preparation was found to be either 2·5 molecules of [¹⁴C]leucine or 2·2 molecules of [¹⁴C]-valine incorporated/ribosome. Assay of the preparation in the complete cell-free system containing 10mm-sodium fluoride indicated that 40% of the incorporation activity is a result of initiation of new polypeptide chains and 60% is due to completion of previously existing chains. Monomeric ribosomes obtained by various treatments of the polyribosomal preparation with sodium fluoride, ribonuclease and potassium deoxycholate had decreased incorporation activity in the cell-free system. However, monomeric ribosomes obtained by treatment with sodium fluoride only had an incorporation activity 50% greater than that of monomers obtained by treatment with ribonuclease only. 3. The results indicate that uterine polymeric and monomeric ribosomes are sites of amino acid incorporation in vivo and in vitro. It is concluded that most polymeric and monomeric ribosomes occurring in the cytoplasmic fraction of the uterus are free and unattached to membranes, and that the polyribosomes are relatively unstable.     

Topographical differences of RNA labelling in rat brain after intraventricular administration of labelled RNA precursors

Summary ¹⁴C-uridine or ¹⁴C-orotic acid was injected into the third or fourth brain ventricle of adult rats. The rate of incorporation of these precursors into the RNA of various brain regions was studied by autoradiography. 0.5 hour, 1 hour, 2 hours and 4 hours after the injection of labelled uridine and/or orotic acid into the third ventricle, a very uneven labelling of different brain regions was observed. The highest grain density was found over the ventricular walls and in the closely adjacent brain tissue; the intensity of labelling decreased sharply with distance from the ventricular lumen. 24 hours after intraventricular injection, a medio-lateral gradient of grain density was no longer observed. An intense labelling of leptomeninx (especially at the base of the brain) and of ependymal cells was observed at all time intervals investigated. At time intervals 0.5–2 hours the grain density of these structures surpassed by a considerable amount the grain density over neurons, glial cells or neuropile.Two hours after the injection of ¹⁴C-orotic acid into the fourth ventricle, the grains were mainly localised over leptomeningeal cells and vessels at the base of the brain and in the adjacent narrow strip of brain tissue. The rest of the brain was only very faintly labelled.     

Purification of reticulocyte ribosomes using polyuridylic acid: sepharose chromatography.

Deoxycholate-KCl washed reticulocyte ribosomes are purified by affinity chromatography using Sepharose columns to which polyuridylic acid has been covalently bound. Upon passage through the column under nonenzymic conditions (high Mg²⁺:K⁺ ratio) approximately 10% of the ribosomes are retained. These ribosomes are then eluted with a buffer containing a high K⁺:Mg²⁺ ratio and are assayed for activity in various steps of the elongation process of protein synthesis. Approximately a 3- to 4-fold increase in the various activities compared with control ribosomes is obtained.     

The role of ribosomal ribonucleic acid in the structure and function of mammalian brain ribosomes

In order to resolve the functional role of intact rRNA in polypeptide chain elongation mouse brain ribosomes were treated with dilute pancreatic or T(1) RNAase (ribonuclease). After RNAase treatment, several physical-chemical properties as well as the functional activity of the ribosomes were measured. RNAase treatment resulted in the extensive hydrolysis of both 18S and 28S rRNA; however, the sedimentation properties of mono-ribosomes were unaltered and more than 90% of the relatively low-molecular-weight RNA fragments remained associated with ribosome particles. Analysis of the ability of RNAase-treated ribosomes to participate in cell-free protein synthesis showed that ribosomes with less than 2% intact rRNA retained more than 85% of their activity in polyphenylalanine incorporation. Proof that the incorporation of phenylalanine by ribosomes with hydrolysed rRNA actually represented active translocation was obtained by the effective inhibition of incorporation by diphtheria toxin. In addition, the oligopeptide products of protein synthesis could be identified by BD (benzoylated diethylaminoethyl)-cellulose column chromatography. Analysis of the size distribution of oligopeptides synthesized by normal and RNAase-treated ribosomes showed no significant differences which indicated that there was no change in the proportion of ribosomes engaged in protein synthesis. Thus strong RNA-protein and protein-protein interactions must serve to maintain the functional integrity of ribosomes even when the rRNA is extensively degraded. The ability of the enzyme-treated ribosomes to efficiently incorporate amino acids clearly demonstrated that ;intact' rRNA is not required for protein-synthetic activity.