The ribonucleic acids of Crithidia fasciculata

Crithidia fasciculata ribosomes were found to be 80S and to dissociate into 58 and 41S subunits; on 5 to 50% sucrose gradients, rRNA was separated into 25, 18, and 5S components. The molecular sizes of the heavier rRNA species, estimated by polyacrylamide gel electrophoresis were 1.24 and 0.84 M (X 10(6) daltons). The 25S RNA has a tendency to interact with the 18S RNA to give a complex that is difficult to separate by sucrose gradient centrifugation. The 25S RNA is also unstable and dissociates into 0.73 and 0.57 M components. The 18S RNA has molecular size (0.84 M) higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba. Ribosomal RNA hybridized 0.29% of the nuclear DNA. Mitochondrial RNA, extracted by a rapid procedure was resolved into 16 and 5S components in sucrose gradients.     

The Ribonucleic Acids of Crithidia fasciculata*

SYNOPSIS. Crithidia fasciculata ribosomes were found to be 80S and to dissociate into 58 and 41S subunits; on 5 to 50% sucrose gradients, rRNA was separated into 25, 18, and 5S components. The molecular sizes of the heavier rRNA species, estimated by polyacrylamide gel electrophoresis were 1.24 and 0.84 M (×10⁶ daltons). The 25S RNA has a tendency to interact with the 18S RNA to give a complex that is difficult to separate by sucrose gradient centrifugation. The 25S RNA is also unstable and dissociates into 0.73 and 0.57 M components. The 18S RNA has molecular size (0.84 M) higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba. Ribosomal RNA hybridized 0.29% of the nuclear DNA. Mitochondrial RNA, extracted by a rapid procedure was resolved into 16 and 5S components in sucrose gradients.     

Metabolism of 5S RNA in the absence of ribosome production

The results presented in this report show that during early development of Xenopus laevis the synthesis of 5S RNA occurs in blastula embryos, whereas the synthesis of 18S and 28S RNA cannot be detected until gastrulation. Thus the initiation of synthesis of the three ribosomal RNAs is not coordinate during early development. Blastula embryos are similar to anucleolate mutants of Xenopus laevis, in that they both synthesize 5S RNA, but are unable to assemble new ribosomes because they do not synthesize 18S and 28S RNA or ribosomal proteins. The blastula and anucleolate embryos thus provide a unique opportunity to determine if newly synthesized soluble 5S RNA can exchange with the 5S RNA present in existing ribosomes. The results show that newly synthesized 5S RNA is not incorporated into the ribosomes of blastula or anucleolate embryos. Furthermore, the 5S RNA synthesized by anucleolate mutants has a shorter half-life than the 5S RNA made by normal embryos. The synthesis of excess 5S RNA and its subsequent degradation in the absence of ribosome production appears to be another example of the phenomenon of wastage of newly synthesized ribosomal RNA.     

Rates of synthesis and degradation of ribosomal ribonucleic acid during differentiation of Dictyostelium discoideum.

Synthesis of ribosomes and ribosomal ribonucleic acid (RNA) continued during differentiation of Dictyostelium discoideum concurrently with extensive turnover of ribosomes synthesized during both growth and developmental stages. We show here that the rate of synthesis of 26S and 17S ribosomal RNA during differentiation was less than 15% of that in growing cells, and by the time of sorocarp formation only about 25% of the cellular ribosomes had been synthesized during differentiation. Ribosomes synthesized during growth and differentiation were utilized in messenger RNA translation to the same extent; about 50% of each class were on polyribosomes. Ribosome degradation is apparently an all-or-nothing process, since virtually all 80S monosomes present in developing cells could be incorporated into polysomes when growth conditions were restored. By several criteria, ribosomes synthesized during growth and differentiation were functionally indistinguishable. Our data, together with previously published information on changes in the messenger RNA population during differentiation, indicate that synthesis of new ribosomes is not necessary for translation of developmentally regulated messenger RNA. We also establish that the overall rate of messenger RNA synthesis during differentiation is less than 15% of that in growing cells.     

Effect of Chloramphenicol on the Synthesis and Stability of Ribonucleic Acid in Bacillus subtilis

The effect of chloramphenicol on the synthesis and accumulation of ribonucleic acid (RNA) in Bacillus subtilis was studied. In the presence of chloramphenicol, transfer RNA and ribosomal RNA were synthesized as rapidly 2 to 3 hr after challenge as they were just prior to the addition of the antibiotic. However, under the same conditions, net RNA accumulation ceased after only 30 to 45 min. The failure to accumulate RNA after this time resulted from a rapid degradation of ribosomal RNA synthesized in the presence of chloramphenicol and a slow degradation of mature ribosomes. Since transfer RNA was not appreciably degraded, the ratio of transfer RNA to total RNA increased during the challenge.     

Binding of mammalian ribosomes to MS2 phage RNA reveals an overlapping gene encoding a lysis function.

The main binding site for mammalian ribosomes on the single-stranded RNA of bacteriophage MS2 is located nine tenths of the way through the coat protein gene. Translation initiated at an AUG triplet in the +1 frame yields a 75 amino acid polypeptide which terminates within the synthetase gene at a UAA codon, also in the +1 frame. Partial amino acid sequence analysis of the product synthesized in relatively large amounts by mammalian ribosomes confirms this assignment of the overlapping cistron. The same protein is made in an E. coli cell-free system, but only in very small amounts. Analysis of the translation products directed by RNA from op3, a UGA nonsense mutant of phage f2, identifies the overlapping cistron as a lysis gene. In this paper we show that the op3 mutation is a C yield U transition occurring in the second codon of the synthetase cistron, which explains the lowered production of phage replicase (as well as lack of lysis) upon op3 infection of nonpermissive cells. We discuss the properties of the overlapping gene in relation to its lysis function, recognition of the lysis initiator region by E. coli versus eucaryotic ribosomes and op3 as a ribosome binding site mutant for the f2 synthetase cistron.     

Escherichia coli ribosomes bind to non-initiator sites of Q beta RNA in the absence of formylmethionyl-tRNA.

Escherichia coli ribosomes and Qβ [³²P]RNA were incubated with or without fMet-tRNA under protein initiation conditions, treated with RNase A, and centrifuged through a sucrose density gradient. The sample incubated with fMet-tRNA gave a main radioactivity peak in the 70 S region, which consisted predominantly of coat cistron initiator fragments. After incubation without fMet-tRNA, equal amounts of radioactivity were found in the 70 S and the 30 S regions, but in both peaks almost all of the radioactivity was duo to three RNase A-resistant oligonucleotides, A-G-A-G-G-A-G-G-Up (P-2a), A-G-G-G-G-G-Up (P-15) and G-G-A-A-G-G-A-G-Cp (P-4). These three oligonucleotides are derived from three different RNA regions, none of which is close to a protein initiation site. All three fragments show striking complementarity to the 3′-terminal region of E. coli 16 S RNA. Ribosomes incubated with an RNase A digest of Qβ [³²P]RNA bound almost exclusively oligonucleotide P-2a; treatment with cloacin DF13 cleaved off a complex consisting of a 49-nucleotide long segment of 16 S rRNA and oligonucleotide P-2a. These experiments show that the interaction of 30 S ribosomes with the “Shine-Dalgarno” region preceding the initiator codon of the Qβ coat cistron is insufficient to direct correct placement of the ribosome on the viral RNA, and that an additional contribution from the interaction of fMet-tRNA with the initiator triplet is required for ribosome binding to the initiator region.     

Non-Continuous Translation of Coat Protein and Ribonucleic Acid Polymerase Cistrons in MS2 Bacteriophage Ribonucleic Acid

In an MS2 phage ribonucleic acid (RNA)-directed in vitro protein-synthesizing system, the coat protein cistron and the adjacent RNA polymerase cistron are translated non-continuously. The ribosomes which have completed the synthesis of coat protein dissociate from the MS2 RNA and do not read through the intercistronic gap. Translation of the adjacent RNA polymerase cistron requires ribosomes other than those translating the coat protein cistron.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. II. General characterization of organelle nucleic acids.

Polytoma obtusum has a main band DNA (alpha) with a buoyant density in CsC1 of rho = 1.711 g/ml and a light DNA satellite (beta) with rho = 1.682 g/ml. beta-DNA was substantially enriched in a fraction containing small leucoplast fragments and some mitochondria, which was obtained in a pellet sedimenting between 3,000 g and 5,000 g. A crude mitochondrial pellet was also obtained by sedimenting at 12,000 g to recover particulates remaining in the supernate after 10 min at 5,000 g. This fraction contained a third DNA component (gamma) with rho = 1.714 g/ml. We have concluded that the leucoplasts of P. obtusum contain the beta-DNA (1.6882) and the mitochondria possess the gamma-component (1.714). Two distinct classess of ribosomes were isolated and separated by sucrose density gradients, a major 79S species and a minor species at 75S. The major species possessed the 25S and 18S ribosomal RNA (rRNA), characteristic of cytoplasmic ribosomes, and these particles co-sedimented in sucrose gradients with the 79S cytoplasmic ribosomes of Chlamydomonas reinhardtii. The minor species was present in about 2% of the total ribosomal population but showed an eight-to-ninefold enrichment in the leucoplast pellet, suggesting that it was of organelle origin. These 73S particles had RNA components migrating very closely with the 18S and 25S species of the 79S ribosomes, but the base composition of the rRNA from these two classes of ribosomes was significantly different; the rRNA from the 79S ribosomes had a G+C mole ratio of 50.0%, while the rRNA from the 73S class had a ratio of 47.5%. By comparison, chloroplast ribosomes of C. reinhardtii were found to sediment at 70S and contain rRNA molecules of 23S and 16S, with a G + C content of 51.0%. These findings support the concept that the Polytoma leucoplast possesses characteristic genetic and protein-forming systems.     

An excess of the small ribosomal subunits and a higher rate of turnover of the 60 S than of the 40 S ribosomal subunits in L cells grown in suspension culture.

A considerable excess of small ribosomal subunits was observed in L cells grown in suspension culture. The ratio between the small and large ribosomal subunits in the cytoplasm was estimated to be 1.17 ± 0.05 for cells dividing every 20 to 24 hours.The 60 S ribosomal subunits were turning over much faster than the 40 S subunits. Half-lives of 155 ± 20 hours for 18 S ribosomal RNA and 82 ± 15 hours for 28 S ribosomal RNA were observed under conditions where the cell number doubled every 24 hours and the viability was 95%. By correcting for cell death the half-lives of 18 S and 28 S ribosomal RNA were estimated to be approximately 300 hours and 110 hours, respectively. During storage of isolated ribosomes the small ribosomal subunits were degraded faster than the large subunits. This shows that the degradation of 60 S subunits was not an artifact taking place during the isolation procedure.It is postulated that the small ribosomal subunits are protected by protein to a greater extent than the 60 S subunits in these rapidly growing cells in suspension culture. The protection may take place both in the nucleus during synthesis, thus avoiding degradation (“wastage”) of nascent subunit precursors, and later in the cytoplasm. A calculation has been carried out to show that the observed excess of small subunits may be accounted for on the basis of a 1:1 synthesis of the small and large ribosomal subunits in the nucleus and different degradation rates in the cytoplasm. The results do not exclude the possibility of a difference in the “wastage” of 18 S and 28 S ribosomal RNA in the nucleus in addition to the difference in the turnover rates in the cytoplasm.