Nucleotide sequence of 5S RNA ofMycobacterium smegmatis

³²P labelled 5S RNA isolated fromMycobacterium smegmatis was digested withT ₁ and pancreatic ribonucleases separately and fingerprinted by two dimensional high voltage electrophoresis on thin-layer DEAE-cellulose plates. The radioactive spots were sequenced and their molar yields were determined. The chain length of the 5S RNA was found to be 120. It showed resemblances to both prokaryotic and eukaryotic 5S RNAs.     

RNA and Macronuclear transcription in the ciliate Stylonychia mytilus

Nuclear and cytoplasmic RNA of Stylonychia mytilus were analyzed on denaturing polyacrylamide gels. The molecular weight of rRNA precursor molecules is within a range of 2.1×10⁶ daltons. A comparison between the electrophoretic pattern of nuclear non-ribosomal RNA and cytoplasmic mRNA indicates that a considerable amount of nuclear RNA sequences is of higher molecular weight than cytoplasmic RNA sequences. The molecular weight distribution of cytoplasmic RNA supports the assumption that also in Stylonychia an average sized mRNA molecule contains 1,200–1,500 nucleotides according to a molecular weight of 4×10⁵ to 5×10⁵ daltons. The size of the polyadenylic acid fragment of poly-A⁺ RNA molecules is about 120 nucleotides. The total mass of cytoplasmic RNA is around 7.5/10¹⁰ g/cell, corresponding to 1.2×10⁷ average sized mRNA molecules per cell. RNA excess hybridization experiments show that 60% of the DNA sequences are transcribed into nuclear RNA and that the cytoplasmic mRNA sequences are homologous to about 40% of macronuclear DNA sequences. There is no indication of different frequency classes within the mRNA. The number of different mRNA species in a Stylonychia cell is 1.2–1.5×10⁴. On the average each of them is present about 1,000 times in every cell.     

Synthesis of Double-Stranded RNA in a Virus-Enriched Fraction from Agaricus bisporus

Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from healthy sporophores. Enzyme activity was dependent upon the presence of Mg²⁺ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The ³H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶; they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs₂SO₄ equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶. The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.     

On the nature of RNA synthesized in pollen tubes ofNicotiana alata

The RNA formed in pollen tubes during 4 hours of growthin vitro was resolved by chromatography on methylated albumine on kieselguhr (MAK) into three principal fractions. Acoording to the labelling from uracil-¹⁴C about 11% was eluted with tRNA and 5 S RNA (low molecular weight RNA), 76% just after rRNA (D-RNA) and nearly 14% was recovered from the column by SDS at 35 °C (TB-RNA). In the presence of actinomycin D at concentration of 30 μg ml^-1 the synthesis of the three classes of RNA was inhibited by 71%, 97% and 70% respectively. On sucrose density gradient the radioactive low molecular weight RNA sedimented at 4 S-5 S which suggests that one or both of these RNA species are synthesized in pollen tubes. The D-RNA eluted from the MAK column is polydisperse in size exhibiting a wide range of sedimentation values up to about 35 S with a large peak at 9 S-10 S and two smaller peaks at 14 S-15 S and at about 23 S. The rapid labelling and the polydisperse rather low molecular weight character suggest that the D-RNA is a heterogeneous population of mRNA. The sedimentation profile of TB-RNA was similar to that of D-RNA. The RNA synthesized in the presence of³²PBO3-₄ or uracil-¹⁴C exhibited no radioactivity peaks corresponding to sedimentation peaks of rRNA.     

Ribosomal RNA contents of maize genotypes with different ribosomal RNA gene numbers

Ribosomal RNA (rRNA) contents were determined in 16 maize genotypes whose individual rRNA gene numbers varied from 5000 to 23,000 per 2C nucleus. Analytical polyacrylamide gel electrophoresis of total RNA showed that no obvious relation existed between rRNA gene number and rRNA content. Only two of nine common inbred lines contained more rRNA than W-23, the inbred with the lowest rRNA gene number. Two of four lines with altered protein content (due to long-term experimental selection) had rRNA contents significantly reduced from those of W-23. A line with an apparent duplication of the nucleolus organizer region of chromosome 6 (called 2-NOR) was expected to possess an elevated quantity of rRNA because it possesses a larger nucleolus; however, we produced a 2-NOR isogenic version and found no difference in rRNA content. The rRNA genes in maize are distributed throughout the NOR-heterochromatin and the NOR-secondary constriction portions of the NOR. The absence of an obvious correlation between rRNA gene number and cellular rRNA content may reflect the presence of a large number of rRNA genes in an inactive state, at least during the stage of growth examined in these experiments.     

Transfer RNA genes ofZea mays chloroplast DNA

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA₂ ^Ile and tRNA^Ala map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAs^Leu and the three for tRNAs^Ser, as well as the two isoaccepting species for tRNA^Asn, tRNA^Gly, tRNAs^Ile, tRNAs^Met, tRNAs^Thr, are shown to be encoded at different loci. Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were³²P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.     

Change in the Template Specificity of RNA Phage SP-Replicase by the <Emphasis Type="Italic">E. coli</Emphasis> Cell Component

Qβ-REPLICASE was isolated from E. coli infected with the RNA bacteriophage Qβ as RNA-dependent RNA polymerase which had template specificity¹. RNA phage SP², which is distinct from RNA phages isolated previously^3,4, has been isolated in our laboratory and SP-replicase⁵ was purified from E. coli infected with SP-phage. SP-replicase has a template specificity different from that of Qβ-replicase. By using this new RNA-replicase, comparison between two distinct replicases has become possible.     

The effect of 2-thiouracil on RNA synthesis in pollen tubesof Nicotiana alata

The level of RNA in pollen is approximately 20 mg g^-1 and remains constant during 6 h pollen germinationin vitro also in the presence of 2-thiouracil which stimulates pollen tube elongation. The synthesis of RNA in pollen tubes was investigated according to the incorporation of the label from uracil-2-¹⁴C, 2-thiouracil-2-¹⁴C, orotic acid-5-³H, fructose-U-¹⁴C and from³²PO₄ ^3- into RNA fractions separated by methylated albumine kieselguhr chromatography. The distribution of radioactivity on elution profiles was different according to the radioactivity source, however it was not changed by the presence of 2-thiouracil in cultivation medium. 2-Thiouracil incorporates into pollen tube RNA at about 50% the rate of uracil. It inhibited the incorporation of orotic acid, of fructose and of phosphate into all RNA fractions. It is suggested that the analogue inhibits the enzymes involved in RNA synthesis essentially as 2-thiouridine-5’-phosphate.     

Electron microscopic autoradiographic study of RNA isolated from apple-tree callus tissue labelled with 6-Benzylaminopurine-8-C14

In the present paper RNA from apple-tree callus tissue labelled with 6-benzyl-minopurine¹⁴) was studied. The RNA isolated from this tissue was prepared as sample for electron microscopic studies and was also used as biochemical control. The electron microscopic autoradiograms obtained showed the labelled structure of RNA, sRNA and rRNA. The incorporation of labelled purine rings was confirmed in all three RNA types by the radioactivity, which was also proved in nucleotides after hydrolysation of RNA fractions. The results were compared with RNA isolated from tissue cultivated on a non-radiactive medium.     

Evidence for ribosomal RNA synthesis in pollen tubes in culture

The evidence is presented that pollen tubes ofNicotiana tabacum L. cultivated in shaken suspension do synthesize 5S, 18S and 28S RNA. Following incubation of pollen tubes in the presence of radioactive uracil or uridine, RNA was isolated from total pollen tube material after the removal of 4S RNA, from polysomes and from 80S ribosomal particles, and fractionated by density gradient centrifugation and MAK column chromatography. The results obtained further suggest a higher rate of 5S RNA synthesis with respect to 18S+28S RNA.     

Changes in DNA-dependent RNA polymerase in sporulating yeast

Diploid yeast cells can be made to undergo sporulation and meiosis in a relatively synchronous fashion. Earlier studies on this process showed that the rate of RNA synthesis reaches a maximum at 6 hr and has declined drastically by 10 hr [5]. Starting at about 6 hr a new peak of RNA polymerase activity appears between polymerases Ib and II upon DEAE Sephadex chromatography. This peak appears to reach a maximum at 8.5 hr and to have decreased by 10 hr. The new peak is intermediate between polymerases Ib and II in its sensitivity to α-amanitin. It does not appear in a non-sporulating (αα) diploid grown under sporulating conditions.