The relationship of the DNA sequences from which polyribosomal messenger RNA (mRNA) and heterogeneous nuclear RNA (NRNA) of mouse L cells are transcribed was investigated by means of hybridization kinetics and thermal denaturation of the hybrids. Hybridization was performed in formamide solutions at DNA excess. Under these conditions most of the hybridizing mRNA and NRNA react at values of D(o)t (DNA concentration multiplied by time) expected for RNA transcribed from the nonrepeated or rarely repeated fraction of the genome. However, a fraction of both mRNA and NRNA hybridize at values of D(o)t about 10,000 times lower, and therefore must be transcribed from highly redundant DNA sequences. The fraction of NRNA hybridizing to highly repeated sequences is about 1.7 times greater than the corresponding fraction of mRNA. The hybrids formed by the rapidly reacting fractions of both NRNA and mRNA melt over a narrow temperature range with a midpoint about 11 degrees C below that of native L cell DNA. This indicates that these hybrids consist of partially complementary sequences with approximately 11% mismatching of bases. Hybrids formed by the slowly reacting fraction of NRNA melt within 4 degrees -6 degrees C of native DNA, indicating very little, if any, mismatching of bases. Hybrids of the slowly reacting components of mRNA, formed under conditions of sufficiently low RNA input, have a high thermal stability, similar to that observed for hybrids of the slowly reacting NRNA component. However, when higher inputs of mRNA are used, hybrids are formed which have a strikingly lower thermal stability. This observation can be explained by assuming that there is sufficient similarity among the relatively rare DNA sequences coding for mRNA so that under hybridization conditions, in which these DNA sequences are not truly in excess, reversible hybrids exhibiting a considerable amount of mispairing are formed. The fact that a comparable phenomenon has not been observed for NRNA may mean that there is less similarity among the relatively rare DNA sequences coding for NRNA than there is among the rare sequences coding for mRNA.

Animals↗

PubMed1970

The 5 s RNA genes of Drosophila melanogaster.

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Animals↗

PubMed1970

Inhibition of RNA synthesis by actinomycin D: characteristic dose-response of different RNA species.

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Cell Nucleolus↗

PubMed1970

Evolution of the transcription unit of ribosomal RNA.

In eukaryotes the two principal RNA components of the ribosomes are initially synthesized as a large complex precursor molecule, which may be thought of as a transcription unit. The precursor is converted, via intermediates, to the mature forms of ribosomal RNA (rRNA). In order to assess the extent of variation in the size of this rRNA transcription unit among different organisms, and to infer its possible mode of evolution, we have determined its molecular weight in several selected species. Pulse-labeled and long-term labeled RNA's were extracted from various types of cells, and analyzed by electrophoresis on acrylamide gels. Identification of particular components as rRNA precursors was made according to several stated criteria. Our results, together with an analysis of previously published data, suggest that in plants and lower animals, up to and including reptiles, the unit of transcription of rRNA is a 2.7-2.8 million dalton molecule, which is only about 25 per cent larger than its combined rRNA products. In contrast, birds, marsupials and placental mammals, exhibit a seemingly less economical form of rRNA synthesis. Their transcription units are 4.0-4.2 million daltons, about 80 per cent larger than the rRNA products. In the organisms with the smaller transcription unit the major intermediate precursor of rRNA is 1.5-1.6 million daltons, as compared to 2.0-2.2 million daltons in birds and mammals. The significance of these findings is discussed in relation to evolutionary changes in the base composition of the ribosomal RNA genes.

Acrylates↗

PubMed1970

Aberrant intranucleolar maturation of ribosomal precursors in the absence of protein synthesis.

To help elucidate the role of protein in the maturation of ribosomal RNA in cultured L cells, we have studied the effects of cycloheximide upon the maturation process and upon the intranucleolar ribonucleoprotein particles containing the "preribosomal RNA's." Five parameters of these particles were analyzed: (a) extractability, (b) sedimentation characteristics in sucrose gradients, (c) RNA composition, (d) buoyant density in CsCl gradients, and (e) effects of increased ionic strength on the buoyant density. When protein synthesis is inhibited, the rate of conversion of the precursor 45S ribosomal RNA is rapidly diminished, falling to less than 30% of the control rate within 1 hr. Nevertheless, in terms of the first three parameters there is no difference between control and cycloheximide nucleolar particles. However, the cycloheximide particles have a lower and more heterogeneous buoyant density and a more variable response to increased ionic strength. The results imply that the protein composition of the cycloheximide particles is different from that of particles from control cells, and that the entire protein complement is not necessary for the first cleavages in the maturation process, although it is necessary for the normal rate of processing and for the eventual appearance of both 18S and 28S rRNA in mature ribosomes.

Cell Nucleolus↗

PubMed1969

Ribosome precursor particles in nucleoli.

Ribonucleoprotein (RNP) particles containing the precursors of ribosomal RNA were extracted from L cell nucleoli and analyzed under conditions comparable to those used in the characterization of cytoplasmic ribosomes. Using nucleoli from cells suitably labeled with (3)H-uridine, we detected three basic RNP components, sedimenting at approximately 62S, 78S, and 110S in sucrose gradients containing magnesium. A fourth particle, sedimenting at about 95S, appears to be a dimer of the 62S and 78S components. When centrifuged in gradients containing EDTA, the 62S, 78S, and 110S particles sediment at about 55S, 65S, and 80S, respectively. RNA was extracted from RNP particles which were prepared by two cycles of zonal centrifugation. The 62S particles yielded 32S RNA and a detectable amount of 28S RNA, the 78S structures, 32S RNA and possibly some 36S RNA, and the 110S particles, a mixture of 45S, 36S, and 32S RNA's. When cells were pulsed briefly and further incubated in the presence of actinomycin D, there was a gradual shift of radioactivity from heavier to lighter particles. This observation is consistent with the scheme of maturation: 110S --> 78S --> 62S. The principal buoyant densities in cesium chloride of the 110S, 78S, and 62S particles are 1.465, 1.490, and 1.545, respectively. These densities are all significantly lower than 1.570, which is characteristic of the mature large subunit of cytoplasmic ribosomes, suggesting that the precursor particles have a relatively higher ratio of protein to RNA, and that ribosome maturation involves, in addition to decrease in the size of the RNA molecules, a progressive decrease in the proportion of associated protein.

Animals↗