Comparative study of the ribosomal RNA from Leishmania and Trypanosoma

The ribosomal RNA from several stocks of the genera Leishmania and Trypanosoma were studied by gel electrophoresis, sedimentation on sucrose density gradients and RNA/DNA hybridization experiments. Three major components were observed after electrophoresis in polyacrylamide gels (PAGE-SDS), the relative molecular masses being respectively: X1 = 0.83 megadaltons, X2 = 0.63 megadaltons and X3 = 0.54 megadaltons for Leishmania RNA; and X1 = 0.86 megaldaltons, X2 = 0.78 megadaltons, and X3 = 0.58 megadaltons for Trypanosoma RNA. Depending upon the isolation procedure, a fourth component, X0 = 1.2 megadaltons (26S), became evident. The later component was purified from Leishmania brasiliensis (Y) by centrifugation on a linear 15-30% sucrose density gradient. This component, after heat denaturation and PAGE-SDS, gave rise to two bands coinciding in molecular mass with those of X2 and X3, indicating that these components are part of the large ribosomal subunit whereas X1 belongs to the small one. The above mentioned differences in mobilities of components X1 and X2 between the two genera were no longer observed after electrophoresis in denaturing agarose-formaldehyde gels, suggesting secondary structural differences among these RNA species. Hybridization experiments with L. brasiliensis (Y) DNA showed that both RNA types compete equally well for the ribosomal sites in this DNA, and that L. brasiliensis (Y) rRNA recognizes the ribosomal sites in DNA of Trypanosoma cruzi (EP), thus indicating that no gross changes occurred in their nucleotide sequences during evolution.     

Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis

Electrophoresis of ribosomal RNA in polyacrylamide-agarose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel electrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes.     

Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis.

Electrophoresis of ribosomal RNA in polyacrylamide-agrose composite gels separates 16S and 23S species into multiple bands. These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA. Gel elctrophoresis was used to test for cation-specific conformational changes in ribosomal RNA. Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change. Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place. The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal. We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes.     

Small RNA species in maize tissue

The low molecular weight RNA components of maize have been analyzed after labeling callus and leaf tissue with [³H]uridine in vitro. Electrophoresis of the isolated RNA on acrylamide slab gels reveals, apart from 5S and transfer RNA, three major and about five minor RNA species with chain lengths between 140 and 280 nucleotides. These RNA molecules are labeled as rapidly as 5S, transfer RNA, and do not represent degradation products of large ribosomal RNA molecules. Furthermore, like 5S and transfer RNA, these small RNA species are stable and show no detectable turnover within forty-eight hours. Fractionation of the tissue into crude subcellular fractions indicates a preferential association of some of the small stable RNA species with the nucleus, while others appear to be located in the cytoplasm. The low molecular weight RNA spectrum from the leaf is similar to that observed in callus, with the major small RNA species equally present in both tissues.Abbreviations tRNA transfer RNA - hnRNA heterogenous nuclear RNA - mRNA messenger RNA - scRNA small cytoplasmic RNA - snRNA small nuclear RNA     

MITOCHONDRIAL RNA FROM CULTURED ANIMAL CELLS : II. A Comparison of the High Molecular Weight RNA from Mouse and Hamster Cells

Discrete RNA fractions sedimenting slightly slower than 18s ribosomal RNA have been found in mitochondrial preparations from both hamster (BHK-21) and mouse (L-929) cells. This RNA could be separated into two components, present in approximately equimolar amounts, by prolonged zonal centrifugation or acrylamide gel electrophoresis. The hamster components had sedimentation constants averaging 16.8 and 13.4, and molecular weights (estimated by gel electrophoresis) averaging 0.74 and 0.42 x 10⁶ daltons. Mixed labeling experiments showed that the mouse components sedimented and electrophoresed 3–6% more slowly than the corresponding hamster components. The RNA from both cell lines resembled mitochondrial ribosomal RNA from yeast and Neurospora in being GC poor, and in addition the larger and smaller components resembled each other in base composition. These results, taken with those of other recent studies, are compatible with the idea that our high molecular weight mitochondrial RNA is ribosomal; such RNA would then constitute a uniquely small size-class of ribosomal RNA.     

Analysis of RNA structure by ultraviolet crosslinking and denaturation gel electrophoresis

Electrophoresis in polyacrylamide gels containing both formamide and urea is a high-resolution technique for the analysis of crosslinked RNA species. Combined with a specific crosslinking agent like uv irradiation, it allows a rapid fingerprint of structural differences between RNA forms. The technique reveals significant differences in the pattern of uv crosslinking of free Escherichia coli 16 S ribosomal RNA compared with the RNA in active or inactive 30 S subunits. Ultraviolet photocrosslinks seen only in the 30 S particle are likely to be tertiary structure contacts.     

Molecular weights of the ribosomal ribonucleic acid of fungi

Summary The molecular weights of the 18s and 25s ribosomal RNA components of fungi from all major classes were determined by electrophoresis in polyacrylamide gels. The molecular weight of the 18s RNA was found to be very similar for all fungi (range 0.71–0.75 million) and about 4–5% larger than the 18s RNA of HeLa cells and soybean. The molecular weight of the 25s RNA ranged between 1.45 million in the Myxomycetes and 1.30–1.31 million in the Ascomycetes and Basidiomycetes. The differences in the 25s RNA molecular weights between various classes of fungi were interpreted as being in agreement with a monophyletic origin of the Chytridiomycetes, Zygomycetes, Ascomycetes and Basidiomycetes, and independent origins for the Myxomycetes and the Oomycetes. The Hyphochytridiomycete examined could not be placed unequivocally in any group on the basis of its 25s RNA. Fungal RNA extracted with a p-aminosalicylate-triisopropylnaphthalene sulfonate-phenol mixture at 40–60°C contained a high molecular weight aggregate of the 18s and 25s ribosomal RNA; this suggested significant base sequence homology between the two ribosomal RNA species in fungi.     

Analysis of a 5S RNA-protein complex isolated from the ribosomes of rye embryos

Rye embryo ribosomes were dissociated into subunits and the large subunit fraction was treated with formamide. A low molecular weight complex of RNA and protein (RNP) was released. Electrophoresis of the RNP in polyacrylamide gels containing sodium dodecyl sulphate yielded an RNA band and a single protein band. The protein had a molecular weight of approximately 41 000 and the RNA of the complex was shown to be 5S ribosomal RNA. Embryos were germinated in the presence of [32P]orthophosphate and the labelled RNP was isolated from their ribosomes. The RNA component was partially digested with pancreatic A ribonuclease and the parts protected from degradation by the protein were determined by sequence analysis. Although the whole 5S RNA molecule was shielded to some extent, the portion most protected was between nucleotides 68 and 108. This is, therefore, probably the part of plant cytosol 5S RNA which is primarily involved in the interaction with protein in the complex and possibly in the ribosome as well.     

Effect of cAMP and related compounds on newt epidermal cell migration both in vivo and in vitro

Mouse testicular cells labelled in vivo with 3H-uridine for 1 hour were separated into enriched cell populations representing different stages of spermatogenesis by centrifugation in an Elutriator rotor. RNA extracted with phenol and chloroform was sized by electrophoresis on 2.4% acrylamide gels. The percentage of newly synthesized RNA which was not ribosomal RNA (or its precursors) and was not transfer RNA, was higher in early postmeiotic, as compared to late premeiotic, stages. RNA was also extracted from fractionated cells in the presence of guanidinium chloride and the proportion of total 3H-RNA-containing poly(A) sequences was determined by binding to an oligo-(dT)-cellulose column. RNA that bound in 0.5 NaCl was eluted with low salt buffer and reapplied twice more, after heating each time in DMSO to disaggregate any non-poly(A)-containing RNA. The percentage of newly synthesized RNA which contained poly(A) did not decrease in early postmeiotic, as compared to late premeiotic, stages. We suggest that at least some part of the 6-15S 3H-RNA and 3H-poly(A)-containing RNA represents mRNA(s) transcribed postmeiotically in haploid germ cells.     

Comparative Study of the Ribosomal RNA from Leishmania and Trypanosoma1

The ribosomal RNA from several stocks of the genera Leishmania and Trypanosoma were studied by gel electrophoresis, sedimentation on sucrose density gradients and RNA/DNA hybridization experiments. Three major components were observed after electrophoresis in polyacrylamide gels (PAGE-SDS), the relative molecular masses being respectively: X₁= 0.83 megadaltons, X₂= 0.63 megadaltons and X₃= 0.54 megadaltons for Leishmania RNA; and X₁= 0.86 megaldaltons, X₂= 0.78 megadaltons, and X₃= 0.58 megadaltons for Trypanosoma RNA. Depending upon the isolation procedure, a fourth component. X₀= 1.2 megadaltons (26S), became evident. The later component was purified from Leishmania brasiliensis (Y) by centrifugation on a linear 15-30% sucrose density gradient. This component, after heat denaturation and PAGE-SDS, gave rise to two bands coinciding in molecular mass with those of X₂ and X₃ indicating that these components are part of the large ribosomal subunit whereas X₁ belongs to the small one. The above mentioned differences in mobilities of components X₁ and X₂ between the two genera were no longer observed after electrophoresis in denaturing agarose-formaldehyde gels, suggesting secondary structural differences among these RNA species. Hybridization experiments with L. brasiliensis (Y) DNA showed that both RNA types compete equally well for the ribosomal sites in this DNA, and that L. brasiliensis (Y) rRNA recognizes the ribosomal sites in DNA of Trypanosoma cruzi (EP), thus indicating that no gross changes occurred in their nucleotide sequences during evolution.     

Transformation in Naegleria gruberi: patterns of RNA synthesis examined by polyacrylamide gel electrophoresis

Naegleria gruberi were grown on bacteria and methods were devised to free the cellular RNA from bacterial RNA contamination. Use of actinomycin D and cycloheximide showed that the transformation of Naegleria from amoeba to flagellate required RNA synthesis for 30 min and protein synthesis for 40 min after the initial stimulus of distilled water. Comparison of the patterns of RNA synthesized during transformation with those during growth indicated a considerable amount of new RNA produced during the phenotypic change. Most marked was the increase in RNA co-migrating on polyacrylamide gels with the small ribosomal sub-unit RNA, together with RNAs between the latter and transfer RNA. These results were compared with other published results using axenically-grown cells cells and sucrose density gradient centrifugation. Cells placed in 80 mM NaCl instead of distilled water fail to transform but the pattern of newly-synthesized RNAs was not significantly different from that seen in transforming cells. This suggested that high salt concentrations inhibit transformation by inhibiting synthesis and/or assembly of certain proteins rather than RNA synthesis. Eluted material from various regions of polyacrylamide gels containing RNA extracted from transforming cells was used in a cell-free system. Incorporation of 3H-glutamic acid but not 3H-tryptophan was stimulated by material extracted from the 18S regions of the gels.     

Molecular interactions of ribosomal components

Summary The formation of a complex between individual 30S ribosomal proteins and 16S ribosomal RNA was studied by three techniques: zone centrifugation, molecular-sieve chromatography and electrophoresis in polyacrylamide gels. Five 30S proteins form a stable complex with the RNA under the conditions used to assemble ribosomes. Specific and nonspecific complex formation can be distinguished by an analysis of the concentration-dependence for complex formation. Similarly, competition experiments between heterologous proteins that bind to RNA can also be used to establish the uniquness of the RNA binding sites for ribosomal proteins. The data show that four of the five proteins bind to unique sites on the RNA. The fifth protein binds nonspecifically to the RNA. In addition, cooperative interactions between several proteins were observed; these enhance the interaction of proteins with the 16S RNA. A partial assembly sequence for the 30S ribosomal subunit is presented.     

Preferential incorporation of an exogenous cytokinin, N6-benzyladenine, into 18S and 25S ribosomal RNA of tobacco cells in suspension culture

Cytokinin-requiring tobacco cells were incubated for 10 h in the presence of a labeled cytokinin. N6-benzyl-[2-3H]Ade, and of [8-14C]Ado. After alkaline hydrolysis of total RNA and fractionation of the resulting nucleotides, 80 per cent of the 3H radioactivity of RNA were recovered as the N6-benzyl-Ado nucleotide, covalently inserted into polynucleotidic chains. The N6-benzyl-Ado nucleotide was not significantly labled by 14C: at most one part of this nucleotide per 10 000 may result from a transfer of the benzyl moiety to adenyl residues in preformed RNA. Thus, the covalent insertion of N6-benzyl-Ade into RNA involves the intact N6-substituted base. Total RNA was fractionated either by sucrose density gradient centrifugation or by polyacrylamide gel electrophoresis. All identified RNA species were shown to contain N6-benzyl-Ade. The insertion frequency, measured as the molecular proportion of N6-benzyl-Ade to the total base content, was 3 to 4 times larger in 25S and 18S rRNA than in 5S and 4S RNA. The amount of N6-benzyl-Ade inserted into cytoplasmic ribosomal RNA accounted for about 90 per cent of the amount incorporated into total RNA. Electrophoresis of denatured RNA in the presence of formamide provided additional evidence that N6-benzyl-Ade was indeed incorporated into RNA molecules.     

Properties of the Deoxyribonucleic Acid Contained in the Defective Particle Coliphage 15

Escherichia coli strain 15 TAU, which requires thymine, arginine, and uracil for growth and harbors an apparently defective prophage, was induced by exposure to ultraviolet light (580 ergs/mm(2)) or to mitomycin C (5 mug/ml). Phage particles (coliphage 15) were recovered from the resulting lysate by treatment with deoxyribonuclease, filtration, and several cycles of differential centrifugation. Analysis of the phage particles obtained by using cesium chloride density gradient centrifugation in a preparative ultracentrifuge resulted in the resolution of three components. The major component had a peak density of 1.52 to 1.53 g/cm(3) followed by components with densities of 1.5 and 1.49 g/cm(3). The guanine plus cytosine content of coliphage 15 deoxyribonucleic acid (DNA) was determined by both analytical ultracentrifugation in cesium chloride and by thermal denaturation in standard saline citrate buffer. Respective values of 46.4 +/- 1% and 46.6 +/- 1% guanine plus cytosine content were obtained. Coliphage 15 DNA formed molecular hybrids with messenger ribonucleic acid (RNA) from both uninduced and ultraviolet-induced cultures of E. coli 15 TAU, but did not hybridize with E. coli ribosomal RNA. The molecular weight of coliphage 15 DNA was determined by constant velocity sucrose density gradient centrifugation to be about 33 x 10(6) daltons.     

Ribosomal-type ribonucleic acid from rodent mitochondria

1. Highly purified mitochondria containing 3.0mug of RNA/mg of mitochondrial protein were prepared from rat liver by differential centrifugation. 2. RNA, labelled with [(32)P]P(i) or [(3)H]orotate, was isolated from these mitochondria by a phenol extraction method. The RNA sedimented at 15S and 13S on sucrose density gradients. Its nucleotide composition was 23% uridylate, 30% adenylate, 22% guanylate and 25% cytidylate. 3. RNA from mouse L cells was labelled with [(3)H]-uridine in the presence of 0.1mug of actinomycin D/ml to suppress the synthesis of cytoplasmic rRNA. The RNA isolated from crude L-cell mitochondria by a cold-phenol-sodium dodecyl sulphate method had components sedimenting at 15S and 12.5S. These components had an electrophoretic mobility on agarose-acrylamide gels of 21 and 12S(E) compared with 28 and 18S(E) for cytoplasmic rRNA. The nucleotide composition was 26% uridylate, 34% adenylate, 18% guanylate and 22% cytidylate. 4. RNA extracted from crude L-cell mitochondria by a hotphenol-sodium dodecyl sulphate method had an additional component sedimenting at 21S and having an electrophoretic mobility of 18S(E). It was probably DNA because of its sensitivity to deoxyribonuclease and its insensitivity to ribonuclease and alkali. It was present in nuclear fragments contaminating the crude mitochondrial fraction and could be removed by deoxyribonuclease or isopycnic-gradient centrifugation.     

Synthesis of messenger ribonucleic acid in excised pea-seedling root segments. Separation of the messenger from microsomes by electrophoresis

1. Electrophoresis on cellulose acetate membrane in a tris-pyrophosphate buffer was used to separate microsomal fractions into three components: (1) the lipoprotein; (2) the nucleoprotein (termed the beta-band); (3) traces of free RNA (termed the alpha-band). In tris buffer containing Mg(2+) the alpha-band was not obtained. 2. The incorporation of uridine and phosphate into RNA by excised pea-seedling root segments was studied by using this electrophoretic technique. 3. It was shown that after a short (;pulse') incubation in the radioactive precursor and a longer (;chase') incubation in the non-radioactive precursor most of the incorporation was into the RNA of the alpha-band and little into that of the beta-band. Previous work showed that in roots of whole seedlings the incorporation is mostly into the ribosomal RNA, corresponding to the material in the beta-band. 4. A pulse-labelled RNA has also been found; this seems to be a cell fraction distinct from the microsomes or ribosomes. 5. The apparent base compositions of labelled RNA in the alpha-band and small amounts of labelled RNA in the beta-band and of unfractionated RNA were very different from the composition of ribosomal or transfer RNA, and somewhat like that of DNA. 6. It is suggested that the excised root segment synthesizes a messenger-RNA fraction labelled after a pulse incubation and a distinct messenger RNA labelled after a pulse and chase incubation, but no ribosomal or transfer RNA. The system is thus similar to the ;step-down' culture conditions in bacteria.     

An argument for the existence of a natural complex between protein L5 and 5 S RNA in rat liver 60-S ribosomal subunits

Electrophoresis of the 60-S ribosomal subunits from rat liver in the presence of citrate ions removes the 7 S ribonucleoprotein complex between protein L5 and 5 S RNA though this complex is not released by dialysis or by centrifugation through a sucrose cushion in the same buffer. Using acetate instead of citrate, the subunits remain intact in all cases. On the other hand, in the presence of EDTA, the complex is always released. The poly(U) directed polyphenylalanine synthesis is correlated in each case with the presence of this complex within the subunits. The melting curves of subunits which have been treated with citrate, acetate or EDTA and then taken back in the buffer in which they were stored suggest that the specific RNA-protein interactions are preserved in the presence of acetate and of citrate but not of EDTA. As a whole, the results support the interpretation that the association of protein L5 and 5 S RNA exists within the active subunits.     

The Relationship between Satellite Deoxyribonucleic Acid, Ribosomal Ribonucleic Acid Gene Redundancy, and Genome Size in Plants

The buoyant density of ribosomal DNA is similar in species with or without satellite DNA, and in all species examined was distinguishable from that of the satellite DNA. In melon tissues (Cucumus melo) the percentage satellite DNA is not correlated with the percentage hybridization to ribosomal RNA. Satellite DNA sequences do not appear to be dispersed between those coding for ribosomal RNA. There is no correlation between the presence of satellite DNA and high ribosomal RNA gene redundancy, but there is a correlation between satellite DNA and small genome size, which results in a correlation between satellite DNA and a high percentage hybridization to ribosomal RNA. Satellite DNAs are defined as minor components after CsCI centrifugation.