Ribosomal proteins in plastids of a mendelian and a nonmendelian streptomycin-resistant mutant of Chlamydomonas reinhardii determined by two-dimensional gel-electrophoresis

The pattern of ribosomal proteins in plastids of a nonmendelian streptomycin resistant mutant does not differ from that of wildtype cells when compared by two-dimensional gel-electrophoresis. The protein pattern of a mendelian resistance mutant is changed, however.Since the resistance in this mendelian mutant is probably caused by a change in the ribosomal proteins, the resistance in the nonmendelian mutant must be considered as a change of the ribosomal RNA.This work was supported by the Deutsche Forschungsgemeinschaft.     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.     

Base composition of ribosomal RNA and evolution

Summary Base composition analysis has been carried out for the two major ribosomal RNA components extracted from ribosomes of plants and animals of various taxonomic position. The high degree of change undergone by these molecules during evolution is evident from the results obtained. Moreover, the evolutionary pattern of therRNA base composition well reflects the phylogenetic relationships of the various taxonomic groups.On leave from the Facultad de Medicina, Universidad Central de Venezuela, Caracas.     

Differentiation of chick embryo cerebral hemispheres: a critical evaluation of a bulk preparation of neuroblasts and spongioblasts

Abstract— A critical evaluation of a previously described method of bulk preparation of neuroblasts and spongioblasts from chick embryo cerebral hemispheres furnished the following results.

• 1 A major loss of total RNA occurs during the preparation of the cell fractions.
• 2 The loss occurred mainly during the dissociation of the tissue and the following centrifugations.
• 3 The missing RNA's were found in the supernatants after cell centrifugation at low speed.
• 4 There was a selective loss of transfer RNA amounting to 70–80 per cent relative to ribosomal RNA.
• 5 The relative proportions of the 29, 18 and 5s ribosomal RNA's were constant, but there was a decrease in their proportion relative to heavy-molecular weight, rapidly-labelled nuclear RNA.
• 6 The ribosomal RNA's in the low-speed supernatant could be resedimented at 35,000 g whereas most transfer RNA could not.

The results are consistent with a loss of fragments of cytoplasm containing ribosomes and with additional extraction of transfer RNA during the preparation.     

Secondary structure of the autoregulatory mRNA binding site of ribosomal protein L1

Summary The secondary structure of the autoregulatory mRNA binding site of Escherichia coli ribosomal protein L1 has been studies using enzymatic methods. The control region of the E. coli L11 operon was cloned into a vector under control of the Salmonella phage SP6 promoter, and RNA transcribed using SP6 RNA polymerase. The secondary structure of this RNA was probed using structure-specific nucleases, and by comparison of the data with computer predictions of RNA folding, secondary structural features were deduced. The proposed model is consistent with elements of some previously proposed models, but differs in other features. Finally, secondary structure information was obtained from two mutant mRNAs and the structural features correlated with observed phenotypes of the mutants.Abbreviations MB mung bean nuclease - V₁ cobra venom nuclease - sss single-strand-specific - dss double-strand-specific     

Identification and germline transformation of the ribosomal protein rp21 gene of Drosophila: complementation analysis with the Minute QIII locus reveals nonidentity

Summary Minute loci represent a class of about 50 different Drosophila genes that appear to be functionally related. These genes may code for components of the protein synthetic apparatus. While one Minute locus has been recently shown to code for a ribosomal protein, it is not yet known whether any of the other Minute loci also code for ribosomal proteins. We have addressed this question by a combined molecular and genetic approach. In this report, a cloned DNA encoding the ribosomal protein rp21 is partially characterized. The rp21 gene maps to the same region (region 80 of chromosome 3L) as the temperature-sensitive Minute QIII gene. Using P-element mediated transformation, the rp21 gene was transformed into the germline of Drosophila. RNA blot experiments revealed that the transformed gene is expressed in transgenic flies. However, genetic complementation analysis indicated that the QIII locus and the rp21 gene are not identical. Implications of these findings for the relationship between Minutes and ribosomal protein genes are discussed.     

Absence of ribosomes in Capsicum chromoplasts

Ribosome development was followed by electron microscopy and gel electrophoresis of ribosomal (r)RNAs in the plastids of fully expanded fruits of Capsicum annuum L. during ripening. Chloroplasts from young Capsicum leaves were used as a structural and electrophoretic standard. Four stages were distinguished on the basis of colour changes during fruit ripening. Chloroplasts of the green fruit had a lower content of 16S and 23S rRNAs than leaf chloroplasts. They contained only a few ribosomes, some more discrete ribosomal particles, and the contrast of ribosomal structures was faint. From the outset of ripening, most of the ribosomal structures in the plastid stroma disappeared. A continuous decrease in plastid rRNAs occurred during ripening. Fully differentiated chromoplasts of the red fruit did not contain rRNAs or ribosomes. Throughout plastid development, DNA nucleoids were evident and there was only a small decrease in the DNA peak on electrophoretograms. The loss of ribosomes during the chloroplast-to-chromoplast conversion in Capsicum fruit is discussed in relation to the variations in pigments and enzymic systems in both plastid types.Abbreviations Developmental stages of leaves and fruits: A four-week-old green leaf - B green fruit - C brownish fruit - D orange fruit - E red fruit - ptRNA, DNA plastid RNA - DNA; rRNA ribosomal RNA     

Inactivation of SSM4, a new Saccharomyces cerevisiae gene, suppresses mRNA instability due to rna14 mutations

Decay rates of mRNAs depend on many elements and among these, the role of the poly(A) tail is now well established. In the yeast Saccharomyces cerevisiae, thermosensitive mutations in two genes, RNA14 and RNA15, result in mRNAs having shorter poly(A) tails and reduced half-life. To identify other components interacting in the same process, we have used a genetic approach to isolate mutations that suppress the thermosensitivity of an rna14 mutant strain. Mutations in a single locus, named SSM4, not only suppress the cell growth phenotype but also the mRNA instability and extend the short mRNA poly(A) tails. The frequency of appearance and the recessive nature of these mutations suggested that the suppressor effect was probably due to a loss of function. We failed to clone the SSM4 gene directly by complementation, owing to its absence from gene banks; it later emerged that the gene is toxic to Escherichia coli, but we have nevertheless been able to clone the SSM4 sequence by Ty element transposition tagging. Disruption of the SSM4 gene does not affect cell viability and suppresses the rna14 mutant phenotypes. The protein encoded by the SSM4 gene has a calculated molecular mass of 151 kDa and does not contain any known motif or show homology with known proteins. The toxicity of the SSM4 gene in E. coli suggests that a direct biochemical activity is associated with the corresponding protein.     

Genetic and biochemical analyses of yeast RNase MRP

RNase MRP cleaves the yeast pre-rRNA at a site in internal transcribed spacer 1 (ITS1) and this cleavage can be reproducedin vitro by the highly purified enzyme. Two protein components (Pop1p and Pop2p) have been identified which are common to yeast RNase MRP and RNase P. Moreover, purified RNase P can also cleave the pre-rRNA substratein vitro, underlining the similarities between these particles. Genetic evidence suggests that RNase MRP functionally interacts with the snoRNPs which are required for other pre-rRNA processing reactions.Abbreviations pre-rRNA ribosomal RNA precursor - snoRNA small nucleolar RNA - snoRNP small nucleolar ribonucleoprotein particle     

Characteristics of ribonucleic acids isolated from Botryodiplodia theobromae pycnidiospores

Nucleic acids isolated from dormant and germinated Botryodiplodia theobromae pycnidiospores contain five distinct species of RNA. They include two ribosomal species, two ribosomal-associated species and transfer RNAs. Sedimentation coefficients of 25.1S and 18S were obtained for the two ribosomal RNA species and 5.8S and 5S for the two ribosomal-associated RNA components. Molecular weights of 1.20, 0.67, 0.054 and 0.035x10⁶ daltons were obtained after formaldehyde treatment and electrophoresis on polyacrylamide gels for these same four RNAs. Methylated nucleotides were present in the transfer RNAs and large and small ribosomal RNAs; in contrast 5.8S and 5S RNAs contained few methylated nucleotides. In addition to the 5 distinct RNA species, polyadenylate-containing RNA was isolated from both dormant and germinated spores.Published with the approval of the Director as paper no. 5006, Journal Series, Nebraska Agricultural Experiment Station. The work was conducted under Nebraska Agricultural Experiment Station Project no. 21-17.     

Minor Components of Rat Liver Ribosomal RNA as Possible Intermediates in the Degradation of 28S RNA in Vivo

The minor RNA components in large ribosomal subunits of rat liver were analyzed by gel electrophoresis. Quantitative analysis showed that these minor components, whose apparent molecular weights ranged from 8.48 × 10⁵ to 11.4 × 10⁵, represented 10 to 13% of the total high-molecular-weight ribosomal RNA.

It was elucidated that they were not artifacts arose during the cell homogenization, sub-cellular fractionation, RNA preparation and gel electrophoresis.

Labeling experiment in vivo showed that they were preferentially present in “old” ribosomes. It was predicted that these minor components were the intermediates in the degradation of 28S ribosomal RNA in vivo.

In the regenerating liver, where the degradation of proteins was reported to be blocked, the relative amount of the minor RNA components was decreased to about a half of that of normal liver. There was, however, no increase in their relative amount in the long-term starved rat liver, where RNA degradation should be going very rapidly.     

An altered ribosomal protein in an edeine-resistant mutant of Saccharomyces cerevisiae

Summary The r-proteins of an edeine-resistant mutant of Saccharomyces cerevisiae were compared to those of the wild-type strain by using two different two-dimensional electrophoretic techniques: (1) the Kaltschmidt-Wittmann method and, (2) the Kaltschmidt-Wittmann system, in the first dimension and the Na Dodecyl-SO₄ system in the second.With the first technique, the results indicate that the patterns of basic ribosomal proteins are similar in the two strains. However, the pattern of acidic ribosomal proteins of the mutant revealed an additional protein band with respect to the normal one. Using the other technique, the patterns of basic and acidic ribosomal proteins of the mutant demonstrated a similarity to the corresponding pattern of the wild-type strain.The data disclose that an acidic ribosomal protein of the mutant may have two forms with different electrophoretic mobilities and similar molecular weights.     

Comparative differential RNA display analysis of arbuscular mycorrhiza in Pisum sativum wild type and a mutant defective in late stage development

In order to analyse gene expression associated with the late stages of arbuscular mycorrhizal development between Pisum sativum and Glomus mosseae, comparative differential RNA display was carried out using wild-type P. sativum and a mutant, RisNod24, where the fungal partner is not able to form functional arbuscules. Comparison of RNA accumulation patterns between controls, G. mosseae-colonized mutant and wild-type roots resulted in the identification of four differentially occurring cDNA fragments. One of the corresponding genes was from the fungus and three of plant origin. One plant gene, Psam4 (P. sativum arbuscular mycorrhiza-regulated), was analysed in more detail. Sequencing of a cDNA clone showed that Psam4 encodes a proline-rich protein. Northern blot analysis and quantitative RT-PCR revealed a higher basal level of Psam4 RNA accumulation in the mutant compared to the wild type. In both pea genotypes, RNA accumulation was reduced after inoculation with mycorrhiza- or nodule-forming symbiotic microorganisms, but enhanced after infection with a root pathogenic fungus.     

Analysis of revertants of a ribosomal mutation in Podospora anserina: Evidence for new ribosomal mutations which confer hypersensitivity to paromomycin

This paper describes the analysis of cold-resistant revertants of a cold-sensitive mutant. Pm1-1 is a ribosomal mutation screened for its paromomycin resistance. Suppression of its cold sensitivity occurs with two kinds of external mutations localized in two different loci. One of them, PmB, is assumed to be a ribosomal gene. PmB mutations confer hypersensitivity to paromomycin in vivo as well as in vitro in a cell-free protein synthesis system.This work was supported by DGRST Grant MRM/P240 and NATO Grant 1637.     

DNA synthesis in UV-irradiated Escherichia coli K-12 strains carrying dnaA mutations.

Summary In this article we describe some in vivo properties of a coldsensitive ribosomal mutant from Escherichia coli. The mutation affects the rplV gene which is the structural gene of ribosomal protein L22.Our work shows that at 22°C, the biosynthesis of both ribosomal subunits and the maturation processing of 16S and 23S ribosomal RNA are impaired. Integration of our results in a general model of in vivo ribosomal assembly in E. coli is presented.     

Morphological and cytochemical analysis of an unusual nucleus-pyrenoid association in a unicellular red alga

Summary Protrusions of the nucleus that extend into the pyrenoid of the unicellular red algaRhodella maculata have been characterised morphologically and cytochemically. Serial reconstructions indicate that cells frequently have two tubular nuclear protrusions that are up to 1.2 m long. Cytochemical analyses were undertaken to investigate the nucleic acid content of the protrusions. DNA was not detected in the nuclear protrusions, though clear labelling was seen in all other DNA-containing zones (viz. the main nuclear compartment, the chloroplast and the mitochondria). High concentrations of RNA were observed in the nuclear protrusion. In situ hybridization experiments indicate that ribosomal RNAs are not a major component of the protrusion RNA. Possible roles of the protrusion and its RNA content are discussed.Abbreviations DNA deoxyribonucleic acid - RNA ribonucleic acid - rRNA ribosomal RNA     

Maturation of ribosomal precursor RNA in Saccharomyces cerevisiae: A mutant with a defect in both the transport and terminal processing of the 20 S species

A mutant (CLP-8) of Saccharomyces cerevisiae possesses an abnormal ratio of native ribosomal subunits since it has an apparent deficiency of cytoplasmic 40 S subparticles. The mutant also has an abnormal anti-association factor activity. The lesion(s) responsible for the ribosomal subunit inbalance is not temperature-sensitive and is incomplete since the mutant still grows, albeit at a reduced rate compared to that of its parent. The lesion(s) in CLP-8 is, however, expressed at the level of 20 S ribosomal precursor RNA maturation. Thus, relative to the wild-type strain, there is both a slowed transport of 20 S ribosomal precursor RNA from the nucleus to the cytoplasm and a slowed cytoplasmic conversion of this RNA component into the mature 18 S form.     

Mitochondrial ribosomal RNA genes of yeast: Their mutations and a common nuclear suppressor

Summary Due to the absence of repetition of the rRNA genes in S. cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system. We describe here a novel thermosensitive mutation, ts1297, localized by rho^- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene. Defective mutations of the small rRNA have not been reported so far.In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced. The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal. However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca²⁺ in place of Mg²⁺. The 50S particles seem to be completely degraded under normal conditions of extraction with Mg²⁺.The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU₁₀₁. The SU₁₀₁ mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene.These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits.     

Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: IV. Effect of Abscisic Acid and Water Content on RNase Activity and RNA

Plants of the wilty tomato (Lycopersicum esculentum) mutant, flacca, and of the normal cultivar Rheinlands Ruhm growing under either “normal” or high humidity were used in this research. Under normal humidity, RNase activity was much higher in mutant plants in which abscisic acid (ABA) and water content were lower than in the normal plant. The mutant also contained less RNA and protein per cell and less soluble RNA relative to ribosomal RNA as compared with the normal genotype. In ABA-treated mutant plants, RNase activity decreased while RNA, protein, the ratio of soluble to ribosomal RNA and water content increased.