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.     

The investigation of the effect of pollination on ribosomal RNA,transfer RNA and DNA contents in styles ofNicotiana alata.

Using the phenol extraction method and MAK column chromatography the contents of nucleic acids in styles ofNicotiana alata were estimated before and up to 48 h after compatible pollination. As a result of pollination, high-molecular-weight rRNA and DNA registered a significant increase in their content approximating 30% and 16% respectively. The change in sRNA (4-S tRNA and 5-S rRNA) level was very slight and non-significant. In pollen grains there is an unusually high amount of rRNA with respect to the content of other nucleic acids and the rRNA/tRNA ratio approximates 14 : 1. On the other hand, in non-pollinated styles the content of rRNA is only about 6 times higher than that of tRNA. The changes in nucleic acid level found in styles after pollination are at least in a major part the result of the addition of the nucleic acids present in the amount of pollen used for pollination. The high rRNA level relatively to tRNA being generally associated with rapidly growing cells, its significance in pollen may be related to the rapid growth of pollen tubes.     

Comparisons of procedures for determining ribosomal ribonucleic acid similarities

Ribosomal ribonucleic acid (rRNA) cistron similarities were determined forClostridium species with labeled preparations of 16S, 23S, or 16S plus 23S rRNA. Similarities were measured by membrane competition and by thermal stability experiments, and the results were correlated. Correlations were also made between 16S rRNA membrane competition homology values and similarities of 16S rRNA oligonucleotides. Very similar results were obtained whether 16S-or 23S-labeled rRNA was used. Clustering patterns were similar for homology values from hybridization experiments incubated at 50° or 60°C, as were the clustering patterns comparing homology values and rRNA hybrid thermal stability values. There was a linear correlation between rRNA homology values and 16S oligonucleotide S_AB values (Tanner et al. [22]) for homology values above 20%, and S_AB values above 0.45.     

Maturation of ribosomal RNA in blue-green algae in light and darkness

The blue-green algaPlectonema boryanum was chosen for a study of the properties of rRNA of procaryotic organisms. The process of rRNA species maturation was studied by labelling RNA with the isotope 32P.23 S and 16 S rRNA molecules each have their own precursors. Molecules with molecular weights of 1.65 X 10⁶ and 1.24xlO⁶ were identified as precursors of23 S rRNA, while the molecules 0.87 X 10⁶; 0.78 X 106 and 0.68 X 10⁶ were found to be precursors of the 16 S rRNA. No common polycistronic precursor could be found. Experiments done with algae cultivated in light and in the dark confirmed the fact, that the rate of rRNA synthesis is reduced in the dark, without however stopping completely.     

Control of RNA level and of RNA ratios in the latex ofHevea brasiliensis Müll. Arg. effect of latex tapping and of growth regulators

The association between latex RNA and latex production was examined using MAK column chromatography techniques. In young untapped trees the introduction of tapping or the treatment of bark with growth regulators resulted in an increase of RNA level and of rRNA/tRNA ratio in the latex. In regularly tapped trees an increase in rRNA but not in tRNA was brought about by increasing the tapping frequency. Treatment with growth regulators had the same effect but essentially only through the related enhancement of latex export from latex vessels. During latex flow, the highest RNA level was registered in latex fractions originating from the most heavily drained areas of bark. Using³²P labeling, evidence was obtained that the export of latex results in an enhancement of rRNA migration into the inner latex containing space of the vessels. This is considered as the reason of the generally observed association of high RNA level and of high rRNA/tRNA ratio with high latex yield. It is proposed that in controlling the RNA level and RNA proportions in the latex an important role is played by changes in turgor pressure associated with the loss of latex which may influence the export of RNA from the nucleus through related induction of pressure disequilibrium between the nucleoplasm and the latex cytoplasm.     

Location of the genes coding for 18S and 28S ribosomal RNA in the human genome

³H-rRNA obtained from Xenopus laevis tissue cultured cells, or a ³H-cRNA made from Xenopus ribosomal DNA, was used for heterologous in situ hybridisation with human lymphocyte metaphase chromosomes. Prior to hybridisation, chromosome spreads were stained with Quinacrine and selected cells showing good Q-banding photographed; the same cells were then rephotographed after autoradiography and pairs of photographs for each cell were used to make dual karyotypes. The chromosomes within each karyotype were divided into equal sized segments (approx. 0.7 μ), with a fixed number of segments for each chromosome type. The distribution of silver grains between segments showed that the ³H-RNAs hybridised specifically to the nucleolar organising regions of the D and G group chromosomes with no other sites of localised labelling in the complement. Control experiments showed no localisation, with insignificant labelling, when metaphase spreads were incubated in a mixture containing Xenopus ³H-rRNA and competing cold human (HeLa) rRNA. Filter hybridisation experiments on isolated human DNA showed that the Xenopus derived ³H-RNAs hybridised to a fraction of human DNA which was on the heavy side of the main DNA peak and that these RNAs were competed out in the presence of excess cold human rRNA, confirming the specificity of the heterologous hybridisation. In situ hybridisation experiments were also carried out on cells from individuals with one chromosome pair showing heteromorphism for either a very long stalk (nucleolar constriction) subtending a satellite, or a large satellite. It was shown that the chromosome with the large stalk hybridised four times as much ³H-rRNA as its homologue, whereas differences in the sizes of the subtended satellites did not materially affect hybridisation levels indicating that rDNA is located in the stalks and not the satellites. The amount of ³H-rRNA hybridised differs between chromosomes and individuals; these differences are heritable and rDNA can be detected by in situ hybridisation in all three chromosomes number 21 in cells from Down's patients and in translocated chromosomes conta.ining a nucleolar constriction. Different D and G group chromosomes which hybridised equal amounts of ³H-rRNA participated in rosette associations at metaphase in a random fashion in some individuals and in a non-random fashion in others. In all individuals studied chromosomes with large amounts of rDNA were not found to be preferentially involved in association. It was therefore concluded that the probability of a chromosome being involved in the formation of a common nucleolus is not a simple function of its rDNA content and other possible factors are considered.     

Dominance of Methanosarcinales Phylotypes and Depth-Wise Distribution of Methanogenic Community in Fresh Water Sediments of Sitka Stream from Czech Republic

The variation in the diversity of methanogens in sediment depths from Sitka stream was studied by constructing a 16S rRNA gene library using methanogen-specific primers and a denaturing gradient gel electrophoresis (DGGE)-based approach. A total of nine different phylotypes from the 16S rRNA library were obtained, and all of them were clustered within the order Methanosarcinales. These nine phylotypes likely represent nine new species and at least 5–6 new genera. Similarly, DGGE analysis revealed an increase in the diversity of methanogens with an increase in sediment depth. These results suggest that Methanosarcinales phylotypes might be the dominant methanogens in the sediment from Sitka stream, and the diversity of methanogens increases as the depth increases. Results of the present study will help in making effective strategies to monitor the dominant methanogen phylotypes and methane emissions in the environment.     

Cytogenetics of the parthenogenetic grasshopper Warramaba virgo and its bisexual relatives

Combinations of DNA-binding fluorescent dyes and counterstains that enhance selectivity and contrast in primary stain fluorescence were used to differentiate types of C-bands in the genus Warramaba. Chromomycin A₃ (in conjunction with two A-T binding counterstains), which identifies chromosome segments enriched in G-C base pair clusters, stains only a minority of the C-bands in Warramaba species, but these include all those known to contain 18S + 26S rRNA cistrons and most of those containing 5S rRNA genes. DAPI/actinomycin D fluorescent staining is positive for a very few bands, including two (in the Standard phylad of W. virgo) that are at or adjacent to sites containing 5S rRNA cistrons. One of the latter regions is also positively stained by DAPI/distamycin A which, in addition, highlights some centromeric bands. The fluorescent staining patterns of the Standard and Boulder-Zanthus phylads of W. virgo are significantly different, confirming their independent origin by hybridization between different races of the ancestral species “P169” and “P196”.     

High Fungal Diversity and Abundance Recovered in the Deep-Sea Sediments of the Pacific Ocean

Knowledge about the presence and ecological significance of bacteria and archaea in the deep-sea environments has been well recognized, but the eukaryotic microorganisms, such as fungi, have rarely been reported. The present study investigated the composition and abundance of fungal community in the deep-sea sediments of the Pacific Ocean. In this study, a total of 1,947 internal transcribed spacer (ITS) regions of fungal rRNA gene clones were recovered from five sediment samples at the Pacific Ocean (water depths ranging from 5,017 to 6,986 m) using three different PCR primer sets. There were 16, 17, and 15 different operational taxonomic units (OTUs) identified from fungal-universal, Ascomycota-, and Basidiomycota-specific clone libraries, respectively. Majority of the recovered sequences belonged to diverse phylotypes of Ascomycota (25 phylotypes) and Basidiomycota (18 phylotypes). The multiple primer approach totally recovered 27 phylotypes which showed low similarities (≤97 %) with available fungal sequences in the GenBank, suggesting possible new fungal taxa occurring in the deep-sea environments or belonging to taxa not represented in the GenBank. Our results also recovered high fungal LSU rRNA gene copy numbers (3.52?×?10⁶ to 5.23?×?10⁷copies/g wet sediment) from the Pacific Ocean sediment samples, suggesting that the fungi might be involved in important ecological functions in the deep-sea environments.     

5.8S rDNA variability in Allium species belonging to the third evolutionary group

Sequence analysis of 5.8S rDNA in 67 accessions of the subgenus Allium and six other subgenera belonging to the third evolutionary group of Allium genus (Friesen et al., 2006) was performed. Nucleotide substitutions in 5.8S rDNA sequences of Allium accessions were identified and studied for the first time. The probable secondary structure of 5.8S rRNA was constructed. It was shown that mutations in 5.8S rDNA do not involve conserved motifs, and they did not significantly affect the secondary structure of the RNA molecule in Allium accessions.     

The nucleolus organizer region of maize (Zea mays L.)

Maize plants were produced partially triploid for the heterochromatic segment of the nucleolus organizer region (NOR) or partially triploid or tetraploid for the site giving rise to the secondary constriction of the NOR. These partially hyperploid plants were characterized in terms of chromosome and/or nucleolar constitution by light microscopy at pachytene, diakinesis, and quartet stages of microsporogenesis. DNA's of the various partially hyperploid plants and appropriate controls were extracted and hybridized with ³H-rRNA. The heterochromatic segment of the NOR was found to contain most of the rRNA cistrons, but has little or no interaction with the nucleolus. In contrast with the heterochromatic segment, the site giving rise to the secondary constriction contains few rRNA cistrons but is active in nucleolar formation as viewed at pachytene, diakinesis and quartet stages.     

Transcription and processing of rRNA in higher plant cells (Daucus carota,Petroselinum crispum,Acer pseudoplatanus)

Actively dividing cells from parsley (Petroselinum crispum) and carrot (Daucus carota) (bothApiaceae) andAcer pseudoplatanus (Aceraceae) were used to detect the primary gene product of the rDNA in plant cells. Parsley and carrot cells were labelled with [³²P] orthophosphate. In both cases only one high molecular weight rRNA precursor was present on polyacrylamide gels under non-denaturing conditions. Its molecular weight did not exceed 2.5 × 10⁶ daltons. The component emerged from the heterogenous material after a labelling period of 5–10 min. In parsley cells 45 min after onset of incubation labelled mature rRNA (25S and 18S) arrived in the cytoplasm. InAcer pseudoplatanus (incubation period 60 min) two rapidly labelled components did emerge from polyacrylamide gels; their molecular weights were 2.3 and 3.2^? 3.4 × 10⁶ daltons. After electrophoresis under denaturing conditions the larger component was no longer present, thus indicating that it was an aggregate of different RNA molecules. The molecular weights of the rRNA precursors ofD. carota andP. crispum determined after electrophoresis in formamide gels were about 2.1 × 10⁶ daltons. From these results we have no evidence for the existence of rRNA precursors exceeding the molecular weight of 2.5 × 10⁶ daltons.     

Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

Transcriptional activity of heterocysts isolated from Anabaena variabilis

Nitrogenase activity, RNA synthesis, and protein synthesis were measured in heterocysts of Anabaena variabilis. Heterocysts labelled in situ for 4 h with [¹⁴C]uracil accumulated label in rRNA and tRNA to the same specific activity as RNA from vegetative cells. With isolated heterocysts, however, assimilation of [³H]uracil into RNa occurred at about 10% the rate in vegetative cells, and ceased 90 min after isolation. Pulse-chase experiments indicated that heterogeneous, high-molecular-weight RNA synthesized during the first 30 min of incubation was turned over during a 2 h chase, howver there was no accumulation of label in rRNA and tRNA as was seen with heterocysts labelled in situ and with vegetative cells. Assimilation of [³H]glycine into protein by isolated heterocysts was linear up to about 60 min, then proceeded at a slower rate for an additional 180 min. Maintenance of protein synsthesis and nitrogen fixation were both blocked by chloramphenicol and rifampicin. The data suggest that differentiated heterocysts continue to synthesize RNA and proteins and that these processes may contribute to the functional lifetime of heterocysts.