Variation in organisation and copy number of ribosomal RNA genes inPetunia hybrida somaclones

The copy number of genes encoding 5S ribosomal RNA has been found to be constant in Petunia hybrida plants regenerated from protoplast and leaf disc-derived callus cultures. However, in one somaclone a heritable change in the length of the major 5S rDNA repeat has arisen. Despite the constant copy number of 5S rRNA genes, that of the 18S–25S rRNA genes is found to very by at least ten-fold. The relevance of these findings to ribosomal RNA gene variability and to somaclonal variation is discussed.     

Size class homogeneity of repeat lengths and evolutionary divergence of ribosomal RNA genes in fishes as studied by restriction fragment length analysis

Fish ribosomal RNA genes (rDNA) have been compared by restriction endonuclease digestion followed by Southern hybridization using rRNA or cloned rRNA genes as labelled probes. In several species belonging to the orders Cypriniformes and Perciformes, the simple restriction patterns revealed a high degree of size class homogeneity among the rDNA repeats and similar restriction map within a species. Different species have different restriction patterns and fragment lengths arising mostly out of different length of the nontranscribed spacer. Polymorphic restriction sites are present in some species. The species-specific differences in fragment lengths produced in rDNA by some restriction enzymes can thus be used to study interspecific fish hybrids.     

The evolutionary pattern of aromatic amino acid biosynthesis and the emerging phylogeny of pseudomonad bacteria

Pseudomonad bacterial are a phylogenetically diverse assemblage of species named within contemporary genera that includePseudomonas, Xanthomonas andAlcaligenes. Thus far, five distinct rRNA homology groups (Groups I through V) have been established by oligonucleotide cataloging and by rRNA/DNA hybridization. A pattern of enzymic features of aromatic amino acid biosynthesis (enzymological patterning) is conserved at the level of rRNA homology, five distinct and unambiguous patterns therefore existing in correspondence with the rRNA homology groups. We sorted 87 pseudomonad strains into Groups (and Subgroups) by aromatic pathway patterning. The reliability of this methodology was tested in a blind study using coded cultures of diverse pseudomonad organisms provided by American Type Culture Collection. Fourteen of 14 correct assignments were made at the Group level (the level of rRNA homology), and 12 of 14 correct assignments were made at the finer-tuned Subgroup levels. Many strains of unknown rRNA-homology affiliation had been placed into tentative rRNA groupings based upon enzymological patterning. Positive confirmation of such strains as members of the predicted rRNA homology groups was demonstrated by DNA/rRNA hybridization in nearly every case. It seems clear that the combination of these molecular approaches will make it feasible to deduce the evolution of biochemical-pathway construction and regulation in parallel with the emerging phylogenies of microbes housing these pathways.     

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.     

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.     

Changes in Sediment Bacterial Community in Response to Long-Term Nutrient Enrichment in a Subtropical Seagrass-Dominated Estuary

Florida Bay exhibits a natural gradient of strong P limitation in the east which shifts to weak P or even N limitation at the western boundary. This nutrient gradient greatly affects seagrass abundance and productivity across the bay. We assessed the effects of N and P additions on sediment bacterial community structure in relation to the existing nutrient gradient in Florida Bay. Sediment samples from 24 permanent 0.25 m² plots in each of six sites across Florida Bay were fertilized with granular N and P in a factorial design for 26 months. Sediment bacterial community structure was analyzed using PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA (rRNA) genes and a cloning strategy from DGGE bands. The phylogenetic positions of 16S rRNA sequences mostly fell into common members found in marine sediments such as sulfate-reducing Deltaproteobacteria, Gammaproteobacteria, Spirochaetes, and Bacteriodetes. Twenty-eight common DGGE bands were found in all sediment samples; however, some DGGE bands were only found or were better represented in eastern sites. Bacterial community diversity (Shannon-Weiner index) showed similar values throughout all sediment samples. The N treatment had no effect on the bacterial community structures across the bay. Conversely, the addition of P significantly influenced the bacterial community structure at all but the most western site, where P is least limiting due to inputs from the Gulf of Mexico. P additions enhanced DGGE band sequences related to Cytophagales, Ectothiorhodospiraceae, and Desulfobulbaceae, suggesting a shift toward bacterial communities with increased capability to degrade polymeric organic matter. In addition, a band related to Deferribacteres was enhanced in eastern sites. Thus, indigenous environmental conditions were the primary determining factors controlling the bacterial communities, while the addition of P was a secondary determining factor. This P-induced change in community composition tended to be proportional to the amount of P limitation obviated by the nutrient additions.     

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.     

Pyrosequencing-based analysis of fecal microbial communities in three purebred pig lines

This study examined the fecal bacterial diversity of 15-weekold pigs from three purebred lines: Duroc, Landrace, and Yorkshire. Taxon-dependent and -independent analyses were performed to evaluate differences in the fecal bacterial communities and to identify bacterial genera that can be used to discriminate breeds, following high-throughput pyrosequencing of 16S rRNA genes. Among the breeds evaluated, Landrace had the most diverse bacterial community composition. Prevotella, Blautia, Oscillibacter, and Clostridium were detected in all samples regardless of breed. On the other hand, Catenibacterium, Blautia, Dialister, and Sphaerochaeta were differentially detected among breeds, as demonstrated by the canonical loading plot. The discriminant analysis of principal components plot also showed clear separation of the three purebred pig lines, with a certain degree of similarity between Landrace and Yorkshire pigs and a distinct separation between Duroc pigs and the other two breeds. Other factors not related to breed, such as season or time of sampling and pen effects, may contribute to shaping the gut microbiota of pigs.     

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.     

Assessment of microbial communities associated with fermentative–methanogenic biodegradation of aromatic hydrocarbons in groundwater contaminated with a biodiesel blend (B20)

A controlled field experiment was conducted to assess the potential for fermentative–methanogenic biostimulation (by ammonium-acetate injection) to enhance biodegradation of benzene, toluene, ethylbenzene and xylenes (BTEX) as well as polycyclic aromatic hydrocarbons (PAHs) in groundwater contaminated with biodiesel B20 (20:80 v/v soybean biodiesel and diesel). Changes in microbial community structure were assessed by pyrosequencing 16S rRNA analyses. BTEX and PAH removal began 0.7 year following the release, concomitantly with the increase in the relative abundance of Desulfitobacterium and Geobacter spp. (from 5 to 52.7 % and 15.8 to 37.3 % of total Bacteria 16S rRNA, respectively), which are known to anaerobically degrade hydrocarbons. The accumulation of anaerobic metabolites acetate and hydrogen that could hinder the thermodynamic feasibility of BTEX and PAH biotransformations under fermentative/methanogenic conditions was apparently alleviated by the growing predominance of Methanosarcina. This suggests the importance of microbial population shifts that enrich microorganisms capable of interacting syntrophically to enhance the feasibility of fermentative–methanogenic bioremediation of biodiesel blend releases.     

Dietary l-glutamine supplementation modulates microbial community and activates innate immunity in the mouse intestine

This study was conducted to determine effects of dietary supplementation with 1 % l-glutamine for 14 days on the abundance of intestinal bacteria and the activation of intestinal innate immunity in mice. The measured variables included (1) the abundance of Bacteroidetes, Firmicutes, Lactobacillus, Streptococcus and Bifidobacterium in the lumen of the small intestine; (2) the expression of toll-like receptors (TLRs), pro-inflammatory cytokines, and antibacterial substances secreted by Paneth cells and goblet cells in the jejunum, ileum and colon; and (3) the activation of TLR4-nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), and phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt) signaling pathways in the jejunum and ileum. In the jejunum, glutamine supplementation decreased the abundance of Firmicutes, while increased mRNA levels for antibacterial substances in association with the activation of NF-κB and PI3K-Akt pathways. In the ileum, glutamine supplementation induced a shift in the Firmicutes:Bacteroidetes ratio in favor of Bacteroidetes, and enhanced mRNA levels for Tlr4, pro-inflammatory cytokines, and antibacterial substances participating in NF-κB and JNK signaling pathways. These results indicate that the effects of glutamine on the intestine vary with its segments and compartments. Collectively, dietary glutamine supplementation of mice beneficially alters intestinal bacterial community and activates the innate immunity in the small intestine through NF-κB, MAPK and PI3K-Akt signaling pathways.     

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.     

Deep-sea water inhibits metastatic potential in HT-29 human colorectal adenocarcinomas via MAPK/NF-κB signaling pathway

A previous investigation showed that deep-sea water (DSW) can affect the expression of genes that regulate metastasis, including cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP-2), urokinase plasminogen activator (uPA) and uPA receptor (uPAR), in HT-29 human colorectal adenocarcinomas. In the present study, we investigated the effects of DSW on inducible nitric oxide synthase (iNOS) expression and cell migration and also explored the mechanism of DSW-induced anti-metastatic potential in HT-29 human colorectal adenocarcinomas. Cytokine-induced expression of iNOS, which is highly expressed in colon cancer and enhances cancer growth and metastasis, was decreased in a hardness-dependent manner by DSW. Also, the wound healing assay revealed that DSW inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration in a hardness-dependent manner. DSW also decreased the phosphorylation of various MAPKs, including p38, ERK and JNK, and suppressed the nuclear translocation of NF-κB but not c-Jun. The results suggest that DSW may inhibit cancer cell growth related to iNOS overexpression and PKC-mediated cell migration in HT-29 human colorectal adenocarcinomas and the antimetastatic potential of DSW may be regulated by prevention of NF-κB nuclear translocation via inhibition of p38, ERK and JNK phosphorylation. In conclusion, the present investigation demonstrates that DSW inhibits cancer growth and metastasis via down-regulation of iNOS expression and the MAPK/NF-κB signaling pathway.     

Basic amino acids and dimethylarginines targeted metabolomics discriminates primary hepatocarcinoma from hepatic colorectal metastases

Hepatocellular carcinoma (HCC) is a very aggressive neoplasia requiring early and accurate diagnosis to improve patient outcomes with timely treatment. The liver is also very frequently colonized by metastases, and the most frequent differential diagnosis is HCC against intrahepatic cholangiocarcinoma or metastatic adenocarcinoma. Metabolomics is a powerful tool for identification of altered biomarkers in cancer, and to evaluate the efficacy of drug treatments. Here we analyzed by HILIC-MS/MS methylated arginines, basic amino acids (Arg, Cit, Orn), and their ratios in the extracts of primary HCC tissues, liver metastases from colorectal carcinoma (MET), cirrhotic related hepatitis-C-virus (CIR), and non-cirrhotic normal liver (NT) adjacent tissues. We found high levels of Arg (p < 0.0001) and Arg/Orn (p < 0.01) in MET compared to other tissues. In MET, compared to NT and CIR, Arg concentration was fivefold higher, while in HCC it was twofold higher. ADMA increased twofold compared to NT and CIR, while in HCC it was 50 % higher. Arg/Cit and ADMA/SDMA ratios were significantly higher in MET compared to NT and CIR (p < 0.005). Arg/Orn, Arg/Cit, and ADMA/SDMA ratios increased progressively from NT, CIR, HCC, to MET tissues. Arg/Cit correlated significantly with Arg/Orn ratios (r = 0.77; p < 0.0001), and discriminates tumor from non-tumor samples. In addition, the discriminant lactate/glucose ratio we previously found by NMR, also correlated significantly with the Arg levels (r = 0.64; p < 0.0001), and discriminated MET from all other tissues. The results indicated that Arg in MET is higher than other tissue classes, suggesting that, together with the lactate/glucose ratio, it can be considered a further biomarker for HCC-metastases differentiation.     

Increased Expression of Angiogenic Factors in Cultured Human Brain Arteriovenous Malformation Endothelial Cells

To compare the mRNA level of angiogenic factor vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-2, and MMP-9 in cultured human brain arteriovenous malformation (AVM) endothelial cells (ECs) and normal brain endothelial cells (BECs). Tissue explants both from deformed vessels of AVM and normal microvessel were put into culture for endothelial cells. After the monolayer adherent ECs reached confluence, they were tested with endothelial specific marker CD34 and von Willebrand factor (vWF) by immunochemical assay. mRNA levels of VEGF-A, MMP-2, and MMP-9 in AVM endothelial cells (AVMECs) and BECs were measured by PCR. Immunostaining confirmed that more than 95 % of the cultured cells were CD34 (Fig. 1b) and/or vWF positive. Expression levels of VEGF-A and MMP-2 mRNAs were significantly higher in AVMECs than in BECs. The MMP-9 level was also increased in AVMECs, but the difference was not statistically significant. Vascular tissue explants adherent method is a better approach for isolation and culture of AVMECs. Cultured AVMECs expressed higher angiogenic factors (VEGF, MMP-2) than the controlled BECs, implicating angiogenesis plays an important role in the pathogenesis of AVM.