Molecular cloning and characterization of ribosomal RNA genes from the brine shrimp: Nucleotide sequence analysis and evolution of the 5.8 S rRNA gene region and its flanking nucleotides

A detailed restriction endonuclease map was prepared for the cloned 5.8 S ribosomal RNA (rRNA) gene region of the brine shrimp Artemia. The nucleotide sequence of the 5.8 S rRNA gene and its flanking nucleotides was determined. This sequence differs in two positions from that of the previously reported 5.8 S rRNA. The primary structure of the Artemia 5.8 S rRNA gene, which, unlike in dipteran insects, is shown to contain no insertion sequence, is conserved according to the relatedness of the species compared. The 5.8 S rRNA gene flanking nucleotides, which were sequenced 176 nucleotide pairs upstream and 70 nucleotide pairs downstream from the gene, show no evidence of sequence conservation between evolutionarily diverse species by computer analysis. Direct nucleotide repeats are present within the flanking sequences at both ends of the gene at about the same distance upstream and downstream, which could serve as processing signals.     

Determination of 16S ribosomal RNA gene copy number in Streptococcus uberis, S. agalactiae, S. dysgalactiae and S. parauberis

Abstract Species-specific oligonucleotide probes and a universal oligonucleotide probe derived from sequences of 16S rRNA were hybridised to chromosomal DNA from Streptococcus agalactiae, S. dysgalactiae, S. parauberis and S. uberis following digestion with Eco RI. Due to the presence of a unique Eco RI site in each 16S rRNA gene, the number of hybridised fragments was indicative of the number of 16S rRNA genes. Southern hybridisation indicated six 16S rRNA genes in ten isolates of S. agalactiae , five genes in ten isolates of S. uberis , five genes in six isolates and six in another isolate of S. dysgalactiae , and six genes in four isolates of S. parauberis . For a fifth isolate of S. parauberis , six 16S rRNA genes were indicated by the universal probe but only five when hybridised to the species-specific probe, indicating sequence variation (microheterogeneity) within the probe target region.     

Ribosomal RNA gene number and sequence divergence in the diploid-tetraploid species pair of north American hylid tree frogs

Hyla chrysoscelis (2n = 24) and H. versicolor (2n = 48) are a diploid-tetraploid species pair of tree frogs. Hybridization saturation of isolated ¹²⁵I-labeled ribosomal RNAs (rRNAs) with filter-immobilized DNA shows that there are twice as many rRNA genes in the tetraploid as in the diploid. For the diploid, saturation occurs at 0.037%, from which it is calculated that there are about 618 copies of the (18 S + 28 S) rRNA genes per haploid genome. Analysis of the extent of hybridization and also the thermal stability of homologous and heterologous hybrids shows that considerably more base substitutions have occurred in the tetraploid rDNA genes than in the diploid since their divergence. This is interpreted to reflect either a relaxation of the gene regulatory correction mechanism hypothesized to be responsible for the maintenance of identical tandem rRNA genes in the tetraploid or a release of one gene set from the normal selective constraints.This research supported by NSF Grants CDP-8002341 and PRM-8106947 to J.C.V. and by research grants from Miami University to L.A.T., D.T.C., R.J.D., and S.W.K.     

Complete sequence and gene organization of the Nosema heliothidis ribosomal RNA gene region

By sequencing the entire ribosomal RNA (rRNA) gene region of Nosema heliothidis isolated from cotton bollworm (Helicoverpa armigera), we showed that its gene organization is similar to the type species, Nosema bombycis: the 5'-large subunit rRNA (2,490 bp)-internal transcribed spacer (192 bp)-small subunit rRNA (1,232 bp)-intergenic spacer (274 bp)-5S rRNA (115 bp)-3'. We constructed two phylogenetic trees, analyzed phylogenetic relationships, examined rRNA organization of microsporidia, and compared the secondary structure of small subunit rRNA with closely related microsporidia. The latter two features may provide important information for the classification and phylogenetic analysis of microsporidia.     

Complete sequence and gene organization of the Nosema spodopterae rRNA gene

By sequencing the entire ribosomal RNA (rRNA) gene of Nosema spodopterae, we show here that its gene organization follows a pattern similar to the Nosema type species, Nosema bombycis, i.e. 5'-large subunit rRNA (2,497 bp)-internal transcribed spacer (185 bp)-small subunit rRNA (1,232 bp)-intergenic spacer (277 bp)-5S rRNA (114 bp)-3'. Gene sequences and the secondary structures of large subunit rRNA, small subunit rRNA, and 5S rRNA are compared with the known corresponding sequences and structures of closely related microsporidia. The results suggest that the Nosema genus may be heterogeneous and that the rRNA gene organization may be a useful characteristic for determining which species are closely related to the type species.     

On the relationship between the sequence conservation and the packing density profiles of the protein complexes

We have recently showed that the weighted contact number profiles (or the packing density profiles) of proteins are well correlated with those of the corresponding sequence conservation profiles. The results suggest that a protein structure may contain sufficient information about sequence conservation comparable to that derived from multiple homologous sequences. However, there are ambiguities concerning how to compute the packing density of the subunit of a protein complex. For the subunits of a complex, there are different ways to compute its packing density – one including the packing contributions of the other subunits and the other one excluding their contributions. Here we selected two sets of enzyme complexes. Set A contains complexes with the active sites comprising residues from multiple subunits, while set B contains those with the active sites residing on single subunits. In Set A, if the packing density profile of a subunit is computed considering the contributions of the other subunits of the complex, it will agree better with the sequence conservation profile. But in Set B the situations are reversed. The results may be due to the stronger functional and structural constraints on the evolution processes on the complexes of Set A than those of Set B to maintain the enzymatic functions of the complexes. The comparison of the packing density and the sequence conservation profiles may provide a simple yet potentially useful way to understanding the structural and evolutionary couplings between the subunits of protein complexes. Proteins 2013; 81:1192–1199. © 2013 Wiley Periodicals, Inc.     

Human NAT10 Is an ATP-dependent RNA Acetyltransferase Responsible for N4-Acetylcytidine Formation in 18 S Ribosomal RNA (rRNA)

Human N-acetyltransferase 10 (NAT10) is known to be a lysine acetyltransferase that targets microtubules and histones and plays an important role in cell division. NAT10 is highly expressed in malignant tumors, and is also a promising target for therapies against laminopathies and premature aging. Here we report that NAT10 is an ATP-dependent RNA acetyltransferase responsible for formation of N⁴-acetylcytidine (ac⁴C) at position 1842 in the terminal helix of mammalian 18 S rRNA. RNAi-mediated knockdown of NAT10 resulted in growth retardation of human cells, and this was accompanied by high-level accumulation of the 30 S precursor of 18 S rRNA, suggesting that ac⁴C1842 formation catalyzed by NAT10 is involved in rRNA processing and ribosome biogenesis.     

Sequence variations in the large-subunit ribosomal RNA gene of ammonia (foraminifera,protozoa) and their evolutionary implications

An unusually high divergence was observed in the ribosomal RNA genes of a free-living population of foraminifera belonging to the genusAmmonia. The sequences of a large-subunit (LSU) rDNA expansion segment D1 and flanking regions were obtained from 20 specimens namedAmmonia sp. 1 andAmmonia sp. 2. The sequence divergence between the two species averages 14%. Within each species it ranges from 0.2% to 7.1% inAmmonia sp. 1 and from 0.7% to 2.3% inAmmonia sp. 2. We did not find two specimens having identical sequences. Moreover, in opposition to the generally acaepted view, rDNA sequence variations were also found within a single individual. The variations among several rDNA copies in a single specimen ofAmmonia may reach up to 4.9%. Most of the observed variations result from multiplication of CA or TA serial repeats occurring in two particularly variable regions. For single base changes, C-T transitions are most frequently observed. We discuss the evolution of expansion segments and their use for phylogenetic studies. Correspondence to: J. Pawlowski     

On the relationship between residue structural environment and sequence conservation in proteins

Residues that are crucial to protein function or structure are usually evolutionarily conserved. To identify the important residues in protein, sequence conservation is estimated, and current methods rely upon the unbiased collection of homologous sequences. Surprisingly, our previous studies have shown that the sequence conservation is closely correlated with the weighted contact number (WCN), a measure of packing density for residue's structural environment, calculated only based on the C_α positions of a protein structure. Moreover, studies have shown that sequence conservation is correlated with environment‐related structural properties calculated based on different protein substructures, such as a protein's all atoms, backbone atoms, side‐chain atoms, or side‐chain centroid. To know whether the C_α atomic positions are adequate to show the relationship between residue environment and sequence conservation or not, here we compared C_α atoms with other substructures in their contributions to the sequence conservation. Our results show that C_α positions are substantially equivalent to the other substructures in calculations of various measures of residue environment. As a result, the overlapping contributions between C_α atoms and the other substructures are high, yielding similar structure–conservation relationship. Take the WCN as an example, the average overlapping contribution to sequence conservation is 87% between C_α and all‐atom substructures. These results indicate that only C_α atoms of a protein structure could reflect sequence conservation at the residue level.     

Silene vulgaris subsp. macrocarpa leaves and roots from Morocco: assessment of the efficiency of different extraction techniques and solvents on their antioxidant capacity,brine shrimp toxicity and phenolic characterization

Abstract

This study was undertaken to investigate the effect of various solvents and techniques on the extractability of antioxidant compounds, particularly phenolics, from leaves and roots of Silene vulgaris subsp. macrocarpa grown wild in Morocco. Maceration and hot extraction with methanol or water and Soxhlet ethanol extraction were utilized. Aimed at establishing the potential safety of the extracts, Artemia salina lethality bioassay was performed. All the extracts were found to be non-toxic, except for the leaf Soxhlet ethanol. The antioxidant potential of the extracts was evaluated in vitro by DPPH, reducing power, and ferrous ions chelating activity assays. The leaf extracts displayed noticeable radical scavenging and chelating activities, and maceration with methanol (Mac-MeOH) resulted the most suitable extraction method for an effective recovery of antioxidants; further, the root Mac-MeOH extract demonstrated good chelating properties (IC₅₀ = 335.49?±?0.70?µg/mL). Thus, leaf and root Mac-MeOH extracts were subjected to phytochemical investigations. The total phenolic, flavonoid and condensed tannin content was determined spectrophotometrically. Thirteen polyphenolic compounds were positively identified, by HPLC-PDA-ESI-MS, in the leaf extract for the first time, with p-coumaric acid derivatives being the most abundant ones (81%), whereas only catechin and procyanidin B1 were found in the root extract.     

静电纺丝的主要参数对PLGA纤维支架形貌和纤维直径的影响

EST(expressed sequence tags,EST)是一段长约150～500bp基因表达的外源序列片段,是由大规模随机挑取的cDNA克隆测序得到的组织或细胞基因组的表达序列标签。一个EST代表生物某一时期的某种组织或细胞的一个表达基因。主要综述了EST技术的原理方法,哺乳动物早期胚胎研究的理论基础以及EST技术在早期胚胎研究方面的应用,并讨论了利用EST进行研究分析的发展趋势。     

Structure of the Large Subunit rDNA from a Diatom, and Comparison Between Small and Large Subunit Ribosomal RNA for Studying Stramenopile Evolution

The aim of this study was to compare the usefulness of complete small and large subunit rRNA, and a combination of both molecules, for reconstructing stramenopile evolution. To this end, phylogenies from species of which both sequences are known Acre constructed with the neighbor-joining, maximum parsimony, and maximum likelihood methods. Also the use of structural features of the rRNAs was evaluated. The large subunit rRNA from the diatom Skeletonema pseudocostatum was sequenced in order to have a more complete taxon sampling, and a group I intron was identified. Our results indicated that heterokont algae are monophyletic, with diatoms diverging first. However, as the analysis was restricted to a particular data set containing merely six taxa, the outcome has limited value for elucidating stramenopile relationships. On the other hand, this approach permits comparison of the performance of both rRNA molecules without interference from other factors, such as a different species selection for each molecule. For the taxa used, the large subunit rRNA clearly contained more phylogenetic information than the small subunit rRNA. Although this result can definitely not be generalized and depends on the phvlogeny to be studied, in some cases determining complete large subunit rRNA sequences certainly seems worthwhile.     

Organization of Ribosomal RNA Genes in Borrelia burgdorferi sensu lato Isolated from Ixodes ovatus in Japan

Borrelia burgdorferi sensu lato was obtained from adult ixodid ticks, Ixodes ovatus, collected in Nagano, Japan, and was named NT112. The genomic DNA was digested with enzymes, electrophoresed, blotted and hybridized with rRNA gene probes obtained from B. burgdorferi sensu stricto B31. The results showed that the borrelial chromosome contains a single rrs (16S rRNA gene) sequence and two copies of rrl/rrf (23S/5S rRNA genes) sequences. The rrl/rrf genes were tandemly repeated at intervals of 3.2 kb and were located separately from the rrs gene on the genome. Our findings indicate that the organization of rRNA genes in Borrelia from I. ovatus ticks is identical to that of B. burgdorferi sensu stricto.     

Ribosomal DNAs: an exception to the conservation of gene order in rice genomes

rDNA (18S-5.8S-25S rDNA) and 5S rDNA loci were visualized on the chromosomes of six species of the genus Oryza by fluorescence in situ hybridization (FISH) and the labeled rice chromosomes were identified based on their condensation patterns. As a result, the chromosomes harboring rDNA and/or 5S rDNA loci were determined in the complement for all the known rice genomes. Variation in the location of the rDNA loci indicated the transpositional nature of the rDNAs in the genus Oryza, as also suggested in Triticeae and Allium. Comparative analysis of the locations of rDNA loci among rice, maize and wheat revealed that variability in the physical location of the rDNA loci was characteristic of the genus Oryza and also of the genera of Gramineae. This variability in the location of the rDNA loci between evolutionarily related species is in sharp contrast to the conservation of the general order of genes in their genomes.     

Sequence of the Small Subunit Ribosomal RNA Gene of Perkinsus atlanticus-like Isolated from Carpet Shell Clam in Galicia, Spain

Parasites identified as Perkinsus atlanticus have been reported infecting carpet shell clams in Galicia (northwest Spain). We have sequenced the 18S ribosomal RNA gene of in vitro cultured Perkinsus atlanticus-like or hypnospores from diseased clams, and compared it with the same genomic region from P. marinus and Perkinsus sp. We have also compared the sequence of internal transcribed spacer (ITS) 1, ITS 2, and 5.8S rRNA from our isolate with the P. atlanticus GenBank sequence. The phylogenetic analysis of our cultured parasite based on the 18S gene led us to conclude that this isolate is not related to the genus Perkinsus but to the protists Anurofeca, Ichthyophonus, and Psorospermium, located near the animal-fungal divergence. These last two genera have been included, together with Dermocystidium, in the newly described DRIPs (Dermocystidium, rossete agent, Ichthyophonus, and Psorospermium) clade, recently named Mesomycetozoa. Received October 25, 1999; accepted February 11, 2000.