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PubMed2025

Molecular phylogeny of some Carangid species from the Egyptian Red Sea using cytochrome c oxidase subunit I (COI) and small (12S rRNA) mitochondrial rRNA genes.

BACKGROUND: Study of five Carangid species to estimate the degree of genetic divergence and draw phylogenetic relationships by using cytochrome c oxidase subunit I (COI), and small (12S rRNA) mitochondrial rRNA genes. AIM: This investigation was designed to evaluate genetic relationships and association analyses in the taxonomy studies of Carangid fishes using mitochondrial sequences. METHODS: The present study analyzed sequence data using two genes to estimate the relationships among five species of the family Carangidae (ray-finned fish), such as Carangoides bajad (gold-spotted trevally), Carangoides malabaricus (Malabar trevally), Caranx melampygus (Bluefin trevally), Caranx sexfasciatus (Bigeye trevally), and Scomberoides lysan (doublespotted queenfish) and to assess the phylogenetic utility of these markers. RESULTS: The classification analysis of the family Carangidae is controversial. Our study was performed to examine the phylogenetic relationships among five Carangid species using 12S rRNA and COI genes, that illustrated certain Carangidae family genera are not monophyletic that does not include all the descendants of a common ancestor (Paraphyletic) refers to a taxonomic grouping that includes a common ancestor and some, but not all, of its descendants. This means that a paraphyletic group consists of the last common ancestor and excludes certain lineages that are part of the broader group, for example, in traditional taxonomy, the class of fish is considered paraphyletic because it does not include all descendants. The data reported here may be employed in study and analysis of the phylogenetic variety and relationships among species and genera of the family Carangidae. CONCLUSION: Our results confirmed the thermostability and environmental adaptation of the five species of the Carangidae family due to higher A+T content. Our results also confirmed the earlier conclusions of other authors that several genera of the Carangidae family are not monophyletic which does not include all the descendants of a common ancestor (Paraphyletic) and demonstrated the usefulness of the 12S rRNA gene and the COI gene in the phylogenetic analysis of the Carangid species.

RNA, Ribosomal↗
PubMed1995

An efficiently expressed 5.8S rRNA 'tag' for in vivo studies of yeast rRNA biosynthesis and function.

Inefficient expression or detrimental markers have limited mutational analyses of eukaryotic 5.8S rRNA and the associated rDNA transcribed spacers. We have found a neutral, 4-base insertion mutation that effectively tags the 5.8S rRNA for improved studies of rRNA expression, processing and function. Cells expressing the tagged rDNA plasmid contain 50-60% mutant 5.8S rRNA, but show a normal growth rate and polysomal profile and a constant distribution of tagged 5.8S rRNA. The high level of expression also demonstrates that plasmid-associated rDNA is preferentially transcribed over chromosomal copies.

Base Sequence↗
PubMed1992

Analysis of DNA encoding 23S rRNA and 16S-23S rRNA intergenic spacer regions from Plesiomonas shigelloides.

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.

Base Sequence↗
PubMed1993

Coordinated decreases in rRNA gene transcription factors and rRNA synthesis during muscle cell differentiation.

rRNA synthesis decreases significantly during the differentiation of rat L6 myoblasts to myotubes. Nuclear run-on assays demonstrated that the decrease was attributable to decreased rates of rRNA gene transcription. Immunoblot analysis indicated a marked reduction in amounts of the RNA polymerase I transcription factors UBF1 and UBF2 (upstream binding factors 1 and 2, respectively). The levels of these factors dropped in parallel with the down-shift in rRNA gene transcription. The amount of UBF does not fall due to a general decrease in cellular protein, as myosin heavy-chain protein accumulates markedly during this same time. RNA blots of total RNA isolated from myoblasts and differentiating myotubes showed a decrease in the mRNA for UBF, at the same time the mRNA for myogenin was accumulating. The down-shift in UBF mRNA levels preceded the decrease in the protein levels for UBF. There have been reports that the acute response of the rRNA gene transcription system to physiological signals in many systems involves an RNA polymerase I-associated factor. However, our results imply that the regulation of rRNA gene DNA transcription in response to physiological processes, such as differentiation, may involve multiple regulatory pathways.

Adenosine Triphosphate↗
PubMed2002

Rpf2p, an evolutionarily conserved protein, interacts with ribosomal protein L11 and is essential for the processing of 27 SB Pre-rRNA to 25 S rRNA and the 60 S ribosomal subunit assembly in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Rrs1p is a nuclear protein that is essential for the maturation of 25 S rRNA and the 60 S ribosomal subunit assembly. In two-hybrid screening, using RRS1 as bait, we have cloned YKR081c/RPF2. Rpf2p is essential for growth and is mainly localized in the nucleolus. The amino acid sequence of Rpf2p is highly conserved in eukaryotes from yeast to human. Similar to Rrs1p, Rpf2p shows physical interaction with ribosomal protein L11 and appears to associate with preribosomal subunits fairly tightly. Northern, methionine pulse-chase, and sucrose density gradient ultracentrifugation analyses reveal that the depletion of Rpf2p results in a delayed processing of pre-rRNA, a decrease of mature 25 S rRNA, and a shortage of 60 S subunits. An analysis of processing intermediates by primer extension shows that the Rpf2p depletion leads to an accumulation of 27 SB pre-rRNA, suggesting that Rpf2p is required for the processing of 27 SB into 25 S rRNA.

Alleles↗
PubMedDate not supplied

The 5S rRNA and the rRNA intergenic spacer of the two varieties of Cryptococcus neoformans.

The intergenic spacers (IGS) separating the 23S-like and 16S-like rDNAs of the two varieties of the human pathogenic fungus Cryptococcus neoformans were amplified, cloned and sequenced. The C. neoformans var. neoformans IGS was 2421 nt with 5S rRNA at positions 1228-1345 3' of the 23S-like rRNA. The C. neoformans var. gattii IGS was 2480 nt with 5S rRNA at positions 1268-1385 3' of the 23S-like rRNA. For both varieties the 5S rDNA genes were in the same orientation as the 16S-5.8-23S genes and encode a 118 nt molecule of identical sequence. Phylogenetic comparison of C. neoformans 5S rDNA with that of other fungi placed this fungus in close relationship with other basidiomycetes including Tremella mesenterica, Bullera alba, and Cryptococcus laurentii. A secondary structure model for the deduced 5S rRNA was constructed by comparative sequence analysis. Polymerase chain reaction-amplified IGS of 12 C. neoformans var. neoformans strains revealed extensive size variation ranging from 100 to 300 nt. Size variation between strains in the length of the IGS may be useful for distinguishing strains. Structurally, the IGS were characterized by the presence of occasional short direct GC-rich 19-nt repeats. Overall IGS sequence identity between the C. neoformans varieties was only 78.5%, in sharp contrast to the identical or nearly identical sequences for the rDNA genes, and suggests rapid evolution for IGS sequences.

Base Sequence↗
PubMed1982

Nucleotide sequence of the region between the 18S rRNA sequence and the 28S rRNA sequence of rat ribosomal DNA.

The DNA sequence of the intragenic region of the rat 45S ribosomal RNA precursor was determined. This sequence contains 2282 nucleotides and extends from the conserved EcoR I site near the 3' terminus of 18S rRNA to 69 nucleotides downstream of the 5' terminus of 28S rRNA. The sequences corresponding to 18S and 5.8S rRNA were identified by comparison with previously published data. The 5' terminus of rat 28S rRNA was identified by S1 nuclease protection and reverse transcriptase elongation assays. The internal transcribed spacers were found to be 1066 and 765 nucleotides long and had little homology with those of Xenopus and yeast. Regions of sequence homology between rat and Xenopus were found at the junctions of the internal transcribed spacers with 18S, 5.8S and 28S rRNA. These homologies suggest that these sequences may function as recognition sites for the processing of the ribosomal precursor RNA.

Animals↗
PubMed1983

Structure of mouse rRNA precursors. Complete sequence and potential folding of the spacer regions between 18S and 28S rRNA.

We have determined the complete nucleotide sequence of the regions of mouse ribosomal RNA transcription unit which separate mature rRNA genes. These internal transcribed spacers (ITS) are excised from rRNA precursor during ribosome biosynthesis. ITS 1, between 18S and 5.8S rRNA genes, is 999 nucleotides long. ITS 2, between 5.8S and 28S rRNA genes, is 1089 nucleotides long. Both spacers are very rich in G + C, 70 and 74% respectively. Mouse sequences have been compared with the other available eukaryotes: while no homology is apparent with yeast or xenopus, mouse and rat ITS sequences have been largely conserved, with homologous segments interspersed with highly divergent tracts. Homology with rat is much more extensive for ITS 1 than for ITS 2. Tentative secondary structure models are proposed for the folding of these regions within rRNA precursor; they are closely related in mouse and rat.

Animals↗
PubMed1984

Nucleotide sequence of the 5'-terminal coding region for pre-rRNA and mature 17S rRNA in Tetrahymena thermophila rDNA.

The 5'-terminus of 35S pre-rRNA and mature 17S rRNA of Tetrahymena thermophila was mapped on cloned rDNA fragments by S1 nuclease protection experiments. A single site for transcription initiation was observed when pre-rRNA prepared by three different methods was used as RNA probe. These mapping results were unambiguously confirmed by sequencing the 5'-terminal region of in vitro capped 35S pre-rRNA. DNA sequence analysis of about 520 nucleotides upstream of the transcription initiation site revealed several distinct sets of highly conserved repeat sequences. In addition, the 840 nucleotides downstream of the transcription initiation site (+ 1) was determined and shown to include the 5'-terminus of the 17S rRNA coding region at position + 647. A region surrounding the position + 195 contains an inverted repeat sequence which could be the structural basis for the recently described premature transcription termination event in this organism (Kister et al. (1983) Nucl. Acids Res. 11, 3487-3502).

Animals↗
PubMed1989

The 5S rRNA-histone repeat in the crustacean Artemia: structure, polymorphism and variation of the 5S rRNA segment in different populations.

5S rRNA genes are linked to the histone genes in the 13 populations of the crustacean Artemia that we have studied. In all cases, two types of repeat units are found. Southern blot analysis of all populations shows that they can be grouped into three classes: a) American bisexuals; b) Eurasian bisexuals, and c) parthenogenetic organisms (all from Eurasia). Restriction analysis of a bisexual population from San Francisco Bay shows that the two repeat units are of 9.0 and 8.5 kb (with minor heterogeneities of restriction sites). In parthenogenetic organisms, the two repeat units are of approximately 12 kb. Sequencing data from the region of the 5S rRNA from the San Francisco Bay population, shows that in both types of units, the single 5S rRNA gene (315 bp in length), is located 430 bp downstream the 3' regulatory sequences of the H2A gene, the last gene in the histone cluster. We have isolated three clones that contain 5S rRNA sequences. Two of them (one from an American bisexual and the other from a parthenogenetic population) contain histone and 5S rRNA genes, both with the same transcriptional polarity. The third clone, lacking histone genes, is likely to be an orphon derived from the parthenogenetic population.

Amino Acid Sequence↗
PubMed1981

The nucleotide sequence at the 3'-end of Neurospora crassa 25S-rRNA and the location of a 5.8S-rRNA binding site.

The sequence of 110 nucleotides adjacent to the 3'-end of Neurospora crassa 25S-rRNA has been derived by chemical sequencing methods. Sequences present between 40 and 85 nucleotides of the 3'-end were found to complement sequences at the 3'- and 5'-ends of 5.8S-rRNA. Interaction was shown to occur between 5.8S-rRNA and a specific 3'-terminal fragment of 85 nucleotides derived from 25S-rRNA. We have also demonstrated that the nucleotide sequence at the 3'-end of N. crassa 5.8S-rRNA (-UCAUUOH) is different from the published sequence (-UUUUOH) which was derived from rDNA.

Base Sequence↗
PubMed1993

Molecular characterization of DNA encoding 23S rRNA and 16S-23S rRNA intergenic spacer regions of Aeromonas hydrophila.

Amplification of the gene encoding 23S rRNA of Aeromonas hydrophila by polymerase chain reaction, with primers complementary to conserved regions of 16S and the 3'-end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli, and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in five clones. Three types of spacer were identified: two clones were identical and encoded tRNA(Ile) and tRNA(Ala) while the remaining three clones contained tRNA(Glu), only two had the same spacer sequences. This variation in sequence indicates that the different clones may be derived from different ribosomal RNA operons.

Aeromonas hydrophila↗
PubMed1985

Unbalanced rRNA gene dosage and its effects on rRNA and ribosomal-protein synthesis.

The synthesis of rRNA was unbalanced by the introduction of plasmids containing rRNA operons with large internal deletions. Significant unbalanced synthesis was achieved only when the deletions affected both 16S and 23S RNA genes or when the deletions affected the 23S RNA gene alone. Although large imbalances in rRNA synthesis resulted from deletions affecting 16S and 23S RNA genes or only 23S RNA genes, excess 16S RNA and defective rRNA species were rapidly degraded. Large imbalances in the synthesis of regions of rRNA did not result in significantly unbalanced synthesis of ribosomal proteins. It therefore is probable that excess intact 16S RNA is degraded because ribosomal proteins are not available for packaging the RNA into ribosomes. Defective RNA species also may be degraded for this reason or because proper ribosome assembly is prevented by the defects in RNA structure. We propose two possible explanations for the finding that unbalanced overproduction of binding sites for feedback ribosomal protein does not result in significant unbalanced translational feedback depression of ribosomal protein mRNAs.

Chromosome Deletion↗
PubMed2001

Changes in rRNA levels during stress invalidates results from mRNA blotting: fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels.

Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value.

Cell Membrane Permeability↗
PubMed1990

[Complementary addressed alkylation of Escherichia coli 16S rRNA with 2',3'-O-[4-N-methyl-N-(2-chloroethyl)aminobenzylidene]m derivatives of oligodeoxyribonucleotides. IV. Determination of binding sites of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA, d(pTTACGACT)rU, d(TTTGCTCCCC)rA].

By site-directed alkylation of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA (II), d(pTTACGACT)rU (III), d(pTTTGCTCCCC)rA (IV) (reagents (II)--(IV] followed by the RNase H treatment a number of 16S rRNA fragments have been obtained. Hybridisation of these fragments with restriction fragments of plasmid pKK 3535, containing operon rrnB of E. coli rRNAs, led to the identification of all reagents' binding sites in 16S rRNA. Good correlation is found between estimated stability of non-perfect 16S rRNA.oligodeoxyribonucleotide duplexes and the level of modification of this site with alkylating derivative of the same oligodeoxyribonucleotide. With high concentration of the reagents (II)--(IV) ((2-5) x 10(-5) M) the site-directed alkylation proceeds not only at the desired site but also at other sites corresponding to non-perfect duplexes between 16S rRNA and the reagents. It should be noted that the modification mainly occurs in the non-perfect duplexes, carrying mismatched bases at the termini. Influence of the secondary structure of 16S rRNA on the site-directed modification is discussed.

Alkylation↗
PubMed2000

Nucleotide sequence of the 235 rRNA from Haloferax mediterranei and phylogenetic analysis of halophilic archaea based on LSU rRNA.

23S rRNA gene from the halophilic archaeon Haloferax mediterranei (strain ATCC 33500) was cloned and sequenced. Proceeding from the 2,912 nucleotides long sequence, the secondary structure of Haloferax genus large subunit rRNA was proposed. Haloferax mediterranei intergenic spacers 16S/23S and 23S/5S were also sequenced, and found to be 382 and 116 nucleotides long respectively. The 16S/23S spacer showed an Ala-tRNA intervening sequence, which is a common feature in Euryarchaeota. Sequence analysis of 23S rRNA and 16S rRNA was performed for the six organisms from the family Halobacteriaceae with both available gene sequences. Phylogenetic trees with completely different topology were obtained using both molecules.

Base Sequence↗
PubMed1996

Heat shock inhibits pre-rRNA processing at the primary site in vitro and alters the activity of some rRNA binding proteins.

The effect of heat shock on pre-rRNA processing at the primary site within external transcribed spacer region 1 (ETS1) was studied in S-100 extract derived from mouse lymphosarcoma cells. In vivo labeling with [32P]orthophosphate showed that the synthesis of the rRNA precursor and its processing to 28S and 18S rRNAs were inhibited significantly due to heat shock. The processing activity was reduced by 50% at 1 h and was completely blocked following 2-h exposure of cells at 42 degrees C. Mixing S-100 extracts from the control and heat-treated cells did not affect the processing activity in the control extract, which proves the absence of a nuclease or other inhibitor(s) of processing in the extract from the heat-shocked cells. Heat shock did not affect interaction between pre-rRNA and U3 snoRNA, a prerequisite for the processing at the primary site, but significantly altered RNA-protein interaction. Three polypeptides of 200, 110, and 52 kDa that specifically cross-link to pre-rRNA spanning the primary processing site were inactivated after heat shock. Hyperthermia did not alter 3' end processing of SV40L pre-mRNA.

Animals↗
PubMed1986

Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes.

In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors lambda EMBL3 and lambda EMBL4. By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined. The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb.

Base Sequence↗