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Self-splicing group I introns have served as a model for RNA catalysis and folding for over two decades. New three-dimensional structures now bring the details into view. Revelations include an unanticipated turn in the RNA backbone around the guanosine-binding pocket. Two metal ions in the active site coordinate the substrate and phosphates from all three helical domains.
The phylogeny of the superfamily Tephritoidea (Diptera: Muscomorpha) was reconstructed from three mitochondrial gene fragments (12S, 16S, and COII) using 49 species representing 19 tephritoid and related families. Phylogenetic signal present in different gene fragments as well as combinations of gene fragments was examined using the interior branch and bootstrap test values from minimum evolution method. The minimum evolution, maximum likelihood, and maximum parsimony trees based on a combined dataset of all three gene fragments provided insight concerning the following phylogenetic relationships: (1) two monophyletic groups (Group-1 and -2) within the superfamily Tephritoidea were clearly recognized; they are compatible with Willi Hennig's Pallopteroidea and Otitoidea that are not used in the contemporary higher classification; (2) the non-monophyletic nature of the family Platystomatidae; and (3) a sister group relationship of Conopidae to Tephritoidea was not supported; instead, our result suggested that Conopidae and Diopsidae might be the basal most groups among the schizophoran families included in this study. The combined data of 12S, 16S, and COII genes was found, therefore, to be a viable genetic marker to resolve divergences among families of the Tephritoidea and other related superfamilies.
We investigated the monophyletic status of the hoplonemertean taxon Tetrastemma by reconstructing the phylogeny for 22 specimens assigned to this genus, together with another 25 specimens from closely related hoplonemertean genera. The phylogeny was based on partial 18S rRNA sequences using Bayesian and maximum likelihood analyses. The included Tetrastemma-species formed a well-supported clade, although the within-taxon relationships were unsettled. We conclude that the name Tetrastemma refers to a monophyletic taxon, but that it cannot be defined by morphological synapomorphies, and our results do not imply that all the over 100 species assigned to this genus belong to it. The results furthermore indicate that the genera Amphiporus and Emplectonema are non-monophyletic.
The genetic study of viruses having dsRNA genomes is hampered by the technical difficulty of complete sequence determination of dsRNA. Optimised methods are described here for sequencing dsRNAs, which meet three different situations: (1) genomes that can be obtained in fairly high amounts (>20 ng per separated segment); (2) genomes with limited amounts of RNA that can be detected by electrophoretic gel separation and staining; (3) genomes that cannot be detected by electrophoretic gel separation and staining. These methods include improved Single Primer Amplification Technique protocols, an adaptation of the SMART methodology, and a new method permitting the selective enzymatic removal of dsRNA segments. Strategies permitting adaptation of these protocols to the full-length determination of dsRNA viral genomes are described. Each of the protocols is described for sequence determination of a chosen dsRNA virus.
The complete nucleotide sequences have been determined for the M segment of 12 California (CAL) serogroup bunyaviruses. A method is described here of long reverse transcription-polymerase chain reaction (RT-PCR) that yields the full-length medium (M) RNA genomic segment. A phylogenetic tree was constructed by comparison of the open reading frames (ORFs) in the M RNA segment of 15 CAL serogroup viruses. Three distinct branches were identified and they are represented by the California encephalitis (CE), Melao (MEL), and Trivittatus (TVT) complexes. These groups correspond to those previously established by small (S) RNA genomic sequences. In addition, except for Inkoo virus, the predicted relationship among these viruses agreed with those found by serology.
Sequences for the spacer regions that separate the 16S and 23S ribosomal RNA genes have been determined for four more (strategically placed) archaebacteria. These confirm the general rule that methanogens and extreme halophiles have spacers that contain a single tRNAala gene, while tRNA genes are not found in the spacer region of the true extreme thermophiles. The present study also shows that the spacer regions from the sulfate reducing Archaeglobus and the extreme thermophile Thermococcus (both of which cluster phylogenetically with the methanogens and extreme halophiles) contain each a tRNAala gene. Thus, not only all methanogens and extreme halophiles show this characteristic, but all organisms on the "methanogen branch" of the archaebacterial tree appear to do so. The finding of a tRNA gene in the spacer region of the extreme thermophile Thermococcus celer is the first known phenotypic property that links this organism with its phylogenetic counterparts, the methanogens, rather than with its phenotypic counterparts, the sulfur-dependent extreme thermophiles.
The sequence of the 16S ribosomal RNA gene from the archaebacterium Thermococcus celer shows the organism to be related to the methanogenic archaebacteria rather than to its phenotypic counterparts, the extremely thermophilic archaebacteria. This conclusion turns on the position of the root of the archaebacterial phylogenetic tree, however. The problems encountered in rooting this tree are analyzed in detail. Under conditions that suppress evolutionary noise both the parsimony and evolutionary distance methods yield a root location (using a number of eubacterial or eukaryotic outgroup sequences) that is consistent with that determined by an "internal rooting" method, based upon an (approximate) determination of relative evolutionary rates.
Traditional ways of identifying slow growing bacteria is slow and often difficult. In this study, a small, Gram-negative, facultative anaerobic, slow growing bacillus was isolated from the blood culture of a 7-year old traffic accident victim. The bacterium was non-hemolytic, catalase and oxidase positive. An attempt to use the Vitek system (GNI+) and the API system (20NE) to identify the strain was unsuccessful as the growth controls showed negative results. 16S ribosomal RNA gene sequencing showed that there was 1 base difference between the isolate and Arcobacter cryaerophilus (GenBank Accession no. U25805), 1 base difference between the isolate and A. cryaerophilus (GenBank Accession no. U34387), 10 base differences between the isolate and A. cryaerophilus (GenBank Accession no. L14624), 34 base differences between the isolate and A. butzleri (GenBank Accession no. U34386), 34 base differences between the isolate and A. butzleri (GenBank Accession no. U34387), and 38 base differences between the isolate and A. butzleri (GenBank Accession no. L14626), indicating that the isolate most closely resembled a strain of A. cryaerophilus. Identification of the isolate in our case by conventional methods was difficult, as the absence of a curved morphology has made it confused with other Gram-negative non-fermentative bacteria, and the slow growth rate has made it unidentifiable by both the Vitek and API systems. Although the exact source of infection and route of transmission in our case remains elusive, we speculate that the bacteria were transmitted through the respiratory tract while the boy was suffocated in the mud. The present report represents an example of showing the usefulness of 16S rRNA gene sequencing for identification of slow growing bacteria.
Standard identification of Streptococcus pneumoniae by optochin and bile solubility testing can lead to ambiguous results for certain isolates. Newer bacteriologic identification techniques (e.g., DNA probes) now exist. In a prospective point prevalence study of oropharyngeal S. pneumoniae carriage rates among outpatients, we compared standard organism identification techniques to DNA probe testing. By standard identification criteria, 35 (4%) of 872 isolates were characterized as presumptive S. pneumoniae. Thirty of 35 presumptive isolates were recoverable for DNA probing; 9 (30%) presumptive isolates were confirmed using a DNA probe. The antimicrobial susceptibility pattern of these DNA probe positive isolates closely paralleled that of clinical blood isolates of S. pneumoniae obtained during the study period. The 21 (70%) DNA probe negative isolates, which may represent phylogenetically related species (such as S. mitis or S. oralis), had significantly reduced antimicrobial susceptibility patterns when compared with the DNA probe positive isolates. In colonization studies, if classic criteria (optochin disc zone and bile solubility) are the sole means of identification, S. pneumoniae penicillin resistance rates may be over-reported.
According to the present classification, the family Agaonidae contains all fig pollinators as well as five subfamilies of non-pollinating fig wasps. The molecular phylogeny of the family was reconstructed using partial sequences of the 28S rRNA (D1 and D2 domains). Our results show that the family Agaonidae is not monophyletic. As a consequence, we restrict the famiy to the pollinator clade, and assign the non-pollinating subfamilies to various chalcid families. Sycoecinae, Otitesellinae and Sycoryctinae are included in Pteromalidae, whereas Sycophaginae and Epichrysomallinae are left unclassified and will require more in-depth morphological studies. Moreover, we proved that the fig pollination syndrome evolved only once, early in group history. The resource due to the fig-pollinator mutualism has secondarily been colonized independently by different Chalcid lineages.
The phylogenetic position of the platyhelminths within the metazoan tree is examined using two independent sets of molecular characters, the evolution of 18S ribosomal RNA sequences and the diversity of the genes belonging to the HOX cluster. Among the various hypotheses that have been considered by zoologists, a position of the platyhelminths within the protostomes, related to the phyla with typical spiral cleavage, appears to be favoured when taking into account all separate lines of evidence. It is in conflict with the traditional hypothesis of an early emergence at the base of the bilaterally symmetrical animals. This relatively late emergence is compatible with the old idea that flatworms are derived from a coelomate ancestor. New evidence from the sequences of Hox genes suggests that the duplicated genes Ultrabithorax/abdominal-A constitute a genetic synapomorphy of the whole protostome clade.
Subclones of neuroblastic (N) and substrate adherent (S) cells have been established from neuroblastoma tumours cultured in vitro which differ in growth characteristics and MYCN expression. N cells derived from the NBL-W cell line (W-N) express 5-fold higher levels of MYCN mRNA and 10-fold higher levels of MYCN protein than S cells (W-S), despite having the same MYCN copy number. In an effort to identify the molecular mechanisms responsible for the disparity in steady-state MYCN levels, the rate of MYCN mRNA degradation was measured in the two subclones. The half-life of MYCN mRNA in the W-N cells was approximately 45 min compared to approximately 6 min in the W-S cells. Similarly, the half-life of another labile mRNA, c-fos, differed in W-N and W-S cells (30 min versus 15 min, respectively). The turnover of labile mRNAs is thought to be mediated by the interactions of trans-acting factors with AU-rich elements within the 3' untranslated region. RNA UV cross-linking assays using W-N cell lysate demonstrated abundant quantities of a protein, 40 kDa in size (p40), that bound specifically to AU-rich elements within the MYCN and c-fos 3' untranslated region. However, p40 was barely detectable in W-S cells. Our studies suggest that p40 may play a role in determining neuroblastoma phenotype by regulating MYCN and c-fos mRNA turnover.
Escherichia coli rRNA contains 10 pseudouridines of unknown function. They are made by synthases, each of which is specific for one or more pseudouridines. Here we show that the sfhB (yfil) ORF of E. coli is a pseudouridine synthase gene by cloning, protein overexpression, and reaction in vitro with rRNA transcripts. Gene disruption by miniTn10(cam) insertion revealed that this synthase gene, here renamed rluD, codes for a synthase which is solely responsible in vivo for synthesis of the three pseudouridines clustered in a stem-loop at positions 1911, 1915, and 1917 of 23S RNA. The absence of RluD results in severe growth inhibition. Both the absence of pseudouridine and the growth defect could be reversed by insertion of a plasmid carrying the rluD gene into the mutant cell, clearly linking both effects to the absence of RIuD. This is the first report of a major physiological defect due to the deletion of any pseudouridine synthase. Growth inhibition may be due to the lack of one or more of the 23S RNA pseudouridines made by this synthase since pseudouridines 1915 and 1917 are universally conserved and are located in proximity to the decoding center of the ribosome where they could be involved in modulating codon recognition.
Unfolding of an mRNA pseudoknot that induces ribosome suppression of the gag gene stop codon in Moloney murine leukemia virus has been studied by UV hyperchromicity and calorimetry. The pseudoknot melts in two steps, corresponding to its two helical stems. The total enthalpy of denaturation is approximately 170 kcal/mol, approximately the value expected for the secondary structure. At low salt concentrations (<50 mM KCl) the unfolding transitions are not two-state, but they approach two-state behavior at higher salt concentrations. The structure is preferentially stabilized by smaller alkali metal ions (Li+ > Na+ > K+ > Rb+ > Cs+) and by NH4+; the same preferences are exhibited by one of the stems in the context of a hairpin. Divalent metal ions are not required to fold the pseudoknot but do stabilize it further. To examine divalent ion effects over a wide concentration range, urea was used to lower the RNA unfolding temperature and was shown not to affect characteristics of the pseudoknot unfolding in other respects. The pseudoknot binds divalent ions somewhat more tightly than a hairpin but shows only weak selectivity for different size ions. It is suggested that a region of "intermediate" divalent ion binding affinity, in between highly ligated specific sites and purely delocalized ion binding in character, is created by the pseudoknot fold but that nonspecific, delocalized ion binding contributes at least half the free energy of pseudoknot stabilization by Mg2+.
Ku protein, a heterodimer of 70 and 83 kDa polypeptides, is the regulatory component of the DNA-dependent protein kinase (DNA-PK). Ku protein binds to DNA ends and is essential for DNA double-strand break repair and V(D)J recombination. Although there is some evidence that Ku protein also binds RNA, its RNA binding properties have not been systematically explored. In the present study, Ku-binding RNAs were identified using systematic evolution of ligands by exponential enrichment (SELEX) technology. These RNAs were assigned to three classes based on common sequence motifs. Most of the selected RNAs bound to Ku protein with a Kd < or = 2 nM, comparable to the affinity of DNA fragments for Ku protein under similar conditions. Many of the RNAs inhibited DNA-PK activity by competing with DNA for a common binding site in Ku protein. None of several RNAs that were tested activated DNA-PK in the absence of DNA. The identification of diverse RNAs that bind avidly to Ku protein is consistent with the idea that natural RNAs may serve as modulators of DNA-PK activity. Moreover, the RNAs identified in this study may have utility as tools for experimental manipulation of DNA double-strand break repair activity in cells and cell extracts.
The definition of the genus Actinomyces relies heavily on traditional methods of taxonomy. This study sought to develop molecular tools for the identification of strains of Actinomyces israelii and Actinomyces gerencseriae. Oligonucleotide probes were designed and one of these successfully differentiated. A. gerencseriae from ten strains of A. israelii and three other Actinomyces species by DNA:DNA hybridization. However, probes based on known 16S rRNA sequences failed to hybridize to all the strains previously identified as A. israelii. Using the PCR technique, a region encoding a portion of the 16S rRNA was amplified from genomic DNA. The results showed that A. israelii can be divided into three different groups based on comparison of the amplified DNA sequences. This information should allow the development of probes that are specific for these newly identified groups of strains within the species A. israelii.
Nucleotide sequences of domain V and domain II regions of the 23S rRNA gene were determined in both in vitro-made mutants and clinical isolates of Mycobacterium avium and M. intracellulare conferring clarithromycin-resistance. All laboratory-made mutants showed high level resistance to clarithromycin (> 150 micrograms ml-1) and mutation at position 2058 (cognate with Escherichia coli base) in domain V region. In the clinical isolates, while the susceptible ones had no mutation in domain V, the resistant strains showed mutation at 2058 or 2059. Six isolates with low level of resistance exhibited no mutation in domain V. All strains tested had no mutation in domain II region. These results suggested that most of the resistance arose from the mutation in domain V of the 23S rRNA gene, but other unknown mechanisms evidently exist in mycobacteria.
The completion of the human genome sequence (International Human Genome Sequence Consortium (2001) Nature 409, 860-921; Venter, J. C., et al. (2001) Science 291, 1304-1351) allows for new ways to analyze global cellular regulatory mechanisms. Here we present a strategy to identify genes regulated by specific transcription factors in the human genome, and apply it to p53. We first collected promoters or introns of all genes available using two methods: GenBank(TM) annotation and a computationally derived transcript map. 4,852 genes analyzed in this way contained at least one p53 consensus binding sequence. Of 13 genes randomly selected for mRNA analysis, 11 were shown to respond to p53 expression. Five promoters were analyzed by chromatin immunoprecipitation, which revealed that all were bound by p53 in vivo. We then analyzed 33,615 unique human genes on cDNA microarrays, identifying 1,501 genes that respond to p53 expression. A parameter was derived that demonstrates that in silico prediction greatly enriches for genes that are activated and repressed by p53 and assists us to suggest other signaling pathways that may be connected to p53. The methods shown here illustrate a novel approach to analysis of global gene regulatory network through the integration of human genomic sequence information and genome-wide gene expression analysis.