Role of the protein moiety of ribonuclease P, a ribonucleoprotein enzyme.

The Bacillus subtilis ribonuclease P consists of a protein and an RNA. At high ionic strength the reaction is protein-independent; the RNA alone is capable of cleaving precursor transfer RNA, but the turnover is slow. Kinetic analyses show that high salt concentrations facilitate substrate binding in the absence of the protein, probably by decreasing the repulsion between the polyanionic enzyme and substrate RNAs, and also slow product release and enzyme turnover. It is proposed that the ribonuclease P protein, which is small and basic, provides a local pool of counter-ions that facilitates substrate binding without interfering with rapid product release.

Analysis of hydrothermal vent-associated symbionts by ribosomal RNA sequences.

Ribosomal RNA (rRNA) sequences were used to establish the phylogenetic affiliations of symbioses in which prokaryotes appear to confer sulfur-based chemoautotrophy on their invertebrate hosts. Two submarine hydrothermal vent animals, the vestimentiferan tube worm Riftia pachyptila and the clam Calyptogena magnifica, and a tidal-flat bivalve, Solemya velum, were inspected. 5S rRNA's were extracted from symbiont-bearing tissues, separated into unique forms, and their nucleotide sequences determined and related to other 5S rRNA's in a phylogenetic tree analysis. The prokaryotic symbionts are related to one another and affiliated with the same narrow phylogenetic grouping as Escherichia coli and Pseudomonas aeruginosa. The sequence comparisons suggest that Riftia is more closely related to the bivalves than their current taxonomic status would suggest.

Evidence from 12S ribosomal RNA sequences that onychophorans are modified arthropods.

The evolutionary relationships of the onychophorans (velvet worms) and the monophyly of the arthropods have generated considerable debate. Cladistic analyses of 12S ribosomal RNA sequences indicate that arthropods are monophyletic and include the onychophorans. Maximum parsimony analyses and monophyly testing within arthropods indicate that myriapods (millipedes and centipedes) form a sister group to all other assemblages, whereas crustaceans (shrimps and lobsters) plus hexapods (insects and allied groups) form a well-supported monophyletic group. Parsimony analysis further suggests that onychophorans form a sister group to chelicerates (spiders and scorpions) and crustaceans plus hexapods, but this relationship is not well supported by monophyly testing. These relationships conflict with current hypotheses of evolutionary pathways within arthropods.

Molecular phylogeny of the animal kingdom.

A rapid sequencing method for ribosomal RNA was applied to the resolution of evolutionary relationships among Metazoa. Representatives of 22 classes in 10 animal phyla were used to infer phylogenetic relationships, based on evolutionary distances determined from pairwise comparisons of the 18S ribosomal RNA sequences. The classical Eumetazoa are divided into two groups. Cnidarians arose from a protist ancestry different from the second group, the Bilateria. Within the Bilateria, an early split gave rise to Platyhelminthes (flatworms) and the coelomate lineage. Coelomates are thus monophyletic, and they radiated rapidly into four groups: chordates, echinoderms, arthropods, and eucoelomate protostomes.

Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.

The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted protein-coding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.

Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process.

The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the following. (i) Their evolutionary relationships mostly conform to established organismal phylogeny: a strong distinction exists between bacterial- and archaeal-type AARSs. (ii) Although the evolutionary profiles of the individual AARSs might be expected to be similar in general respects, they are not. It is argued that these differences in profiles reflect the stages in the evolutionary process when the taxonomic distributions of the individual AARSs became fixed, not the nature of the individual enzymes. (iii) Horizontal transfer of AARS genes between Bacteria and Archaea is asymmetric: transfer of archaeal AARSs to the Bacteria is more prevalent than the reverse, which is seen only for the "gemini group. " (iv) The most far-ranging transfers of AARS genes have tended to occur in the distant evolutionary past, before or during formation of the primary organismal domains. These findings are also used to refine the theory that at the evolutionary stage represented by the root of the universal phylogenetic tree, cells were far more primitive than their modern counterparts and thus exchanged genetic material in far less restricted ways, in effect evolving in a communal sense.

Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales.

Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.

The RDP-II (Ribosomal Database Project).

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [Nucleic Acids Res. (2000), 28, 173-174], continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 8.0 (June 1, 2000) consisted of 16 277 aligned prokaryotic small subunit (SSU) rRNA sequences while the number of eukaryotic and mitochondrial SSU rRNA sequences in aligned form remained at 2055 and 1503, respectively. The number of prokaryotic SSU rRNA sequences more than doubled from the previous release 14 months earlier, and approximately 75% are longer than 899 bp. An RDP-II mirror site in Japan is now available (http://wdcm.nig.ac.jp/RDP/html/index.h tml). RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/). Analysis services include rRNA probe checking, approximate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment polymorphism experiments. The RDP-II email address for questions and comments has been changed from curator@cme.msu.edu to rdpstaff@msu.edu.

Identification and mapping of a 60 bp EcoRI fragment in the Dictyostelium discoideum ribosomal DNA.

We have re-examined the organization of the transcribed sequences in the ribosomal DNA repeat unit of Dictyostelium discoideum. In addition to the four EcoRI fragments previously reported, we have identified and cloned a fifth fragment which defines a small portion of the 25S ribosomal RNA. The fragment is 60 bp long and is located between the 1.5 kbp and the 3 kbp EcoRI fragments of the ribosomal DNA repeat unit.

Phylogenetic comparative analysis and the secondary structure of ribonuclease P RNA--a review.

The most incisive a priori approach to inferring the higher order structure of large RNAs has proven to be the use of phylogenetic comparisons. This article provides guidelines to the method, using as an illustration the elucidation of the secondary structure of the catalytic RNA subunit of ribonuclease P (RNase P). The resultant structure is compared to the possibilities that are predicted thermodynamically for the RNase P RNA sequences of nine eubacteria.

A reconstruction of the metabolism of Methanococcus jannaschii from sequence data.

The interpretation of the Methanococcus jannaschii genome will inevitably require many years of effort. This initial attempt to connect the sequence data to aspects of known biochemistry and to provide an overview of what is already apparent from the sequence data will be refined. Numerous issues remain that can be resolved only by direct biochemical analysis. Let us draw the reader's attention to just a few that might be considered central: (1) We are still missing key enzymes from the glycolytic pathway, and the conjecture is that this is due to ADP-dependency. The existence of glycolytic activity in the cell-free extract should be tested. (2) The issue of whether the Calvin cycle is present needs to be examined. (3) We need to determine whether the 2-oxoglutarate synthase (ferredoxin-dependent) (EC 1.2.7.3) activity is present. (4) The issue of whether cyclic 2,3-bisphosphate is detectable in the cell-free extracts needs to be checked. If it is, this result would confirm our assertion of the two pathways controlling synthesis and degradation of cyclic 2,3-bisphosphate.

Cloning, characterization and sequence comparison of the gene coding for IMP dehydrogenase from Pyrococcus furiosus.

We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Pyrococcus furiosus (Pf), a hyperthermophillic archeon. Sequence analysis of the Pf gene indicated an open reading frame specifying a protein of 485 amino acids (aa) with a calculated M(r) of 52900. Canonical Archaea promoter elements, Box A and Box B, are located -49 and -17 nucleotides (nt), respectively, upstream of the putative start codon. The sequence of the putative active-site region conforms to the IMPDH signature motif and contains a putative active-site cysteine. Phylogenetic relationships derived by using all available IMPDH sequences are consistent with trees developed for other molecules; they do not precisely resolve the history of Pf IMPDH but indicate a close similarity to bacterial IMPDH proteins. The phylogenetic analysis indicates that a gene duplication occurred prior to the division between rodents and humans, accounting for the Type I and II isoforms identified in mice and humans.

Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana.

We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Arabidopsis thaliana (At). The transcription unit of the At gene spans approximately 1900 bp and specifies a protein of 503 amino acids with a calculated relative molecular mass (M(r)) of 54,190. The gene is comprised of a minimum of four introns and five exons with all donor and acceptor splice sequences conforming to previously proposed consensus sequences. The deduced IMPDH amino-acid sequence from At shows a remarkable similarity to other eukaryotic IMPDH sequences, with a 48% identity to human Type II enzyme. Allowing for conservative substitutions, the enzyme is 69% similar to human Type II IMPDH. The putative active-site sequence of At IMPDH conforms to the IMP dehydrogenase/guanosine monophosphate reductase motif and contains an essential active-site cysteine residue.

Similar subunit architecture of archaeal and eukaryal RNA polymerases.

Protein interactions among RNA polymerase small subunits from the archaeon Methanococcus jannaschii were investigated using affinity pulldown assays in pairwise and higher-order combinations. In the most extensive study of archaeal RNA polymerase subunit interactions to date, including 37 pairs of proteins, 10 ternary combinations, and three quaternary combinations, we found evidence for pairwise interactions of subunit D with subunits L and N, and a ternary complex of subunits D, L and N. No other small subunit interactions occurred. These results are consistent with interactions observed in a crystal structure of eukaryotic RNA polymerase II and support a common archaeal/eukaryal RNA polymerase architecture. We further propose that subunit E" is not an integral member of archaeal RNA polymerases. Finally, we discuss the relative accuracy of the various methods that have been used to predict protein-protein interactions in RNA polymerase.

Archaebacterial phylogeny: perspectives on the urkingdoms.

Comparisons of complete 16S ribosomal RNA sequences have been used to confirm, refine and extend earlier concepts of archaebacterial phylogeny. The archaebacteria fall naturally into two major branches or divisions, I--the sulfur-dependent thermophilic archaebacteria, and II--the methanogenic archaebacteria and their relatives. Division I comprises a relatively closely related and phenotypically homogeneous collection of thermophilic sulfur-dependent species--encompassing the genera Sulfolobus, Thermoproteus, Pyrodictium and Desulfurococcus. The organisms of Division II, however, form a less compact grouping phylogenetically, and are also more diverse in phenotype. All three of the (major) methanogen groups are found in Division II, as are the extreme halophiles and two types of thermoacidophiles, Thermoplasma acidophilum and Thermococcus celer. This last species branches sufficiently deeply in the Division II line that it might be considered to represent a separate, third Division. However, both the extreme halophiles and Tp. acidophilum branch within the cluster of methanogens. The extreme halophiles are specifically related to the Methanomicrobiales, to the exclusion of both the Methanococcales and the Methanobacteriales. Tp. acidophilum is peripherally related to the halophile-Methanomicrobiales group. By 16S rRNA sequence measure the archaebacteria constitute a phylogenetically coherent grouping (clade), which excludes both the eubacteria and the eukaryotes--a conclusion that is supported by other sequence evidence as well. Alternative proposals for archaebacterial phylogeny, not based upon sequence evidence, are discussed and evaluated. In particular, proposals to rename (reclassify) various subgroups of the archaebacteria as new kingdoms are found wanting, for both their lack of proper experimental support and the taxonomic confusion they introduce.

A phylogenetic analysis of Aquifex pyrophilus.

The 16S rRNA of the bacterion Aquifex pyrophilus, a microaerophilic, oxygen-reducing hyperthermophile, has been sequenced directly from the the PCR amplified gene. Phylogenetic analyses show the Aq. pyrophilus lineage to be probably the deepest (earliest) in the (eu)bacterial tree. The addition of this deep branching to the bacterial tree further supports the argument that the Bacteria are of thermophilic ancestry.