Bioequivalence in dogs of a meloxicam formulation administered as a transmucosal oral mist with an orally administered pioneer suspension product.

A mucosal mist formulation of meloxicam, administered as a spray into the mouth (test article), was compared for bioequivalence to a pioneer meloxicam suspension for oral administration (reference article). Pharmacokinetic profiles and average bioequivalence were investigated in 20 dogs. The study design comprised a two-period, two-sequence, two-treatment cross-over design, with maximum concentration (C(max)) and area under plasma concentration-time curve to last sampling time (AUC(last)) used as pivotal bioequivalence variables. Bioequivalence of the products was confirmed, based on relative ratios of geometric mean concentrations (and 90% confidence intervals within the range 0.80-1.25) for C(max) of 101.9 (97.99-106.0) and for AUC(last) of 97.24 (94.44-100.1). The initial absorption of meloxicam was more rapid for the test article, despite virtually identical C(max) values for the two products. Mean elimination half-lives were 29.6 h (test article) and 30.0 h (reference article). The meloxicam plasma concentration-time profiles were considered in relation to published data on the inhibition of the cyclooxygenase-1 (COX-1) and COX-2 isoenzymes by meloxicam.

Cleavage of mitochondria-like transfer RNAs expressed in Escherichia coli.

Mitochondrial (mt) transfer RNAs (tRNAs) often harbor unusual structural features causing their secondary structure to differ from the conventional cloverleaf. tRNAs designed with such irregularities, termed mt-like tRNAs, are active in Escherichia coli as suppressors of reporter genes, although they display low steady-state levels. Characterization of fragments produced during mt-like tRNA processing in vitro and in vivo suggests that these RNAs are not fully processed at their 5' ends and are cleaved internally. These abnormal processing events may account for the low levels of mature mt-like RNAs in vivo and are most likely related to defective processing by RNase P.

Identification and characterization of a Plasmodium falciparum RNA polymerase gene with similarity to mitochondrial RNA polymerases.

Nearly all mitochondrial RNA polymerase genes identified to date are encoded in the nucleus and have similarities to T3 and T7 bacteriophage RNA polymerases. Some chloroplast genes are also transcribed by T3/T7 phage-like RNA polymerases, raising the possibility that the apicomplexan parasites, which have both a mitochondrion and a plastid, might have two such genes. As part of an investigation of Plasmodium falciparum organelle transcription, we initiated a search for T3/T7 bacteriophage-like RNA polymerase genes. We employed degenerate primers based on highly conserved plant, animal and fungal mitochondrial RNA polymerase sequences to amplify corresponding P. falciparum sequences by polymerase chain reaction (PCR). Less well-conserved flanking sequences were obtained by inverse PCR. The resulting sequence predicts a 1503 amino acid open reading frame with similarity to other T3/T7 phage-like RNA polymerases. Essential amino acids that have been identified in T7 mutant analyses are conserved in the P. falciparum RNA polymerase gene. Comparison of the sequence with preliminary data from the P. falciparum genome sequencing project revealed strain heterogeneity within two regions of the gene. The amino-terminal predicted amino acid sequence of the RNA polymerase gene has similarities to mitochondrial targeting sequences. Taken together, these points suggest that we have identified the P. falciparum mitochondrial RNA polymerase gene.

Hammerhead-mediated processing of satellite pDo500 family transcripts from Dolichopoda cave crickets.

This work reports the discovery and functional characterization of catalytically active hammerhead motifs within satellite DNA of the pDo500 family from several DOLICHOPODA: cave cricket species. We show that in vitro transcribed RNA of some members of this satellite DNA family do self-cleave in vitro. This self-cleavage activity is correlated with the efficient in vivo processing of long primary transcripts into monomer-sized RNA. The high sequence conservation of the satellite pDo500 DNA family among genetically isolated DOLICHOPODA: schiavazzii populations, as well as other DOLICHOPODA: species, along with the fact that satellite members are actively transcribed in vivo suggests that the hammerhead-encoding satellite transcripts are under selective pressure, perhaps because they fulfil an important physiological role or function. Remarkably, this is the third example of hammerhead ribozyme structures associated with transcribed repetitive DNA sequences from animals. The possibility that such an association may not be purely coincidental is discussed.

Distribution of hammerhead and hammerhead-like RNA motifs through the GenBank.

Hammerhead ribozymes previously were found in satellite RNAs from plant viroids and in repetitive DNA from certain species of newts and schistosomes. To determine if this catalytic RNA motif has a wider distribution, we decided to scrutinize the GenBank database for RNAs that contain hammerhead or hammerhead-like motifs. The search shows a widespread distribution of this kind of RNA motif in different sequences suggesting that they might have a more general role in RNA biology. The frequency of the hammerhead motif is half of that expected from a random distribution, but this fact comes from the low CpG representation in vertebrate sequences and the bias of the GenBank for those sequences. Intriguing motifs include those found in several families of repetitive sequences, in the satellite RNA from the carrot red leaf luteovirus, in plant viruses like the spinach latent virus and the elm mottle virus, in animal viruses like the hepatitis E virus and the caprine encephalitis virus, and in mRNAs such as those coding for cytochrome P450 oxidoreductase in the rat and the hamster.

The distribution of RNA motifs in natural sequences.

Functional analysis of genome sequences has largely ignored RNA genes and their structures. We introduce here the notion of 'ribonomics' to describe the search for the distribution of and eventually the determination of the physiological roles of these RNA structures found in the sequence databases. The utility of this approach is illustrated here by the identification in the GenBank database of RNA motifs having known binding or chemical activity. The frequency of these motifs indicates that most have originated from evolutionary drift and are selectively neutral. On the other hand, their distribution among species and their location within genes suggest that the destiny of these motifs may be more elaborate. For example, the hammerhead motif has a skewed organismal presence, is phylogenetically stable and recent work on a schistosome version confirms its in vivo biological activity. The under-representation of the valine-binding motif and the Rev-binding element in GenBank hints at a detrimental effect on cell growth or viability. Data on the presence and the location of these motifs may provide critical guidance in the design of experiments directed towards the understanding and the manipulation of RNA complexes and activities in vivo.

The recognition of a noncanonical RNA base pair by a zinc finger protein.

BACKGROUND: The zinc finger (ZF) is the most abundant nucleic-acid-interacting protein motif. Although the interaction of ZFs with DNA is reasonably well understood, little is known about the RNA-binding mechanism. We investigated RNA binding to ZFs using the Zif268-DNA complex as a model system. Zif268 contains three DNA-binding ZFs; each independently binds a 3 base pair (bp) subsite within a 9 bp recognition sequence. RESULTS: We constructed a library of phage-displayed ZFs by randomizing the alpha helix of the Zif268 central finger. Successful selection of an RNA binder required a noncanonical base pair in the middle of the RNA triplet. Binding of the Zif268 variant to an RNA duplex containing a G.A mismatch (rG.A) is specific for RNA and is dependent on the conformation of the mismatched middle base pair. Modeling and NMR analyses revealed that the rG.A pair adopts a head-to-head configuration that counterbalances the effect of S-puckered riboses in the backbone. We propose that the structure of the rG.A duplex is similar to the DNA in the original Zif268-DNA complex. CONCLUSIONS: It is possible to change the specificity of a ZF from DNA to RNA. The ZF motif can use similar mechanisms in binding both types of nucleic acids. Our strategy allowed us to rationalize the interactions that are possible between a ZF and its RNA substrate. This same strategy can be used to assess the binding specificity of ZFs or other protein motifs for noncanconical RNA base pairs, and should permit the design of proteins that bind specific RNA structures.

A small nucleolar RNA:ribozyme hybrid cleaves a nucleolar RNA target in vivo with near-perfect efficiency.

A hammerhead ribozyme has been localized to the yeast nucleolus by using the U3 small nucleolar RNA as a carrier. The hybrid small nucleolar RNA:ribozyme, designated a "snorbozyme," is metabolically stable and cleaves a target U3 RNA with nearly 100% efficiency in vivo. This is the most efficient in vivo cleavage reported for a trans-acting ribozyme. A key advantage of the model substrate featured is that a stable, trimmed cleavage product accumulates. This property allows accurate kinetic measurements of authentic cleavage in vivo. The system offers new avenues for developing effective ribozymes for research and therapeutic applications.

tRNA nucleotide 47: an evolutionary enigma.

A previous analysis of tRNA sequences suggested a correlation between the absence of a nucleotide at position 47 (nt 47) in the extra loop and the presence of a U13:G22 base pair in the D-stem. We have evaluated the significance of this correlation by determining the in vivo activity of tRNAs containing either a C13:G22 or a U13:G22 pair in tRNA molecules with or without nt 47. Although this correlation might reflect some malfunction of tRNAs lacking nt 47, but containing the C13:G22, assays of the in vivo suppressor activity showed that this tRNA is actually more active than the tRNA with the features found in the database, i.e., a U13:G22 base pair and no nt 47. Moreover, analogous constructs with a GGC anticodon permitted the growth of an Escherichia coli strain deleted for tRNA(Ala)GGC genes equally well. On the other hand, long-term growth experiments with competing E. coli strains harboring the tRNA lacking nt 47, either with the C13:G22 or the U13:G22 base pair demonstrated that the U13:G22 tRNA overtook the C13:G22 strain even when the starting proportion of strains favored the C13:G22 strain. Thus, the preference for the U13:G22 tRNA lacking nt 47 in the sequence database is most likely due to factors that come into play during extended growth or latency rather than to the ability of the tRNA to engage in protein synthesis.

Modeling active RNA structures using the intersection of conformational space: application to the lead-activated ribozyme.

The Pb2+ cleavage of a specific phosphodiester bond in yeast tRNA(Phe) is the classical model of metal-assisted RNA catalysis. In vitro selection experiments have identified a tRNA(Phe) variant, the leadzyme, that is very active in cleavage by Pb2+. We present here a three-dimensional modeling protocol that was used to propose a structure for this ribozyme, and is based on the computation of the intersection of conformational space of sequence variants and the use of chemical modification data. Sequence and secondary structure data were used in a first round of computer modeling that allowed identification of conformations compatible with all known leadzyme variants. Common conformations were then tested experimentally by evaluating the activity of analogues containing modified nucleotides in the catalytic core. These experiments led to a new structural hypothesis that was tested in a second round of computer modeling. The resulting proposal for the active conformation of the leadzyme is consistent with all known structural data. The final model suggests an in-line SN2 attack mechanism and predicts two Pb2+ binding sites. The protocol presented here is generally applicable in modeling RNAs whenever the catalytic or binding activity of structural analogues is known.

Schistosome satellite DNA encodes active hammerhead ribozymes.

Using a computer program designed to search for RNA structural motifs in sequence databases, we have found a hammerhead ribozyme domain encoded in the Smalpha repetitive DNA of Schistosoma mansoni. Transcripts of these repeats are expressed as long multimeric precursor RNAs that cleave in vitro and in vivo into unit-length fragments. This RNA domain is able to engage in both cis and trans cleavage typical of the hammerhead ribozyme. Further computer analysis of S. mansoni DNA identified a potential trans cleavage site in the gene coding for a synaptobrevin-like protein, and RNA transcribed from this gene was efficiently cleaved by the Smalpha ribozyme in vitro. Similar families of repeats containing the hammerhead domain were found in the closely related Schistosoma haematobium and Schistosomatium douthitti species but were not present in Schistosoma japonicum or Heterobilharzia americana, suggesting that the hammerhead domain was not acquired from a common schistosome ancestor.

Amber suppression in Escherichia coli by unusual mitochondria-like transfer RNAs.

The "cloverleaf" base-pairing pattern was established as the structural paradigm of active tRNA species some 30 years ago. Nevertheless, this pattern does not accommodate the folding of certain mitochondrial tRNAs. For these recalcitrant tRNAs, we have proposed structures having from 5 to 10 base pairs in the anticodon stem rather than the canonical 6. The absence of these types of tRNAs in cytoplasmic translation systems, however, raises the possibility that they may not be bona fide alternate folding patterns for active tRNA molecules. For this reason, we have designed new tRNA genes based on our model of unusual mitochondrial tRNAs, having 7, 8, 9, and 10 base pairs in the anticodon stem with other modifications to the D-stem and connector regions. We show here that these synthetic genes produce tRNAs that actively suppress amber codons in vivo.

Genome structure and gene content in protist mitochondrial DNAs.

Although the collection of completely sequenced mitochondrial genomes is expanding rapidly, only recently has a phylogenetically broad representation of mtDNA sequences from protists (mostly unicellular eukaryotes) become available. This review surveys the 23 complete protist mtDNA sequences that have been determined to date, commenting on such aspects as mitochondrial genome structure, gene content, ribosomal RNA, introns, transfer RNAs and the genetic code and phylogenetic implications. We also illustrate the utility of a comparative genomics approach to gene identification by providing evidence that orfB in plant and protist mtDNAs is the homolog of atp8 , the gene in animal and fungal mtDNA that encodes subunit 8 of the F0portion of mitochondrial ATP synthase. Although several protist mtDNAs, like those of animals and most fungi, are seen to be highly derived, others appear to be have retained a number of features of the ancestral, proto-mitochondrial genome. Some of these ancestral features are also shared with plant mtDNA, although the latter have evidently expanded considerably in size, if not in gene content, in the course of evolution. Comparative analysis of protist mtDNAs is providing a new perspective on mtDNA evolution: how the original mitochondrial genome was organized, what genes it contained, and in what ways it must have changed in different eukaryotic phyla.

Modeling RNA-ligand interactions: the Rev-binding element RNA-aminoglycoside complex.

An approach to the modeling of ligand-RNA complexes has been developed by combining three-dimensional structure-activity relationship (3D-SAR) computations with a docking protocol. The ability of 3D-SAR to predict bound conformations of flexible ligands was first assessed by attempting to reconstruct the known, bound conformations of phenyloxazolines complexed with human rhinovirus 14 (HRV14) RNA. Subsequently, the same 3D-SAR analysis was applied to the identification of bound conformations of aminoglycosides which associate with the Rev-binding element (RBE) RNA. Bound conformations were identified by parsing ligand conformational data sets with pharmacophores determined by the 3D-SAR analysis. These "bioactive" structures were docked to the receptor RNA, and optimization of the complex was undertaken by extensive searching of ligand conformational space coupled with molecular dynamics computations. The similarity between the bound conformations of the ligand from the 3D-SAR analysis and those found in the docking protocol suggests that this methodology is valid for the prediction of bound ligand conformations and the modeling of the structure of the ligand-RNA complexes.

PolI-driven integrative expression vectors for yeast.

A novel expression vector for yeast has been constructed from the regulatory elements present in the polI promoter and the enhancer/termination region (E/T) of rDNA. Under some conditions, this promoter/vector combination produces small RNAs such as the hammerhead RNA sequence at levels comparable to polII- and polIII-dependent systems. No stable transcription product can be demonstrated with this vector when the enhancer/termination sequence is less than 100 nucleotides downstream from the promoter. On the other hand, high expression of a stable, hammerhead RNA molecule can be obtained from this vector by inserting a 400-bp fragment containing the ADH1 transcription termination region upstream of the E/T. RNAs produced by this vector are polyadenylated and multiple copies of this plasmid can be stably integrated into the yeast chromosome.

An ancestral mitochondrial DNA resembling a eubacterial genome in miniature.

Mitochondria, organelles specialized in energy conservation reactions in eukaryotic cells, have evolved from eubacteria-like endosymbionts whose closest known relatives are the rickettsial group of alpha-proteobacteria. Because characterized mitochondrial genomes vary markedly in structure, it has been impossible to infer from them the initial form of the proto-mitochondrial genome. This would require the identification of minimally derived mitochondrial DNAs that better reflect the ancestral state. Here we describe such a primitive mitochondrial genome, in the freshwater protozoon Reclinomonas americana. This protist displays ultrastructural characteristics that ally it with the retortamonads, a protozoan group that lacks mitochondria. R. americana mtDNA (69,034 base pairs) contains the largest collection of genes (97) so far identified in any mtDNA, including genes for 5S ribosomal RNA, the RNA component of RNase P, and at least 18 proteins not previously known to be encoded in mitochondria. Most surprising are four genes specifying a multisubunit, eubacterial-type RNA polymerase. Features of gene content together with eubacterial characteristics of genome organization and expression not found before in mitochondrial genomes indicate that R. americana mtDNA more closely resembles the ancestral proto-mitochondrial genome than any other mtDNA investigated to date.