Long range base-pairing in the leftward transcription unit of bacteriophage lambda. Characterization by electron microscopy and computer-aided sequence analysis.

Restriction fragments of bacteriophage lambda DNA corresponding to the major leftward transcription unit were purified, denatured to form single-stranded DNA, self-annealed, and examined by electron microscopy. Three intrastrand stem and loop secondary structures were observed reproducibly and the locations of the paired regions were determined. A method for computer-aided sequence analysis of these regions is presented and used to identify sets of base-pairings likely to account for the observed structures. One loop observed within gene Ea47 is postulated to involve pairing of sequences which include the polypeptide initiation and termination codons. Another loop is postulated to involve pairing of sequences in gene int with sequences located in the gam-cIII region. A third loop appears to involve sequences in and to the right of gene Ea22 paired with sequences located in the bet-gam region. A general discussion of base-pairing which gives rise to long range interactions is presented along with possible effects of the postulated models on gene expression.

Ribosomal RNA of the primitive eukaryote Giardia lamblia: large subunit domain I and potential processing signals.

The cytoplasmic ribosomal RNA (rRNA) from the intestinal protozoan, Giardia lamblia, is unusually short; the large subunit (LS) and small subunit RNA and the 5.8S RNA are only 70-80% of the length found in typical protozoa, and are even smaller than most of their prokaryotic counterparts. Flanking regulatory DNA and processed rRNA sequences are similarly compact in size. To shed light on the origins and implications of this 'minimal' rRNA, the nucleotide sequence encoding the 5.8S RNA and domain I of LS RNA was determined. Secondary structure analysis revealed that an evolutionarily variable internal hairpin is partially 'deleted' in G. lamblia 5.8S RNA; the 3'-terminal pairing with LS RNA is conserved. Previously characterized eukaryotic 'expansion' regions are extensively shortened within the LS RNA; in one case, a hairpin is precisely 'deleted'. The short sequences flanking the mature 5.8S RNA that are removed by RNA processing (ITS1 and ITS2) are C-rich; our analysis suggests that the sequence GCGCCCC, in a hairpin configuration, may function as the processing signal.

Comparisons of ribosomal RNA sequences from amitochondrial protozoa: implications for processing, mRNA binding and paromomycin susceptibility.

The amitochondrial (a-mt) protozoa include four groups of organisms that are of interest as important human parasites and as probable descendents of the earliest branches of eukaryotic evolution. These organisms have not been directly compared in terms of structure and function of a specific molecule. We sequenced portions of their rRNA-encoding genes coding for the internal transcribed spacers (ITS1 and 2) and adjoining small subunit (SS), 5.8S and large subunit (LS) rRNAs. Included are sites for RNA processing, mRNA interaction and aminoglycoside binding, as well as potential protein-encoding genes. The ITS of all a-mt protozoa examined are relatively short, but otherwise diverse. They include one or two predominant nucleotides (A in Entamoeba and Trichomonas, T in Encephalitozoon and C in Giardia) and have minimal potential secondary structure, which may form the basis for the preferential processing of ITS sequences. The mechanism employed by a-mt protozoa to bind mRNA may be unique, since Giardia, Trichomonas and Entamoeba mRNAs have usually short 5' non-coding regions. In bacteria, the 3' terminus of the SS rRNA is involved in mRNA binding; analysis of Entamoeba and Trichomonas mRNA 5' non-coding sequences suggests an analogous mechanism involving potential base pairing to the loop of the terminal SS rRNA hairpin. Giardia sensitivity to paromomycin was previously correlated with the presence of a C:G bp near the decoding region of SS rRNA. This bp is also present in Entamoeba and Trichomonas, consistent with their susceptibility. Its absence in Encephalitozoon and other microsporidia predicts paromomycin resistance, and suggests a distinct evolutionary origin for this group.

Domain V of Giardia lamblia large-subunit rRNA: structure of the peptidyl transferase loop from an early-branching eukaryote and correlation with antibiotic sensitivity.

Large subunit rRNA (LSR) sequences that have been implicated in peptide bond formation form a specific secondary structure called the peptidyl transferase loop (PTL). Although well conserved, the PTLs of eubacteria, archaebacteria, and eukaryotes have several distinct differences. These differences correlate with different sensitivities to peptidyl transferase and translocase inhibitors. To shed light on the basis for these kingdom-specific differences in PTL structure and function, we have analyzed the sequence and secondary structure of LSR domain V, which contains the PTL, from Giardia lamblia. This parasitic protozoan derives from a very early branch in eukaryotic evolution, and its rRNA was previously shown to have bacteria-like features. In vitro and cell-free systems were also used to test the sensitivity of G. lamblia protein synthesis to specific PTL-targeted inhibitors. Our results indicate that the PTL structure and inhibitor sensitivity typical of higher eukaryotes is conserved in G. lamblia. However, several adjacent domain V sequences more closely resemble archaebacterial rRNA, confirming the 'primitive' nature of G. lamblia rRNA. Thus, the eukaryotic PTL has been conserved over a vast evolutionary period. We speculate that the eukaryotic PTL is primordial and employs specific RNA-RNA interactions to catalyze protein synthesis. Three potential interactions were identified.

The DNA between Rz and cosR in bacteriophage lambda is nonessential.

Near the right end of phage lambda DNA, between gene Rz and the cos site, are 2050 bp of apparently non-coding DNA. We have cloned a lambda DNA fragment containing this DNA into a plasmid and constructed a deletion, omega l, extending from a site within the Rz gene to a site about 560 bp from cos. This deletion could be recombined into viable lambda phage at a frequency equal to that observed for the undeleted sequence. Recombinant phage lambda carrying the omega l deletion were demonstrated to have the same burst size and kinetics of phage production as undeleted lambda. The omega l deletion can be used to extend the capacity of lambda cloning vectors and to provide a region for the insertion of heterologous DNA which should exhibit controllable high level expression from the lambda late promoter, p'R.

Site-directed mutagenesis of Saccharomyces cerevisiae beta-tubulin: interaction between residue 167 and benzimidazole compounds.

Benzimidazoles are widely used as anthelmintic agents and systemic fungicides. In susceptible organisms, benzimidazoles bind to beta-tubulin and block microtubule polymerization. To further characterize this interaction, site-directed mutagenesis followed by gene replacement was used to change Saccharomyces cerevisiae beta-tubulin residue Phe-167 to Tyr. Consistent with previous studies, this mutation resulted in at least 3-4-fold decreased sensitivity to the benzimidazole derivatives carbendazim and nocodazole. The Tyr-167 mutant was cold sensitive, implying a direct effect on benzimidazole binding rather than a nonspecific increase in microtubule stability. Surprisingly, the mutant had 8-fold increased sensitivity to the derivative benomyl, which is structurally identical to carbendazim except at position 1. This suggests that residue 167 interacts with benzimidazoles in the vicinity of the 1-position.

Beta-tubulin genes of Trichomonas vaginalis.

Microtubules, formed by polymerization of alpha and beta-tubulins, are major structural components of the mitotic spindle, cytoskeleton, and flagella, and are also an important target for the antiparasitic benzimidazole drugs. Trichomonas vaginalis, a flagellated protozoan responsible for urogenital tract infections in humans, is highly sensitive to certain benzimidazoles in vitro. As a first step towards defining the roles of microtubules in this organism, the regulation of their expression, and the basis for their benzimidazole sensitivity, we have characterized the genes encoding T. vaginalis beta-tubulin. A combination of genomic DNA cloning using bacteriophage lambda and PCR amplification using conserved beta-tubulin gene primers was employed. Southern blots of DNA from two different T. vaginalis strains suggest there are 6-7 beta-tubulin gene copies. Sequencing identified three distinct genes: btub1, btub2, and btub3. Amplification of cDNA with gene-specific primers indicated that the relative expression of RNA transcripts was btub1 > btub2 >> btub3. The promoter region from btub1 includes a 15-bp repeat also found (with 1-bp difference) upstream of the T. vaginalis ferredoxin gene. Primer extension suggests the 5' leader of the mRNA transcribed from btub1 is only 10 nucleotides long, similar to the lengths found in other anaerobic protozoa. In 152 residues examined by PCR, btub2 and btub3 differed by 1 and 12 amino acids, respectively, from btub1. All three sequences, however, have diverged considerably (20-24%) from beta-tubulins of other protozoa. T. vaginalis beta-tubulins include residues Tyr167 and Phe200, previously implicated in resistance and sensitivity, respectively, to the benzimidazole derivative benomyl.

Heat shock and stress response in Giardia lamblia.

The heat shock response was studied as a model for control of gene expression and protein synthesis in Giardia lamblia. Cultured trophozoites were metabolically labelled with [35S]methionine, and proteins were analysed by SDS-polyacrylamide gel electrophoresis and fluorography. A temperature shift from 37 degrees C to 43 degrees C resulted in the depression of normal protein synthesis, and the enhanced synthesis of four major heat shock proteins of 100, 83, 70 and 30 kDa. This response resembles that seen in other organisms of wide phylogenetic diversity. An examination of the kinetics of induction and recovery from heat shock suggests that the individual heat shock proteins are independently regulated. In vitro translation of messenger RNA isolated from heat shocked cells further indicates that regulation occurs at both transcriptional and translational levels. The response of G. lamblia to other stresses including cysteine deprivation, exposure to oxygen, ethanol, hydrogen peroxide, and the chemotherapeutic drugs metronidazole and quinacrine was also investigated. The induction of two or more of the heat shock proteins was generally observed; however, certain treatments inhibited synthesis of all proteins including heat shock proteins.

Role of glutathione peroxidase in rheumatoid arthritis: analysis of enzyme activity and DNA polymorphism.

Aberrant expression of the antioxidant enzyme glutathione peroxidase (GPx) could contribute to the etiology of rheumatoid arthritis (RA). However, previous enzyme activity studies examining this relationship were inconclusive. Indirect evidence for this relationship derives from the known efficacy of gold therapy in RA, since gold compounds specifically inhibit GPx. The hypothesis that variants of GPx are associated with RA was examined by two approaches: enzyme activity analysis and restriction fragment length polymorphism (RFLP) association analysis. No significant difference was found in whole blood GPx activity between 28 RA patients and 36 controls. GPx activity appeared to be independent of sex, race, or type of drug treatment. However, a statistically significant difference was found with respect to treatment responsiveness. RA patients classified as good responders to gold therapy, but who were no longer taking gold, had a significantly higher GPx activity compared to both the controls and good responders currently on gold therapy. Aberrantly high GPx activity could contribute to RA by generating excess oxidized glutathione, a potent collagenase activator. Gold therapy would reduce GPx activity to normal levels. The restriction enzyme Pvu II in conjunction with a GPx gene probe identified a useful RFLP (Al, 22 kbp; A2, 15 kbp) with allelic frequencies of A1 and A2 equal to 0.11 and 0.89, respectively, in the control population. No statistically significant association, however, could be demonstrated between this allelic variant of the GPx gene and RA.

Detection of Plasmodium falciparum using a synthetic DNA probe.

A labeled synthetic polynucleotide representing a repetitive sequence from Plasmodium falciparum was hybridized with genomic DNA spotted on nitrocellulose. After an overnight exposure, 0.1 ng of P. falciparum DNA was specifically detected and 0.01 ng was detected after an exposure of 1 week. The synthetic probe showed no cross-hybridization with host DNA or with DNA isolated from other species in the phylum Apicomplexa, P. vivax and Babesia species. Since synthetic DNA is easily prepared, the observed sensitivity and specificity suggests that synthetic DNA probes would be generally useful in diagnosis.

Susceptibility of Giardia lamblia to aminoglycoside protein synthesis inhibitors: correlation with rRNA structure.

The very limited development of antiparasitic agents targeting protein synthesis stems in part from the belief that parasite and host ribosomes are sufficiently similar to preclude selective toxicity. However, recent studies have revealed that Giardia lamblia rRNA has an unusual size and sequence; consequently, this organism and its homogeneous rRNA provide a useful model for the development of protein synthesis inhibitors with antiparasitic activity. In this study, I determined the sequence and secondary structure of the 3' end of the small-subunit RNA, the target for aminoglycoside inhibitory activity. The primary structure of these 140 nucleotides includes two blocks of sequence highly conserved among other organisms; the remaining sequence, although not conserved, can be folded into a secondary structure common to all rRNAs. The presence of U-1495 within one of the conserved blocks predicts hygromycin susceptibility. Also, a specific base pair (C-1409.G-1491) implicated in paromomycin susceptibility is present; whereas all procaryotes have this base pair, it is absent in many eucaryotes (including mammals). Conversely, kanamycin and apramycin resistance can be predicted from substitution of A-1408 with G. A growth inhibition assay was used to test the susceptibility of G. lamblia to a variety of aminoglycosides. After 48 h, 8 of 11 aminoglycosides tested failed to inhibit growth at a concentration of 200 micrograms/ml. Paromomycin and hygromycin, however, inhibited growth of three strains tested by 50% at 50 to 60 micrograms/ml and by close to 90% at 120 micrograms/ml. These results correlate well with the sequence and secondary-structure analyses. Paromomycin may be clinically useful when the toxicity of standard antigiardial drugs is of concern.

Tetracyclines as antiparasitic agents: lipophilic derivatives are highly active against Giardia lamblia in vitro.

Comparisons of the inhibitory activities of different tetracyclines have been reported for Plasmodium falciparum but no other parasites. The in vitro response of the intestinal parasite Giardia lamblia to six tetracyclines in current use was determined. In addition, the experimental drug thiacycline (EMD 33,330) was evaluated. Three groups were discerned, with representative 50 and 90% inhibitory concentrations of, respectively, 36 and 130 (tetracycline), 6.4 and 22 (doxycycline), and 1.8 and 3.4 (thiacycline) micrograms/ml. These dramatic differences in activity correlate with increased lipophilicity.

Enhanced antiparasitic activity of lipophilic tetracyclines: role of uptake.

It was previously noted that the inhibitory activities of lipophilic tetracyclines against the growth of Giardia lamblia in vitro were up to 40-fold greater than those of nonlipophilic tetracyclines (50% inhibitory concentration [IC50] = 1.8 to 71 micrograms/ml) (T. D. Edlind, Antimicrob. Agents Chemother. 33:2144-2145, 1989). We have now extended this observation to Trichomonas vaginalis (IC50 = 2.9 to 200 micrograms/ml), Entaoeba histolytica (IC50 = 3.8 to 36 micrograms/ml), and Leishmania major promastigotes (IC50 = 21 to 250 micrograms/ml; one strain only). The basis for these differential tetracycline activities was investigated with G. lamblia. In a cell-free protein synthesis system, lipophilic and nonlipophilic tetracyclines had similar, relatively low activities (IC50 = 170 to 500 micrograms/ml). On the other hand, tetracycline uptake into intact cells after a 1-h incubation varied dramatically: the ratios of intracellular to extracellular drug concentrations were 1.7 to 7.2 for nonlipophilic tetracyclines and 47 to 112 for lipophilic derivatives. Thus, the variable effects of tetracyclines on the growth of G. lamblia can be fully accounted for by differences in uptake. Passive diffusion probably plays a more important role than active transport in uptake of lipophilic tetracyclines, since similar results were obtained with cells rendered nonviable by metronidazole treatment.

Intrastrand base pairing in single-stranded deoxyribonucleic acid from ColE1-derived plasmid pCR1.

Single strands of EcoRI-cleaved pCR1 deoxyribonucleic acid were examined by electron microscopy for intrastrand base pairing by using partial denaturing conditions. The locations of three stem and loop structures were mapped relative to the inverted repeat of Tn903. Potential roles and origins of these loops are discussed.

Entamoeba histolytica encodes a highly divergent beta-tubulin.

The microtubules of the amitochondrial parasite Entamoeba histolytica are atypical in certain respects. Consistent with this, we report that E. histolytica encodes the most divergent beta-tubulin identified to date, with only 54% to 58% identity to beta-tubulins from various species. A similarly divergent beta-tubulin is encoded by the related Entamoeba invadens; single gene copies appear to be present in both organisms. The Entamoeba sequences were compared with a database of 101 beta-tubulins, including the highly divergent sequence from another amitochondrial protozoan, Trichomonas vaginalis. A total of 81 residues were universally conserved, and 76 residues varied only once. Correlations with previous studies indicate that microtubule function is altered when most, but not all, conserved residues are mutated.

Electron microscopic mapping of secondary structures in bacterial 16S and 23S ribosomal ribonucleic acid and 30S precursor ribosomal ribonucleic acid.

Electron microscopy revealed reproducible secondary structure patterns within partially denatured 16S and 23S ribosomal ribonucleic acid (rRNA) from Escherichia coli. When prepared with 50% formamide-100 mM ammonium acetate, 16S rRNA included two small hairpins that appeared in over 50% of all molecules. Three open loops were observed with frequencies of less than 25%. In contrast, 23S rRNA included a terminal open loop and two additional large structures in over 75% of all molecules. These secondary structure patterns were conserved in the 16S and 23S rRNA from Pseudomonas aeruginosa. The secondary structure of the 30S precursor rRNA from the ribonclease III-deficient E. coli mutant AB105 was mapped after partial denaturation in 70% formamide-100 mM ammonium acetate. Two large open loops were superimposed on the 16S and 23S rRNA secondary structure patterns. These loops were the most frequent structures found on the precursor, and their stems coincided with ribonuclease III cleavage sites. A tentative 5'-3 orientation was determined for the secondary structure patterns of 16S and 23S rRNA from their relative locations within 30S precursor rRNA. The relation of secondary structure to ribosomal protein binding and ribonuclease III cleavage is discussed.

In vitro susceptibilities of the AIDS-associated microsporidian Encephalitozoon intestinalis to albendazole, its sulfoxide metabolite, and 12 additional benzimidazole derivatives.

Recent reports have described the successful treatment of Encephalitozoon intestinalis infection in AIDS patients with albendazole. However, this compound is rapidly metabolized in vivo to albendazole sulfoxide, and furthermore it is only 1 of about 15 commercially developed benzimidazole derivatives. To compare the activities of albendazole, albendazole sulfoxide, and other benzimidazoles, an in vitro system involving infection of green monkey kidney cell (E6) monolayers with E. intestinalis spores was developed. After 14 days, the effects of benzimidazoles on spore production were determined. Ten of fourteen derivatives tested, including albendazole, were inhibitory at concentrations of 1 to 10 ng/ml. Derivatives modified at the 1 or 2 position were less active. Albendazole sulfoxide was 1.7-fold more inhibitory than albendazole but significantly less toxic to E6 cells, a finding that explains the clinical efficacy of this compound. Potential alternatives to albendazole are discussed. No albendazole-resistant E. intestinalis mutants were obtained following in vitro selection.