Steve N. Gagnon

Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii: primary and secondary structure of the 5S gene

The ribosomal DNA (rDNA encoding rRNA) of the obligately intracellular protozoan parasite, Toxoplasma gondii, was identified, cloned, physically mapped, its copy number determined, and the 5S gene sequenced. Using total RNA as a probe, a collection of recombinant lambda phages containing copies of rDNA were isolated from a lambda 2001 tachyzoite genomic library. Northern gel hybridization confirmed specific homology of the 7.5-kb rDNA unit, subcloned into pTZ18R, to T. gondii rRNA. The mapped rDNA found in pTOX1 contained small ribosomal subunit (SS; 18S)- and large ribosomal subunit (LS; 26S)-encoding genes localized using intragenic heterologous probes from the conserved sequences of the SS (18S) and LS (28S) Xenopus laevis genes. the physical mapping data, together with partial digestion experiments and Southern gel hybridization, confirmed a 7.5-kb rDNA unit arranged in a simple head-to-tail fashion that is tandemly repeated. We estimated the rDNA repeat copy number in T. gondii to be 110 copies per haploid tachyzoite genome. Parts of the SS gene and the complete 5S gene were sequenced. The 5S gene was found to be within the rDNA locus, a rare occurrence found only in some fungi and protozoa. Secondary-structure analysis revealed an organization remarkably similar to the 5S RNA of eukaryotes.

SECONDARY STRUCTURES AND FEATURES OF THE 18S, 5.8S AND 26S RIBOSOMAL RNAS FROM THE APICOMPLEXAN PARASITE TOXOPLASMA GONDII

The two major subunits of the ribosomal RNA (rRNA) of Toxoplasma gondii, 18S and 26S, as well as 5.8S, have been sequenced and folded according to known consensus and established secondary structures. Conserved and variable nucleotide (nt) regions were identified using multiple alignments with rRNA sequences of selected organisms. The 18S rRNA showed a well conserved core structure of 48 stems and a hypervariable V4 region identified four additional stems including a pseudoknot. The 18S rRNA contained an additional helix in the V2 region located between nt 204 to 258. We noted that T. gondii 18S does not have a true V6 region, but was organized as a motif of a simple stem. T. gondii 26S had a conserved core structure of 83 stems and its expansion segments, so-called divergent domains, demonstrated a high degree of similarity with secondary structures from rRNA of dinoflagellates and ciliates. For the T. gondii 26S sequence, we found two additional stems, D3d and D3e, composed of 140 nt having a higher deltaG value. These segments are absent from the prokaryotic rRNA structures, whereas the hypervariable V4 region of the small subunit is not as variable. The well preserved structures could indicate an additional function for the eukaryotic ribosome.

Molecular cloning, complete sequence of the small subunit ribosomal RNA coding region and phylogeny of Toxoplasma gondii

Traditionally, the taxonomy of macroorganisms, animals, higher plants and fungi, etc., has been based upon morphological criteria, whereas for microorganisms, biochemical and biophysical properties were commonly used. The comparative studies of phenotypes were found to be untenable for protists, mainly because of their remarkable diversity. During the last 10 years, studies of molecular evolution have demonstrated that genetic diversity in the eukaryotic domain exceeds that seen in the entire prokaryotic world [1]. Toxoplasma gondii is an obligate intracellular protozoanparasite of the phylum Apicomplexa and class Sporozoa. It infects a wide variety of birds and mammals, including the felidae, which serve as host to the sexual phase of the life cycle [2]. The apicomplexans are

Caenorhabditis elegans LET-767 is able to metabolize androgens and estrogens and likely shares common ancestor with human types 3 and 12 17 -hydroxysteroid dehydrogenases

Mutations that inactivate LET-767 are shown to affect growth, reproduction, and development in Caenorhabditis elegans. Sequence analysis indicates that LET-767 shares the highest homology with human types 3 and 12 17beta-hydroxysteroid dehydrogenases (17beta-HSD3 and 12). Using LET-767 transiently transfected into human embryonic kidney-293 cells, we have found that the enzyme catalyzes the transformation of both 4-androstenedione into testosterone and estrone into estradiol, similar to that of mouse 17beta-HSD12 but different from human and primate enzymes that catalyze the transformation of estrone into estradiol. Previously, we have shown that amino acid F234 in human 17beta-HSD12 is responsible for the selectivity of the enzyme toward estrogens. To assess whether this amino acid position 234 in LET-767 could play a role in androgen-estrogen selectivity, we have changed the methionine M234 in LET-767 into F. The results show that the M234F change causes the loss of the ability to transform androstenedione into testosterone, while conserving the ability to transform estrone into estradiol, thus confirming the role of amino acid position 234 in substrate selectivity. To further analyze the structure-function relationship of this enzyme, we have changed the three amino acids corresponding to lethal mutations in let-767 gene. The data show that these mutations strongly affect the ability of LET-767 to convert estrone in to estradiol and abolish its ability to transform androstenedione into testosterone. The high conservation of the active site and amino acids responsible for enzymatic activity and substrate selectivity strongly suggests that LET-767 shares a common ancestor with human 17beta-HSD3 and 12.

Photoactivated multivitamin preparation induces poly(ADP-ribosyl)ation, a DNA damage response in mammalian cells

Multivitamin preparation (MVP) is part of total parenteral nutrition given to premature infants. Photoactivated MVP carries an important load in peroxides, but their cellular effects have not yet been determined. We hypothesized that these peroxides may elicit a DNA-damage response. We found that photoactivation of MVP and the resulting peroxide production were time-dependent and required the simultaneous presence of ascorbic acid and riboflavin. Cells treated with photoactivated MVP showed strongly stimulated poly(ADP-ribosyl)ation, an early DNA-damage response in mammals. Poly(ADP-ribosyl)ation stimulation was dependent on the presence of ascorbic acid and riboflavin in the photoactivated MVP. It did not occur in the presence of a specific PARP inhibitor nor in mouse fibroblasts deficient in PARP-1. Photoactivated MVP was able to induce single- and double-strand breaks in DNA, with a predominance of single-stand breaks. The presence of double-strand breaks was further confirmed using a 53PB1 focus analysis. Finally, photoactivated MVP was shown to be toxic to human cells and induced caspase-independent cell death. These results suggest that photoactivated MVP carries an important toxic load able to damage DNA and induce cell death. This study also emphasizes the importance of protecting MVP solution from light before use in preterm infants.

Single amino acid substitution enhances bacterial expression of PARP-1D214A

Poly(ADP-ribose) polymerase-1 (PARP-1) is the canonical member of the PARP family of enzymes and modulates many crucial nuclear functions. PARP-1 is involved in apoptosis and is the substrate of caspase-3, a protease that cleaves PARP-1 at the conserved sequence 211DEVD214. To generate a caspase-3-uncleavable PARP-1, we introduced an amino acid substitution D214→A214 at the site of cleavage. We observed that following over-expression in bacteria, the mutant protein HIS-PARP-1D214A was expressed several-fold more than a unmutated copy, HIS-PARP-1. The specific activity of HIS-PARP-1 enzyme in total bacterial extracts was 6.94 and 4.61 U/mg for HIS-PARP-1D214A. This approach should provide new avenues for crystallographic study of PARP-1 as well as new information for drug design targeting PARP-1.