Hexyltrimethylammonium ion enhances potential copper-chelating properties of ammonium thiomolybdate in an in vivo zebrafish model.

Ammonium and hexyltrimethylammonium thiomolybdates (ATM and ATM-C6) and thiotungstates (ATT and ATT-C6) were synthesized. Their toxicity was evaluated using both in vitro and in vivo approaches via the zebrafish embryo acute toxicity assay (ZFET), while the copper-thiometallate interaction was studied using cyclic voltammetry, as well as in an in vivo assay. Cyclic voltammetry suggests that all thiometallates form complexes with copper in a 2:1 Cu:thiometallate ratio. Both in vitro and in vivo assays demonstrated low toxicity in BALB/3T3 cells and in zebrafish embryos, with high IC50 and LC50 values. Furthermore, the hexyltrimethylammonium ion played a crucial role in enhancing viability and reducing toxicity during prolonged treatments for ATM and ATT. In particular, the ZEFT assay uncovered the accumulation of ATM in zebrafish yolk, averted by the incorporation of the hexyltrimethylammonium ion. Notably, the copper-thiometallate interaction assay highlighted the improved viability of embryos when cultured in CuCl2 and ATM-C6, even at high CuCl2 concentrations. The hatching assay further confirmed that copper-ATM-C6 interaction mitigates inhibitory effects induced by thiomolybdates and CuCl2 when administered individually. These results suggest that the incorporation of the hexyltrimethylammonium ion in ATM increase its ability to interact with copper and its potential application as a copper chelator.

Roles for ligases in the RNA editing complex of Trypanosoma brucei: band IV is needed for U-deletion and RNA repair.

Trypanosome RNA editing utilizes a seven polypeptide complex that includes two RNA ligases, band IV and band V. We now find that band IV protein contributes to the structural stability of the editing complex, so its lethal genetic knock-out could reflect structural or catalytic requirements. To assess the catalytic role in editing, we generated cell lines which inducibly replaced band IV protein with an enzymatically inactive but structurally conserved version. This induction halts cell growth, showing that catalytic activity is essential. These induced cells have impaired in vivo editing, specifically of RNAs requiring uridylate (U) deletion; unligated RNAs cleaved at U-deletion sites accumulated. Additionally, mitochondrial extracts of cells with reduced band IV activity were deficient in catalyzing U-deletion, specifically at its ligation step, but were not deficient in U-insertion. Thus band IV ligase is needed to seal RNAs in U-deletion. U-insertion does not appear to require band IV, so it might use the other ligase of the editing complex. Furthermore, band IV ligase was also found to serve an RNA repair function, both in vitro and in vivo.

Trypanosome RNA editing: simple guide RNA features enhance U deletion 100-fold.

Trypanosome RNA editing is a massive processing of mRNA by U deletion and U insertion, directed by trans-acting guide RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPase (A6) pre-mRNA and gRNA. Here we examine which gRNA features are important for this U deletion. We find that, foremost, this editing depends critically on the single-stranded character of a few gRNA and a few mRNA residues abutting the anchor duplex, a feature not previously appreciated. That plus any base-pairing sequence to tether the upstream mRNA are all the gRNA needs to direct unexpectedly efficient in vitro U deletion, using either the purified editing complex or whole extract. In fact, our optimized gRNA constructs support faithful U deletion up to 100 times more efficiently than the natural gRNA, and they can edit the majority of mRNA molecules. This is a marked improvement of in vitro U deletion, in which previous artificial gRNAs were no more active than natural gRNA and the editing efficiencies were at most a few percent. Furthermore, this editing is not stimulated by most other previously noted gRNA features, including its potential ligation bridge, 3' OH moiety, any U residues in the tether, the conserved structure of the central region, or proteins that normally bind these regions. Our data also have implications about evolutionary forces active in RNA editing.

Trypanosoma brucei U insertion and U deletion activities co-purify with an enzymatic editing complex but are differentially optimized.

RNA editing, the processing that generates functional mRNAs in trypanosome mitochondria, involves cycles of protein catalyzed reactions that specifically insert or delete U residues. We recently reported purification from Trypanosoma brucei mitochondria of a complex showing seven major polypeptides which exhibits the enzymatic activities inferred in editing and that a pool of fractions of the complex catalyzed U deletion, the minor form of RNA editing in vivo . We now show that U insertion activity, the major form of RNA editing in vivo , chromatographically co-purifies with both U deletion activity and the protein complex. Furthermore, these editing activities co-sediment at approximately 20 S. U insertion does not require a larger, less characterized complex, as has been suggested and could have implied that the editing machinery would not function in a processive manner. We also show that U insertion is optimized at rather different and more exacting reaction conditions than U deletion. By markedly reducing ATP and carrier RNA and increasing UTP and carrier protein relative to standard editing conditions, U insertion activity of the purified fraction is enhanced approximately 100-fold.

T. brucei RNA editing: adenosine nucleotides inversely affect U-deletion and U-insertion reactions at mRNA cleavage.

In the currently envisioned mechanism of trypanosome mitochondrial RNA editing, U-insertion and U-deletion cycles begin with a common kind of gRNA-directed cleavage. However, natural, altered, and mutationally interconverted editing sites reveal that U-deletional cleavage is inefficient without and activated by ATP and ADP, while U-insertional cleavage shows completely reverse nucleotide effects. The adenosine nucleotides' effects appear to be allosteric and determined solely by sequences immediately adjacent to the anchor duplex. Both U-deletional and U-insertional cleavages are reasonably active at physiological mitochondrial ATP concentration. Notably, ATP and ADP markedly stimulate complete U-deletion and inhibit U-insertion reactions, reflecting their effects on cleavage. These plus previous results suggest that U deletion and U insertion are remarkably distinct.

Unexpected electrophoretic migration of RNA with different 3' termini causes a RNA sizing ambiguity that can be resolved using nuclease P1-generated sequencing ladders.

It has been widely believed that the electrophoretic migration difference of otherwise identical RNAs with a P versus OH terminus would be the same as occurs for DNA, a fairly reproducible approximately 1/2 nucleotide (nt) offset. RNA with a 5'-OH indeed migrates

Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria.

Kinetoplastid mitochondrial RNA editing, the insertion and deletion of U residues, is catalyzed by sequential cleavage, U addition or removal, and ligation reactions and is directed by complementary guide RNAs. We have purified a approximately 20S enzymatic complex from Trypanosoma brucei mitochondria that catalyzes a complete editing reaction in vitro. This complex possesses all four activities predicted to catalyze RNA editing: gRNA-directed endonuclease, terminal uridylyl transferase, 3' U-specific exonuclease, and RNA ligase. However, it does not contain other putative editing complex components: gRNA-independent endonuclease, RNA helicase, endogenous gRNAs or pre-mRNAs, or a 25 kDa gRNA-binding protein. The complex is composed of eight major polypeptides, three of which represent RNA ligase. These findings identify polypeptides representing catalytic editing factors, reveal the nature of this approximately 20S editing complex, and suggest a new model of editosome assembly.

Resolution of the RNA editing gRNA-directed endonuclease from two other endonucleases of Trypanosoma brucei mitochondria.

RNA editing in kinetoplastids, the specific insertion and deletion of U residues, requires endonuclease cleavage of the pre-mRNA at each cycle of insertion/deletion. We have resolved three endoribonuclease activities from Trypanosoma brucei mitochondrial extracts that cleave CYb pre-mRNA specifically. One of these, which sediments at approximately 20S and is not affected substantially by DTT, has all the features of the editing endonuclease. It cleaves CYb pre-edited or partially edited mRNA only when annealed to the anchor region of a cognate guide RNA (gRNA), and it cleaves accurately just 5' of the duplex region. Its specificity is for the 5' end of extended duplex RNA regions, and this prevents cleavage of the gRNA or other positions in the mRNA. This gRNA-directed nuclease is evidently the same activity that functions in A6 pre-mRNA editing. However, it is distinct and separable from a previously observed DTT-requiring endonuclease that sediments similarly under certain conditions, but does not cleave precisely at the first editing site in either the presence or absence of a gRNA. The editing nuclease is also distinct from a DTT-inhibited endonuclease that cleaves numerous free pre-mRNAs at a common structure in the region of the first editing site.

Trypanosome U-deletional RNA editing involves guide RNA-directed endonuclease cleavage, terminal U exonuclease, and RNA ligase activities.

We have studied the mechanism of accurate in vitro RNA editing of Trypanosoma brucei ATPase 6 mRNA, using four mRNA-guide RNA (gRNA) pairs that specify deletion of 2, 3, or 4 U residues at editing site 1 and mitochondrial extract. This extract not only catalyzes deletion of the specified number of U residues but also exhibits a novel endonuclease activity that cleaves the input pre-mRNA in a gRNA-directed manner, precisely at the phosphodiester bond predicted in a simple enzymatic model of RNA editing. This cleavage site is inconsistent with a chimera-based editing mechanism. The U residues to be deleted, present at the 3' end of the upstream cleavage product, are then removed evidently by a 3' U-specific exonuclease and not by a reverse reaction of terminal U transferase. RNA ligase can then join the mRNA halves through their newly formed 5' P and 3' OH termini, generating mRNA faithfully edited at the first editing site. This resultant, partially edited mRNA can then undergo accurate, gRNA-directed cleavage at editing site 2, again precisely as predicted by the enzymatic editing model. All of these enzymatic activities cofractionate with the U-deletion activity and may reside in a single complex. The data imply that each round of editing is a four-step process, involving (i) gRNA-directed cleavage of the pre-mRNA at the bond immediately 5' of the region base paired to the gRNA, (ii) U deletion from or U addition to the 3' OH of the upstream mRNA half, (iii) ligation of the mRNA halves, and (iv) formation of additional base pairing between the correctly edited site and the gRNA that directs subsequent nuclease cleavage at the next editing site.

A novel transcribed repeat element from Entamoeba histolytica.

We have identified an unusual 0.55-kb DNA repeat element specific to Entamoeba histolytica (Eh) which we call interspersed element (IE). The IE is a common feature in independently isolated genomic and cDNA fragments. Hybridization of labeled IE sequences to trophozoite DNA, RNA and first-strand cDNA prepared from poly(A)-enriched mRNA indicate that the IE are reiterated about 500 times per Eh trophozoite and that one or more can be found as RNA transcripts. These features and the degree of conservation of IE suggest a possible role for these sequences.

Cloning, characterization and expression of two Xenopus bcl-2-like cell-survival genes.

We describe two cloned cDNAs, termed xR1 and xR11, isolated from a Xenopus laevis stage 28-30 embryonic head cDNA library. Comparison of amino acid (aa) sequences derived from nucleotide (nt) sequences of xR1 and xR11 cDNAs revealed substantial homology with bcl-2-related genes, especially with bcl-xL. In particular, there was a marked conservation of the BH1 and BH2 domains considered to be important for the anti-cell death and heterodimerisation properties of bcl-2. Constitutive expression of xR11 in cultured rat fibroblast (Rat-1) cells conferred a strong protection against cell death induced by the cytotoxic agents staurosporine and cycloheximide, by serum deprivation and specific deregulation of c-myc. Measurement of xR1 and xR11 mRNAs by RNase protection assay revealed similar widespread expression in Xenopus embryos and tadpoles. Except for an abrupt increase in the accumulation of xR1 and xR11 mRNAs in brains of mid-metamorphic and post-metamorphic tadpoles and adults, there was insignificant modulation of their expression in tissues undergoing total regression (tail) or morphogenesis (limb) during natural or thyroid hormone-induced metamorphosis. These findings raise the possibility of continuing expression of cell survival genes in tissues undergoing total regression during post-embryonic development.

Ribosomal DNA sequences in the differentiation of pathogenic and non-pathogenic isolates of Entamoeba histolytica.

Recombinant ribosomal DNA sequences were amplified by PCR and used as probes to perform a fingerprint analysis of total DNA from different Entamoeba histolytica isolates. RFLPs obtained with one of the probes, R-1, support previous proposals that pathogenic and non-pathogenic E. histolytica are closely related, yet genotypically distinct. Another probe, R-2, while not distinguishing between the two forms of E. hystolytica, was able to differentiate between them and E. moshkovskii, which has morphologically identical cysts and trophozoites. A third probe, BR-1, identified strain-specific RFLPs.

A DNA probe specific to pathogenic Entamoeba histolytica.

A DNA sequence, IE-gen1 (3.1 kb), was isolated from the pathogenic strain of E. histolytica NIH-200. IE-gen1 was identified by the subtractive hybridization of a genomic library to a cDNA probe prepared from NIH-200 trophozoites. The IE-gen1 probe specifically detected pathogenic E. histolytica in slot blots of genomic DNA and Northern blots, but not other Entamoeba species and additional human parasites. This genomic probe could detect with complete specificity DNA from about 10(3) organisms. The IE-gen1 probe could be related to highly specialized loci in pathogenic E. histolytica, and is likely to be a valuable DNA reagent for clinical diagnosis and epidemiological investigations.