A metabolic prototype for eliminating tryptophan from the genetic code.

We set out to reduce the chemical constitution of a living organism to 19 amino acids. A strain was constructed for reassigning the tryptophan codon UGG to histidine and eliminating tryptophan from Escherichia coli. Histidine codons in the gene for an essential enzyme were replaced with tryptophan codons and the restoration of catalytic activity by missense suppressor His-tRNA bearing a CCA anticodon was selected. We used automated cultivation to assess the stability of this genetic construct during evolution. Histidine to tryptophan mutation at codon 30 in the transketolase gene from yeast and its cognate suppressor tRNA were stably propagated in a tktAB deletant of E. coli over 2500 generations. The ratio of histidine misincorporation at tryptophan sites in the proteome increased from 0.0007 to 0.03 over 300 days of continuous culture. This result demonstrated that the genetic code can be forced to evolve by permanent metabolic selection.

Artificially ambiguous genetic code confers growth yield advantage.

A primitive genetic code is thought to have encoded statistical, ambiguous proteins in which more than one amino acid was inserted at a given codon. The relative vitality of organisms bearing ambiguous proteins and the kinds of pressures that forced development of the highly specific modern genetic code are unknown. Previous work demonstrated that, in the absence of selective pressure, enforced ambiguity in cells leads to death or to sequence reversion to eliminate the ambiguous phenotype. Here, we report the creation of a nonreverting strain of bacteria that produced statistical proteins. Ablating the editing activity of isoleucyl-tRNA synthetase resulted in an ambiguous code in which, through supplementation of a limited supply of isoleucine with an alternative amino acid that was noncoding, the mutant generating statistical proteins was favored over the wild-type isogenic strain. Such organisms harboring statistical proteins could have had an enhanced adaptive capacity and could have played an important role in the early development of living systems.

Hypermutagenic in vitro transcription employing biased NTP pools and manganese cations.

In vitro DNA-dependent RNA transcription using bacteriophage T3 RNA polymerase may be rendered hypermutagenic by employing biased nucleoside triphosphate (NTP) concentrations and manganese cations. Using the E. coli R67 plasmid-encoded dihydrofolate reductase (DHFR) gene as target substitution rates approaching 4 x 10(-2) per base per reaction could be achieved, on a par with hypermutagenic reverse transcription. In all cases the majority of substitutions was that expected from the NTP pool bias. The addition of manganese ions increased the frequency of mutations, particularly the proportion of transversions. Functional DHFR hypermutants with up to 8% amino acid substitutions were readily obtained from a single reaction which, given the unique mutation matrix allows exploration of sequence space complementary to that accessed by other hypermutagenic protocols.

Ambiguous base pairing of the purine analogue 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide during PCR.

In principle the hydrogen bonding capacities of 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide (dY), and its N-propyl derivative (dYPr), allow them to pair to all four deoxynucleosides. Their triphosphate derivatives (dYTP and dYPrTP) are preferentially incorporated as dATP analogues in a PCR reaction. However, once incorporated into a DNA template their ambiguous hydrogen bonding potential gave rise to misincorporation at frequencies of approximately 3 x 10(-2) per base per amplification. Most of the substitutions were transitions resulting from rotation about the carboxamide bond when part of the template. Between 11-15% of transversions were noted implying rotation of purine or imidazole moieties about the glycosidic bond. As part of a DNA template, dYPr behaved in the same way as dY, despite its propyl moiety. These deoxyimidazole derivatives are among the most radical departures from the canonical bases used so far as substrates in PCR and could be used to generate mutant gene libraries.

Exploring the functional robustness of an enzyme by in vitro evolution.

The evolution of natural proteins is thought to have occurred by successive fixation of individual mutations. In vitro protein evolution seeks to accelerate this process. RNA hypermutagenesis, cDNA synthesis in the presence of biased dNTP concentrations, delivers elevated mutant and mutation frequencies. Here lineages of active enzymes descended from the homotetrameric 78 residue dihydrofolate reductase (DHFR) encoded by the Escherichia coli R67 plasmid were generated by iterative RNA hypermutagenesis, resulting in >20% amino acid replacement. The 22 residue N-terminus could be deleted yielding a minimum functional entity refractory to further changes, designating it as a determinant of R67 robustness. Complete substitution of the segment still allowed fixation of mutations. By the facile introduction of multiple mutations, RNA hypermutagenesis allows the generation of active proteins derived from extant genes through a mode unexplored by natural selection.

Fate of direct and inverted repeats in the RNA hypermutagenesis reaction.

RNA hypermutagenesis results from cDNA synthesis in the presence of highly biased dNTP precursor concentrations and preferentially exploits human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Such reaction conditions slow down DNA synthesis, which might be conducive to strand transfer and deletion. This has been investigated. A 6 bp inverted repeat nested between 10 bp repeats was efficiently deleted at dCTP concentrations typically used. Inter- or intramolecular strand transfer between 10 bp repeated sequences separated by runs of templated G residues occurred, but at lower concentrations. If RNA hypermutagenesis of a sequence containing direct and inverted repeats is unavoidable, avian myeloblastosis virus (AMV) reverse transcriptase could be used, as strand transfer occurs with much diminished dCTP substrate dependence.

Envenenamiento tipo atropínico en niños por ingestión accidental de productos vegetales. presentación de casos clínicos / Atropine-like poisoning in children due to ingestion of vegetable products

El presente es un reporte de tres casos clínicos acerca de la ingestión de la planta conocida como "toé" en una región de la selva de nuestro país. Se revisa la literatura al respecto y los efectos que generan sus componentes luego de su ingestión, finalizando con recomendaciones generales acerca de las plantas utilizadas en medicina tradicional y sus posibles riesgos.