Biomedical subjects

Paul Gaytán

Publications and source records attributed to Paul Gaytán.

4 recordsLinked to original sources

PubMed2005

The genome project of Taenia solium.

We have constituted a consortium of key laboratories at the National Autonomous University of Mexico to carry out a genomic project for Taenia solium. This project will provide powerful resources for the study of taeniasis/cysticercosis, and, in conjunction with the Echinococcus granulosus and Echinococcus multilocularis genome project of expressed sequence tags (ESTs), will mark the advent of genomics for cestode parasites. Our project is planned in two consecutive stages. The first stage is being carried out to determine some basic parameters of the T. solium genome. Afterwards, we will evaluate the best strategy for the second stage, a full blown genome project. We have estimated the T. solium genome size by two different approaches: cytofluorometry on isolated cyton nuclei, as well as a probabilistic calculation based on approximately 2000 sequenced genomic clones, approximately 3000 ESTs, resulting in size estimates of 270 and 251 Mb, respectively. In terms of sequencing, our goal for the first stage is to characterize several thousand EST's (from adult worm and cysticerci cDNA libraries) and genomic clones. Results obtained so far from about 16,000 sequenced ESTs from the adult stage, show that only about 40% of the T. solium coding sequences have a previously sequenced homologue. Many of the best hits are found with mammalian genes, especially with humans. However, 1.5% of the hits lack homologues in humans, making these genes immediate candidates for investigation on pharmaco-therapy, diagnostics and vaccination. Most T. solium ESTs are related to gene regulation, and signal transduction. Other important functions are housekeeping, metabolism, cell division, cytoskeleton, proteases, vacuolar transport, hormone response, and extracellular matrix activities. Preliminary results also suggest that the genome of T. solium is not highly repetitive.

Animals↗

PubMed2004

Combinatorial codon-based amino acid substitutions.

Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region.

Amino Acid Substitution↗

PubMed2004

Protein evolution by codon-based random deletions.

A method to delete in-phase codons throughout a defined target region of a gene has been developed. This approach, named the codon-based random deletion (COBARDE) method, is able to delete complete codons in a random and combinatorial mode. Robustness, automation and fine-tuning of the mutagenesis rate are essential characteristics of the method, which is based on the assembly of oligonucleotides and on the use of two transient orthogonal protecting groups during the chemical synthesis. The performance of the method for protein function evolution was demonstrated by changing the substrate specificity of TEM-1 beta-lactamase. Functional ceftazidime-resistant beta-lactamase variants containing several deleted residues inside the catalytically important omega-loop region were found. The results show that the COBARDE method is a useful new molecular tool to access previously unexplorable sequence space.

Amino Acids↗

PubMed2002

Novel ceftazidime-resistance beta-lactamases generated by a codon-based mutagenesis method and selection.

Four known and nine new ceftazidime-resistance beta-lactamases were generated by a novel, contaminating codon-based mutagenesis approach. In this method, wild-type codons are spiked with a set of mutant codons during oligonucleotide synthesis, generating random combinatorial libraries of primers that contain few codon replacements per variant. Mutant codons are assembled by tandem addition of a diluted mixture of five Fmoc-dimer amidites to the growing oligo and a mixture of four DMTr-monomer amidites to generate 20 trinucleotides that encode a set of 18 amino acids. Wild-type codons are assembled with conventional chemistry and the whole process takes place in only one synthesis column, making its automation feasible. The random and binomial behavior of this approach was tested in the polylinker region of plasmid pUC19 by the synthesis of three oligonucleotide libraries mutagenized at different rates and cloned as mutagenic cassettes. Additionally, the method was biologically assessed by mutating six contiguous codons that encode amino acids 237-243 (ABL numbering) of the TEM(pUC19) beta-lactamase, which is functionally equivalent to the clinically important TEM-1 beta-lactamase. The best ceftazidime-recognizing variant was a triple mutant, R164H:E240K: R241A, displaying a 333-fold higher resistance than the wild-type enzyme.

Amino Acid Substitution↗