RESUMEN
N-Methylated amino acids (N-MeAAs) are privileged residues of naturally occurring peptides critical to bioactivity. However, de novo discovery from ribosome display is limited by poor incorporation of N-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the N-methyl-aminoacyl-tRNA. By reconfiguring the tRNA's T-stem region to compensate and tune the EF-Tu affinity, we conducted Random nonstandard Peptides Integrated Discovery (RaPID) display of a macrocyclic peptide (MCP) library containing six different N-MeAAs. We have here devised a "pool-and-split" enrichment strategy using the RaPID display and identified N-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate mutases. The enriched MCPs reached 57% N-methylation with up to three consecutively incorporated N-MeAAs, rivaling natural products. Potent nanomolar inhibitors ranging in ortholog selectivity, strongly mediated by N-methylation, were identified. Co-crystal structures reveal an architecturally related Ce-2 Ipglycermide active-site metal-ion-coordinating Cys lariat MCP, functionally dependent on two cis N-MeAAs with broadened iPGM species selectivity over the original nematode-selective MCPs. Furthermore, the isolation of a novel metal-ion-independent Staphylococcus aureus iPGM inhibitor utilizing a phosphoglycerate mimetic mechanism illustrates the diversity of possible chemotypes encoded by the N-MeAA MCP library.
Asunto(s)
Transferasas Intramoleculares , Factor Tu de Elongación Peptídica , Aminoácidos/química , Transferasas Intramoleculares/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Biblioteca de Péptidos , Péptidos/química , Péptidos Cíclicos/química , ARN de TransferenciaAsunto(s)
Caballos/genética , Complejos Multienzimáticos/genética , Familia de Multigenes , Proteínas Serina-Treonina Quinasas/genética , Mapeo de Híbrido por Radiación , Proteínas Quinasas Activadas por AMP , Animales , Cromosomas de los Mamíferos , Hibridación Fluorescente in Situ , Escala de Lod , Análisis de Secuencia de ADNRESUMEN
A comparative approach that utilizes information from more densely mapped or sequenced genomes is a proven and efficient means to increase our knowledge of the structure of the horse genome. Human chromosome 2 (HSA2), the second largest human chromosome, comprising 243 Mb, and containing 1246 known genes, corresponds to all or parts of three equine chromosomes. This report describes the assignment of 140 new markers (78 genes and 62 microsatellites) to the equine radiation hybrid (RH) map, and the anchoring of 24 of these markers to horse chromosomes by FISH. The updated equine RH maps for ECA6p, ECA15, and ECA18 resulting from this work have one, two, and three RH linkage groups, respectively, per chromosome/chromosome-arm. These maps have a three-fold increase in the number of mapped markers compared to previous maps of these chromosomes, and an increase in the average marker density to one marker per 1.3 Mb. Comparative maps of ECA6p, ECA15, and ECA18 with human, chimpanzee, dog, mouse, rat, and chicken genomes reveal blocks of conserved synteny across mammals and vertebrates.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 2/genética , Caballos/genética , Animales , Cromosomas Artificiales Bacterianos , Cricetinae/genética , Cartilla de ADN , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Metafase , Hibridación de Ácido NucleicoRESUMEN
Expressed sequence tags (EST) containing microsatellites have been used in the development of both genetic linkage maps and syntenic maps for various species and, thus, offer the advantage of being a convenient tool in comparative mapping studies. A turkey embryonic cDNA library was constructed and screened with (CA/TG)15, (GA/CT)15, (AGG)10, and (AAAC)7 probes for the development of polymorphic microsatellite markers. Sequences of 128 cDNA revealed 42 new loci containing microsatellites. BLAST nucleotide analysis demonstrated significant homology to known mammalian or avian coding regions for 15 of the turkey EST, five of which matched chicken transcripts. The remaining 27 EST represented novel sequences. Of the 42 new loci, 31 were polymorphic when tested on commercial turkey lines, including the founding individuals of a new resource population developed for genetic linkage mapping. Comparative mapping of these markers will provide new information toward the evolutionary divergence of turkey and chicken as well as other species.