RESUMEN
The objective of the current study was to analyze Ty1-copia group retrotransposons in cultivated G. barbadense L. cottons. DNA sequence analysis of 27 partial reverse transcriptase sequences revealed that these elements are heterogeneous and this heterogeneity is resolved into 11 distinct families. Phylogenetic analyses provided strong bootstrap support for a monophyletic origin of plant Ty1-copia group retrotransposons, yet showed high diversity within and between Gossypium species. Furthermore, G. Barbadense element topologies are incongruent with Gossypium phylogeny. The high ratio of synonymous to nonsynonymous changes indicates that the reverse transcriptase domain of these families is evolving under purifying selection. The antiquity and wide distribution of Ty1-copia group retrotransposons illustrate their active role in shaping and evolution of the Gossypium genome.
Asunto(s)
ADN Polimerasa Dirigida por ARN/genética , Retroelementos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Molecular , Genoma de Planta , Gossypium , Datos de Secuencia Molecular , Filogenia , Mutación Puntual/genética , Reacción en Cadena de la Polimerasa , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/normas , Alineación de Secuencia , Análisis de Secuencia , Especificidad de la EspecieRESUMEN
A colorimetric assay for reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) was developed using a double labelled (biotin and digoxigenin) deoxyuridine triphosphate mixture instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from oligodeoxythymidylic acid (oligo-dT) contained both biotin and digoxigenin labels. This nucleotide was bound to streptavidin-magnetic beads by the reaction to biotin. At the detection step, an alkaline phosphatase conjugated antibody to digoxigenin was added, followed by the reaction of a colorimetric substrate for this enzyme. This RT assay was comparable to the isotopic RT assay using purified AMV-RT and two strains of HIV grown in cell lines. In addition it was equivalent to the isotopic RT assay for analysis of the time course of in vitro infection of human peripheral blood lymphocytes by HIV-1. The total assay time after the RT reaction step was less than one hour.