RESUMEN
A novel simple nonradioactive method for detection of specific nucleotide sequences has been developed. This method consists of the hybridization of a target DNA with a DNA probe modified with trans-diamminedichlorplatinum(II) (trans-DDP) followed by detection of DNA/DNA hybrids with affinity-isolated anti-DNA-trans-DDP antibodies and poly-horseradish peroxidase-protein A conjugate. Major advantages of this approach are the low cost and the extreme simplicity of the labeling procedure, which involves only mixing of the reagents. The sensitivity of the proposed technique is sufficient to detect 0.8 pg of DNA in Southern blot hybridization and 25 fg in dot hybridization and permits colony screening.
Asunto(s)
Cisplatino , Sondas de ADN , ADN/análisis , Microquímica/métodos , Anticuerpos , Bacteriófago lambda , Southern Blotting , ADN Bacteriano/análisis , ADN Bacteriano/genética , ADN Recombinante/análisis , ADN Recombinante/genética , ADN Viral/análisis , ADN Viral/genética , Estabilidad de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Haptenos , Técnicas para Inmunoenzimas , Hibridación de Ácido Nucleico/métodos , Radioisótopos de Fósforo , Sensibilidad y EspecificidadRESUMEN
The paper describes a sensitive latex hybridization assay (LHA) method applied for indirect detection of biotinylated nucleic acid hybrids immobilized on a synthetic membrane. The biotinylated hybrids were visualized by means of latex particles containing the fluorescent dye pyronine G and coated with streptavidin; 1.6 and 0.3 pg of lambda-phage DNA was detected by dot blot hybridizations on nylon membrane and polyethyleneimine-cellophane, respectively. The assay sensitivity was increased by three orders of magnitude over that with fluorescently labeled probes due to encapsulation of the fluorescent dye in polymer particles. LHA is simple (single-stage detection procedure), fast, and more sensitive than any of the other nonradioactive hybridization methods.
Asunto(s)
ADN/química , Látex , Hibridación de Ácido Nucleico , Coloración y Etiquetado , Acroleína , Proteínas Bacterianas , Biomarcadores , Biotina , Celofán , Colorantes Fluorescentes , Membranas Artificiales , Nylons , Polietileneimina , Polímeros , EstreptavidinaRESUMEN
Ultraviolet irradiation (lambda = 254 nm) of ternary complexes of E. coli 70 S ribosomes with poly(U) and either Phe-tRNAPhe (in the A-site) or NAcPhe-tRNAPhe (in the P-site) effectively induces covalent linking of tRNA with a limited number of ribosomal proteins. The data obtained indicate that in both sites tRNA is in contact with proteins of both 30 S and 50 S subunits (S5, S7, S9, S10, L2, L6 and L16 proteins in the A-site and S7, S9, S11, L2, L4, L7/L12 and L27 proteins in the P-site). Similar sets of proteins are in contact with total aminoacyl-tRNA and N-acetylaminoacyl-tRNA. However, here no contacts of tRNA in the P-site with the S7 and L25/S17 proteins were revealed, whereas in the A-site total aminoacyl-tRNA contacts L7/L12. Proteins S9, L2 and, probably, S7 and L7/L12 are common to both sites.
Asunto(s)
Escherichia coli/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Sitios de Unión , Reactivos de Enlaces Cruzados , Escherichia coli/genética , Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo , Rayos UltravioletaRESUMEN
Direct contacts between 16-S RNA and split proteins S2, S3, S5, S14 and S21 inside the 30-S subunit of Escherichia coli ribosomes were evidenced by the formation of ultraviolet-induced (lambda = 254 nm) RNA-protein cross-links. 30-S subunits were reassembled from core particles and a mixture of split proteins containing in each case a single 125I-labelled protein. All the proteins tested are cross-linked as a result of a single-hit process; proteins S3 and S21 were cross-linked at the highest rate.
Asunto(s)
Escherichia coli/metabolismo , Biosíntesis de Proteínas , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Relación Dosis-Respuesta en la Radiación , Cinética , Peso Molecular , Unión Proteica , ARN Ribosómico/efectos de la radiación , Proteínas Ribosómicas/efectos de la radiación , Ribosomas/efectos de la radiación , Rayos UltravioletaRESUMEN
UV (lambda = 254 nm) irradiation of bacteriophage MS2 or its treatment with bisulfite induce covalent crosslinkage of the RNA to the coat protein. epilsonN-(2-oxopyrimidyl-4)-lysine was found in the phage hydrolysates after either type of treatment. An equimolar mixture of 0-methylhydroxylamine and bisulfite causes complete disappearance of the cross-links. This led to the conclusion that one of the factors responsible for the UV-induced polynucleotide-protein crosslinkage and the main factor in treatment with bisulfite is substitution of the exocyclic amino group of the activated cytosine nucleus by the lysine residue epilson-amino group of the protein.