RESUMEN
Culture conditions were developed that permit efficient incorporation of [(32)P]phosphate into nucleic acids of baker's yeast. RNA sufficiently labeled for sequence determination was obtained in this way. Pure "denaturable" leucine acceptor transfer RNA (tRNA(Leu)(3)) was isolated for this purpose.
Asunto(s)
Ácidos Nucleicos/biosíntesis , Fosfatos/metabolismo , ARN de Transferencia/aislamiento & purificación , Saccharomyces/metabolismo , Cromatografía DEAE-Celulosa , Electroforesis Discontinua , Leucina/metabolismo , Métodos , Desnaturalización de Ácido Nucleico , Isótopos de Fósforo , ARN de Transferencia/análisisRESUMEN
Conditions for laboratory growth of the thermophilic alga Cyanidium caldarium at elevated temperatures have been developed. Growth characteristics of the organisms are described.
RESUMEN
The nucleotide sequence of " denaturable"leucine acceptor transfer RNA (tRNA(Leu)(3)) from baker's yeast was determined on (32)P-labeled material. The molecule is 85 nucleotides long and can be folded into the "cloverleaf" model for secondary structure. The basis on which the sequence was deduced from the products of complete enzymatic digestion, prior to its unambiguous determination, is presented.
Asunto(s)
Nucleótidos/análisis , ARN de Transferencia/análisis , Saccharomyces/metabolismo , Secuencia de Bases , Cromatografía , Electroforesis , Escherichia coli , Código Genético , Leucina/metabolismo , Modelos Estructurales , Desnaturalización de Ácido Nucleico , RibonucleasasRESUMEN
A microsatellite-containing 359-bp restriction fragment, isolated from the rat Pigr gene (murine polymeric immunoglobulin receptor gene) 3'-untranslated region (3'-UTR) and inserted into 3'-UTR or 3' flanking positions in transcription units of supercoiled plasmids, attenuates luciferase reporter gene expression in orientation- and position-dependent ways following transient transfection of human 293 cells. The same fragment stimulates orientation-dependent gene expression in a 5' flanking position. Plasmid linearization abrogates both orientation- and position-dependent responses. Cell-free translation reveals that 5' and 3' flanking expression responses are proportional to increased and decreased luciferase mRNA levels, whereas 3'-UTR expression is associated with control mRNA levels. Hypersensitivity to nucleases S1 and P1, gel mobility differences between supercoiled plasmids carrying opposing microsatellite orientations, and anomalous melting profiles of this fragment are also observed. These results suggest that functional pleiotropy of this fragment depends on the DNA context of its purine-rich microsatellite strand and on DNA supercoiling. Intramolecular triplexes stabilized by supercoiling and secondary structures of purine repeat-rich mRNAs may also confer regulatory properties to similar genomic elements.
Asunto(s)
Regiones no Traducidas 3'/genética , ADN/química , ADN/genética , Regulación de la Expresión Génica , Repeticiones de Microsatélite/genética , Conformación de Ácido Nucleico , Receptores de Inmunoglobulina Polimérica/genética , Regiones no Traducidas 3'/metabolismo , Animales , Secuencia de Bases , Línea Celular , ADN Superhelicoidal/química , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , Elementos de Facilitación Genéticos/genética , Genes Reporteros/genética , Humanos , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Especificidad de Órganos , Plásmidos/química , Plásmidos/genética , Plásmidos/metabolismo , Biosíntesis de Proteínas , Ratas , Reproducibilidad de los Resultados , Especificidad de la Especie , Termodinámica , Transcripción Genética/genéticaAsunto(s)
Conformación de Ácido Nucleico , Fotoquímica , Polinucleótidos/efectos de la radiación , Rayos Ultravioleta , Nucleótidos de Adenina/efectos de la radiación , Bacteriófagos/efectos de la radiación , Ciclobutanos , Nucleótidos de Citosina/efectos de la radiación , Desoxirribonucleótidos/efectos de la radiación , Metilación , Nucleótidos/efectos de la radiación , ARN/efectos de la radiación , ARN de Transferencia/efectos de la radiación , ARN Viral/efectos de la radiación , Efectos de la Radiación , Ribonucleótidos/efectos de la radiación , Ribosomas/efectos de la radiación , Tiouridina/efectos de la radiación , Timidina/efectos de la radiación , Nucleótidos de Timina/efectos de la radiación , Virus del Mosaico del Tabaco/efectos de la radiación , Nucleótidos de Uracilo/efectos de la radiaciónAsunto(s)
ARN , Secuencia de Bases , Sitios de Unión , Escherichia coli , Código Genético , Calor , Matemática , Modelos Moleculares , Conformación de Ácido Nucleico , Oligonucleótidos , Concentración Osmolar , Polinucleótidos , Purinas , Pirimidinas , ARN/metabolismo , ARN Bacteriano , ARN de Transferencia , Sodio , Espectrofotometría UltravioletaAsunto(s)
Polinucleótidos/efectos de la radiación , Rayos Ultravioleta , Dicroismo Circular , Ciclobutanos , Estabilidad de Medicamentos , Cinética , Conformación Molecular , Concentración Osmolar , Fotoquímica , Poli A-U/efectos de la radiación , Poli U/efectos de la radiación , Efectos de la Radiación , Sodio , Espectrofotometría , Nucleótidos de Uracilo , Uridina , AguaAsunto(s)
Nucleótidos de Adenina , Polinucleótidos , Nucleótidos de Uracilo , Fenómenos Químicos , Química , Cinética , Sodio , EspectrofotometríaRESUMEN
A significant level of correction of the mutation responsible for sickle cell anemia has been achieved in monkey COS-7 cells on a plasmid containing a beta-globin gene fragment. The plasmid was treated in vitro with a nucleic acid 'third strand' bearing a terminal photoreactive psoralen moiety that binds immediately adjacent to the mutant base pair. Following covalent attachment of the psoralen by monoadduct or diadduct formation to the mutant T-residue on the coding strand, the treated plasmid was transfected into the cells, which were then incubated for 48 h to allow the cellular DNA repair mechanisms to remove the photoadducts. Upon re-isolation and amplification of the transfected plasmid, sickle cell mutation correction, as determined by sequence analysis of both complementary strands, was established in a full 1%. This result encourages extension of the approach to correct the mutation directly on the chromosome.
Asunto(s)
Anemia de Células Falciformes/terapia , Furocumarinas/genética , Terapia Genética/métodos , Globinas/genética , Plásmidos/farmacología , Transfección/métodos , Anemia de Células Falciformes/sangre , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Aductos de ADN , Reparación del ADN , Ingeniería Genética , Humanos , Mutación PuntualRESUMEN
On the basis of chemical considerations and model building, the Watson-Crick concept of complementary base pairing is extended to a wider range of DNA pairs that A-T and G-C (including A-C, G-T, A-A, G-G and G-A) by invoking imino or enol tautomers (or protonated species) and synisomers. The virtual absence of these additional base pairs from DNA is explained in terms of the low frequency with which these unfavoured forms occur and the two-step mechanism of DNA synthesis, whereby residues are first incorporated by the DNA polymerase and then checked. This base-pairing hypothesis is used to explain the origin, nature and level of spontaneous substitution mutations, their enhancement by base analogues, and the unique effects of certain mutator alleles.
Asunto(s)
ADN , Modelos Biológicos , Mutación , 2-Aminopurina/metabolismo , Bromodesoxiuridina/metabolismo , Fenómenos Químicos , Química , ADN Nucleotidiltransferasas/metabolismo , Replicación del ADN , Exonucleasas/metabolismo , Enlace de Hidrógeno , Moldes GenéticosRESUMEN
Base pairing in codon-anticodon interaction has been investigated in order to understand the basis on which particular base pairs have been selected for or against participation at the wobble position and the basis for codon-anticodon infidelity.
Asunto(s)
Anticodón , Codón , Biosíntesis de Proteínas , ARN Mensajero , ARN de Transferencia , Adenina , Enlace de Hidrógeno , Inosina , Modelos Biológicos , Conformación Molecular , Conformación de Ácido Nucleico , Ribosomas/metabolismo , AguaRESUMEN
Characteristic fluorescence excitation and emission is induced by either acetone-sensitized 313 nm irradiation of mixtures of 8-bromoadenosine and adenosine or 254 nm irradiation of oligo- and polynucleotides containing adenine neighbors. The acetone-sensitized reaction involves cleavage of bromine from 8-bromoadenosine with activation of C-8, leading to formation of an 8,8-adenosine dehydrodimer. Comparable fluorescence properties arise in the unsensitized photoreaction of dApdA, pdApdA, ApA, poly(dA), poly(A), poly(dA.dT), and poly(dA.U). The previously unidentified adenine ultraviolet photoproduct described by Porschke has been isolated as several variants from solutions of pdApdA and poly(dA) irradiated at 254 nm. Based upon fluorescence spectra and mass spectra, these variants are shown to contain the 8,8-adenine dehydrodimer moiety.