RESUMEN
Hb G-Philadelphia, an alpha-globin allele, is expressed as either 20%, 30%, or 40% of the total hemoglobin. Restriction analyses published thus far have shown that among persons with 30% and 40% hemoglobin (Hb) G the alpha G allele is seen only in a single-locus haplotype. We now report the identification of a second haplotype in which the alpha G allele is found in tandem with an alpha A allele. This haplotype has been found present in DNA from the members of one family in which Hb G is expressed as 20% of the total hemoglobin, determined by both cellulose acetate electrophoresis and high-performance liquid chromatography (HPLC). Synthesis was balanced in all individuals. The identification of a variant alpha-globin allele in two distinct haplotypes presents the possibility of independent mutation. However, an alternative explanation cannot be ruled out; namely, that the original allele may have become distributed among the two haplotypes by unequal crossing-over.
Asunto(s)
Alelos , alfa-Globulinas/genética , Mapeo Cromosómico , Regulación de la Expresión Génica , Hemoglobinas Anormales/genética , Intercambio Genético , ADN/genética , Femenino , Humanos , Masculino , Mutación , Hibridación de Ácido Nucleico , LinajeRESUMEN
The incorporation into a 7% polyacrylamide gel of a nucleic acid-specific photochemical reagent 4,5',8-trimethylpsoralen at a concentration of 1 mg/dl of acrylamide solution improves both the sensitivity and the efficiency of the transfer of plasmid DNA fragments from the gel onto nitrocellulose filters. This improvement allows detection by blot hybridization of DNA fragments as small as 57 bp long.
Asunto(s)
ADN/aislamiento & purificación , Desoxirribonucleasas de Localización Especificada Tipo II , Plásmidos , Colodión , Enzimas de Restricción del ADN , Electroforesis en Gel de Poliacrilamida , Microquímica , Hibridación de Ácido Nucleico , PapelRESUMEN
Only certain DNA fragments are taken up efficiently by component Haemophilus cells; this implies that efficient uptake requires the presence of a specific nucleotide sequence on the incoming DNA (Sisco and Smith, 1979). To determine the structure of this "uptake site", we have isolated and sequenced four small fragments of cloned H. parainfluenzae DNA which retain the ability to be taken up by cells. These fragments have a sequence of eleven base pairs in common, 5'-AAGTGCGGTCA-3' and ethylation of certain phosphoryl groups in this sequence causes significant decreases in fragment uptake. We conclude that this is the sequence of the uptake site.
Asunto(s)
ADN/metabolismo , Haemophilus influenzae/metabolismo , Transformación Genética , Secuencia de Bases , Transporte Biológico Activo/efectos de los fármacos , Clonación Molecular , Enzimas de Restricción del ADN , Escherichia coli/genética , Etilnitrosourea/farmacología , Haemophilus/genética , Haemophilus influenzae/genética , Plásmidos , Transformación Genética/efectos de los fármacosRESUMEN
Haemophilus cells efficiently take up Haemophilus DNA from the medium during transformation but do not take up other DNAs. To study the mechanism of this specificity we have cloned an 8.1-kilobase (kb) fragment of H. parainfluenzae DNA in the escherichia coli--pBR322 host--vector system and reisolated the DNA fragment for use as a defined probe. The 5'32P end-labeled 8.1-kb DNA is efficiently absorbed by competent Haemophilus cells whereas vector DNA present in the mixture is not, implying that the 8.1-kb DNA contains sequence-specific recognition sites that are needed for DNA uptake. Absorbed DNA can be recovered from cells as a 32P-labeled duplex of unaltered size for several minutes after uptake. We have determined the number and location of uptake sites in the 8.1-kb DNA by constructing a restriction endonuclease cleavage map and assaying fragments for uptake. Only two small fragments retain the ability to be absorbed. These fragments, 120 and 140 base pairs long, are 3.8 kb apart on the 8.1-kb fragment. We assume that each of these fragments contains a short common sequence, perhaps 8--12 base pairs long, that is the actual recognition site. We have shown by DNA competition assays, with the 8.1-kb DNA as a standard, that about 600 copies of the uptake sites are present in the Haemophilus genome.