RESUMO
A new design for pulsed field gradient (PFG) gel electrophoresis of large (greater than 50 kb) DNA fragments is described. The method eliminates distortion of migration of DNA because of the geometry of the applied electric field, requires a single power supply and a simple switching device, and is extremely simple to use. Parameters investigated include variation in pulse time, conditions of restriction enzyme digestion and DNA concentration, and the use of different restriction endonucleases. The method has been applied to restriction enzyme digested mammalian DNA from Chinese hamster and human/Chinese hamster hybrid sources and has allowed examination of the minimum separation of two DNA markers known to be on the same human chromosome, chromosome 21.
Assuntos
Enzimas de Restrição do DNA , DNA/isolamento & purificação , Animais , Linhagem Celular , Cricetinae , Cricetulus , DNA Fúngico/isolamento & purificação , Eletroforese/instrumentação , Eletroforese/métodos , Feminino , Humanos , Células Híbridas/análise , Hibridização de Ácido Nucleico , Ovário , Saccharomyces cerevisiae/genéticaRESUMO
The isolation and characterization of a new pyrimidine requiring mutant of Chinese hamster ovary cells (CHO-Kl, pro-) is described. This mutant (P192) shows an absolute requirement for uridine for growth but is fully capable of pyrimidine nucleotide synthesis. P192 cells accumulate exceedingly large amounts of a compound identified by a variety of chromatographic, chemical, and labelling criteria as UDP glucuronic acid (UDPglcUA). This compound comprises approximately 60% of the acid-soluble nucleotides of P192 cells. Possible genetic mechanisms leading to the phenotype of P192 are examined, and the role of UDPglcUA in cellular metabolism is discussed.
