DNA Replication in Permeabilized Cells of Sea Urchin Embryos: (DNA replication/permeabilized embryonic cells/sea urchin).

ABSTRACT

For study of the regulation of DNA replication in sea urchin embryos during the early stages of development, an embryonic cell system that was permeable to exogenously supplied nucleotides was established. Embryos were permeabilized by incubating them in hypotonic buffer containing 0.3 M glucose. The permeabilized embryonic cells maintained their morphological integrity, and synthesized DNA when supplied with exogenous dNTPs. DNA synthesis in these permeabilized embryonic cells required the presence of ATP and three other deoxyribonucleoside triphosphates in addition to labeled dTTP. DNA synthesis was almost completely inhibited by N-ethylmaleimide, and proceeded in a discontinuous fashion. Only cells permeabilized during the S phase could incorporate nucleoside triphosphates into DNA cells permeabilized during other phases did not synthesize DNA. During a 60 min-incubation period, over 10% of the genomic DNA was replicated under the experimental conditions used.