Abstract
The amounts of the various forms of DNA polymerase (alpha 1, alpha 2, beta, and gamma) have been determined in oocytes, eggs, and embryos of the frog, Xenopus laevis. During oogenesis the relative proportions and absolute levels of all forms changed dramatically. In stage I (early) oocytes, DNA polymerase-gamma, the "mitochondrial" polymerase, was the predominant form. During oocyte growth, DNA polymerase-alpha 1 and -alpha 2 increased by more than 100-fold, DNA polymerase-beta by 15-fold, and DNA polymerase-gamma by only 8-fold. During oocyte maturation and ovulation, the levels of all forms of DNA polymerase roughly doubled. The mature stage VI oocyte contained 5 orders of magnitude more DNA polymerase activity than is found in an individual somatic cell. DNA polymerase-alpha 1 and -alpha 2, the "replicative" polymerases, were the predominant forms in mature oocytes and ovulated unfertilized eggs. During fertilization, the relative proportions and absolute levels of the four forms remained constant. During subsequent stages of embryogenesis, the total amounts of DNA polymerase-alpha 1 and -alpha 2 declined slightly from cleavage through gastrulation, the stages of most rapid chromosomal DNA replication. The rapid increase in cell number during early embryogenesis establishes the same levels of DNA polymerase/cell as are present in adult somatic cells. After neurulation, the absolute levels of DNA polymerase-alpha 1 and -alpha 2 increased in proportion to increases in cell number. The absolute levels of DNA polymerase-beta remained constant, and the levels of DNA polymerase-gamma increased 2-fold throughout embryogenesis.
Highlights
DNA polymerases which have distinct subcellular localizationsand which replicate specific DNAs at specific times during development [1,2,3,4,5]
Our results suggest that the presumptive replicative DNA polymerase-al and -a2are synthesized at very high levelsand stored throughout oogenesis, in the absence of chromosomal
It should be noted that there is no chromosomal DNA replication throughout oogenesis, ribosomal DNA amplification is observed in stage I and I1 oocytes
Summary
DNA Polymerase-cu Activity-Assays contained 50 mM Tris-HC1 (pH 8.0), 0.1 mM EDTA, 200 pg/ml bovine serum albumin, 6 mM MgC12,1 mM dithiothreitol, 25 mM NaC1,25 mM KCl, 10% glycerol (v/v), 200 p~ poly(dT), 50 p~ oligo(rAlo),and 100pCi/ml [3H]dATP (1000 dpm/pmol) in a total volume of 50 pl. DNA Polymerase-0 Activity-Assays contained 50 mM Tris-HC1 (pH 8.9), 30 pg/ml bovine serum albumin, 0.8 mM MnC12,200 mM KCl, 50 p M poly(rA), 25 p M oligo(dT12-18)ar nd 100 pCi/ml [3H]dTTP (1650dpm/pmol) in a total volume of 100 pl. After correcting for inhibition, the absolute number of DNA polymerase molecules of a given type per oocyte, egg, or embryo can be calculated from these values since the assays used in this paper are identical to those used during the purification to homogeneity of the four X . We assume that DNA polymerase(Y activities and DNA polymerase-a protein levels are proportional over the ranges tested
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