EFFECT OF GIBBERELLIC ACID ON ELONGATION AND NUCLEIC ACID SYNTHESIS IN ETIOLATED ALASKA PEA EPICOTYL

The following results were obtained using etiolated Alaska pea epicotyls. Gibberellic acid (GA) had the remarkable effect on the elongation in part I (elongating region) of epicotyls, whereas it had little effect on that in part II (mature region) of epicotyls. In cortex of part I and II of epicotyls, the cell number in longitudinal direction was hardly affected by GA. On the increase in width of epicotyls, GA was hardly effective in any parts of epicotyls. In both part I and II GA enhanced the incorporation of ³²P into all nucleic acid fractions prepared by methylated albumin kieselguhr (MAK) columns, i.e. sRNA, DNA and rRNA + mRNA. In part I the net increase in DNA and RNA content during the incubation period was slightly promoted by GA, whereas in part II the net decrease in both nucleic acids content was slightly promoted by GA. The relationship between GA-induced growth and nucleic acid synthesis is discussed.     

MOUSE MUSCLE CELL DIFFERENTIATION IN CLONAL CULTURE

Clonal culture of muscle cells from thigh muscles of the term fetus of the mouse was undertaken. Myoblasts were spherical or spindle-shaped, and proliferated exponentially until day 4 in culture. The generation time of the muscle cells from 2 to 4 days' culture was 9.1 to 13.4 hr. The fusion of myoblasts began on day 4 in culture; many myotubes had been formed by day 6 and spontaneous contraction was observed on day 7. Clonal efficiency was 30%, and the proportion of muscle colonies in all the colonies was 72%.     

Purification of GVBD-Inducing Protein from the Ciliate Tetrahymenathermophila

Germinal-vesicle-breakdown (GVBD) was induced if a 132,000-g supernatant of Tetrahymena thermophila homogenates was injected into Xenopus oocytes. Using this induction of GVBD as a bioassay system, a GVBD-inducing substance was purified from the Tetrahymena by ultra-filtration, liquid chromatography, and electroelution from a band on native-PAGE gel. Proteins eluted from the single band on the native-PAGE gel induced GVBD in the absence of oocyte protein synthesis. This band resolved into two bands on SDS-PAGE: 60 and 112 kDa. The 60 kDa protein was the active fraction inducing GVBD. Immunoprecipitation of the 60 kDa protein prevented the GVBD-inducing activity, supporting the conclusion that the 60 kDa protein is the GVBD-inducing substance. An immunoblot with anti-60 kDa monoclonal antibody and PSTAIR antibody showed that p13suc1-beads could remove cdc2 homologues from T. thermophila supernatant but could not remove the GVBD-inducing activity. The 60-kDa protein appeared at the same time as micronuclear division and disappeared at the beginning of the macronuclear division during synchronous cell division. The cyclic appearance of the 60-kDa protein in the T. thermophila cell cycle suggests that this protein has a cell cycle function.     

Metabolism of Round Spermatids: Pyruvate cannot Maintain the ATP Level

Round spermatids (steps 1–8) were isolated from rat testes and the effect of pyruvate on their intracellular ATP level was examined. Results showed that although the spermatids consumed a considerable amounts of pyruvate, this substrate alone did not maintain their ATP level. However, their ATP level was maintained in the presence of both pyruvate and α-ketovalerate or α-ketobutyrate. Maintenance of the ATP level by these substrates was associated with electron trasnport and oxidative phosphorylation. α-Ketoacid inhibited pyruvate reduction to lactate in the lactate dehydrogenase (LDH) reaction, but increased pyruvate oxidation to CO₂. The NADH level in spermatids was too low to be detectable, but the NAD level remained unchanged in the presence of pyruvate and α-ketovalerate. These results suggest that pyruvate by itself is not an adequate energy-yielding substrate for spermatids and that a high NADH/NAD ratio may be essential for maintenance of their ATP level.