Bungo Sakaguchi

Egg-yolk trypsin inhibitor identical to albumen ovomucoid

Abstract Two preparations of trypsin inhibitor were purified from yolk, one from unincubated hens eggs and the other from those at day 16 of incubation. Both were indistinguishable from albumen ovomucoid with respect to molecular weight, isoelectric point, peptide map after limited proteolysis, immunological reactivity and so forth. Although a gap in specific activity was observed, it was concluded that the yolk inhibitors were species of ovomucoid. This suggested a flow of ovomucoid from egg white to yolk during embryonic development.

Purification and characterization of a novel chymotrypsin inhibitor controlled by the chymotrypsin inhibitor A (Ict-A) gene from the larval hemolymph of the silkworm, Bombyx mori

Abstract 1. 1. On polyacrylamide gel electrophoresis, chymotrypsin inhibitors in the larval hemolymph of the silkworm were found as 15 electrophoretically distinct bands. 2. 2. One of them, CI-13 migrating fastest toward the anode, was purified from the hemolymph. 3. 3. The inhibitor was a monomeric protein with a molecular mass of 14,000 and showed stability for both heat and a wide range of pH. The isoelectric point was pH 4.1. 4. 4. CI-13 was able to inhibit chymotrypsin completely and trypsin slightly, but was ineffective against other proteinases such as papain, ficin, carboxypeptidase A, V8 proteinase, serratia peptidase, cocoonase and proteinases derived from gut juice of the silkworm.

Nucleolar size in parallel with ribosomal RNA synthesis at diapause termination in the eggs of Bombyx mori.

The eggs of Bombyx mori, both in diapause and nondiapause, were subjected to cytological examination of nucleoli and measurement of RNA precursor incorporation (2 hours) into ribosomal RNA. In diapause eggs, the nucleoli were very small and the rate of ribosomal RNA synthesis was the lowest of the samples tested. Most cells in diapause possessed nuclei with one nucleolus. In contrast, the eggs activated from diapause by long chilling attained the largest size of nucleoli and the highest rate of ribosomal RNA synthesis. A significant proportion of the cell nuclei still had only one nucleolus at this stage. Three days after activation, the eggs exhibited intermediate levels in both the size of nucleoli and the rate of ribosomal RNA synthesis. At this stage, about half of the egg cell nuclei had two nucleoli.

RNA content and RNA synthesis in diapause and non-diapause eggs of Bombyx mori

Abstract Changes in amount of RNA and its synthesis were studied in diapause and non-diapause eggs of Bombyx mori. In non-diapause eggs, the amount of total RNA began to increase at the stage of late gastrula and increased constantly thereafter. The synthesis of total and ribosomal RNA commenced at the onset of gastrulation, and after two days it levelled off. In contrast, the amount of total RNA in diapause eggs did not change essentially, and only a low level of total and ribosomal RNA synthesis was detected. On the other hand, if diapause eggs were activated by hot HCl treatment 24 hr after oviposition, the results obtained were essentially similar to those for non-diapause eggs, except that there was a lag of two days after acid treatment. These findings indicate that a significant control of ribosomal and other RNA occurs in relation to diapause and development.

Does the rate of ribosomal RNA synthesis vary depending on the number of nucleoli in a nucleus

Abstract Cultured kidney cells of Xenopus laevis were pulse-labeled with [ 3 H]uridine for 10, 20 and 30 min during their logarithmic growth phase and then processed for autoradiography. The labeled cells were assigned into two categories, one- and two-nucleolated cells, and the rate of ribosomal RNA (rRNA) synthesis was measured by counting the number of grains in nucleoli. The results obtained revealed that a two-nucleolated cell incorporated significantly much more radioactivity into its nucleoli than did a one-nucleolated partner for all the periods examined. Cells of these different nucleolar types, however, contained essentially the same amount of rDNA (DNA complementary to rRNA) as estimated by in situ hybridization with [ 125 I]rRNA. Although it remains to be proved that the observed increase in incorporation represents the increased rate of rRNA synthesis in two-nucleolated cells, the present findings seem to be very interesting, since they might indicate that the activity of rRNA genes is in some way regulated or affected by their spatial relationship in a cell nucleus.

Translation of RNA from Eggs During Post-Diapause Development of Bombyx mori

Eggs of Bombyx mori are aroused from diapause by long‐term chilling and develop when transferred to 25°C. During the first 20 hr of post‐diapause development, the polysome content and the presumed rate of protein synthesis increase about 3‐fold, while the ribosome content and the total RNA content increase only 1.1‐fold. In this study, total RNAs were extracted from chilled eggs (termed 0 hr of development), and post‐diapause eggs at 10 and 20 hr of development. The RNAs were purified further by high pressure liquid chromatography to remove RNA‐like oligonucleotides. On translation in a protein‐synthesizing system derived from wheat germ with a subsaturating amount of RNA, no difference was found in the relative amounts of translatable mRNA activity at 10 and 20 hr of development from that at 0 hr. Moreover, the translation products of the different RNA preparations in a rabbit reticulocyte lysate system appeared very similar when separated by gel electrophoresis and located by fluorography. These facts suggest that protein synthesis in early post‐diapause development is controlled at a translational level.

Differential changes in nucleolar size and ribosomal RNA synthesis during diapause break by prolonged chilling in Bombyx eggs

The activity of ribosomal RNA (rRNA) genes as judged by nucleolar size and rRNA synthesis has been shown to depend upon the phase of diapause in the eggs of Bombyx mori. In the present study, we found that nucleolar size in diapausing eggs was enlarged at a very early stage during cold treatment, a procedure necessary for the termination of diapause. In contrast, the intrinsic capacity of ribosomal RNA synthesis in the chilled eggs, as examined at 25°C by radioactive precursor incorporation into rRNA, increased much later, in parallel with the break of diapause. The early phase of cold treatment is the period when the eggs undergo some important changes (the so-called diapause development), preparing for diapause termination. Thus we infer that the above mentioned increase in nucleolar size may be one of the features of diapause development.

Presence of translatable mRNA in diapause eggs of Bombyx mori

Abstract Total RNA extracted from diapause eggs of Bombyx mori was found to be translatable in a cell-free heterologous protein synthesis system derived from wheat germ or rabbit reticulocyte lysate. This supports the idea that active mRNAs are contained in the polysomes of diapause eggs.

Changes in polysome content during development after diapause of Bombyx mori embryos

Undegraded polysomes could be extracted from eggs of Bombyx mori by cutting egg shells with a blade in a buffer containing high salt. The polysome content as measured by this method increased steeply during post‐diapause development, which was commenced by long term chilling followed by hot HCl treatment of diapausing eggs. At the 24th hr of the post‐diapause development the polysome content became 2.6‐times the level at 0 h. The alteration of the polysome content paralleled that of the incorporation of [14C]leucine into acid‐insoluble fractions investigated by modified Takamis embryonic culture system.

INSTABILITY OF MESSENGER AND RIBOSOMAL RNA IN A GLUE-PROTEIN MUTANT OF Bombyx mori

The mucous glands of Bombyx pupae secrete glue proteins which attach deposited eggs to the mounting sheet. A mutant of a dominant gene, named no glue (Ng), produces nonadhesive eggs which have a low capacity for glue‐protein synthesis. In the present study it was shown that the mucous glands of Ng silkworms showed rapid degradation of mRNA as well as rRNA during development; this may cause the low capacity for glue‐protein synthesis in the mutant organ. In contrast, the mucous glands of normal silkworms showed a significant increase in content of RNAs until the maximum rate of glue‐protein synthesis was achieved. The degradation of RNA in the Ng mucous gland was inhibited by actinomycin D injected into the body fluid. Thus it is supposed that the Ng gene codes for a presumptive controller RNA, which would be the mediator of RNA instability in the mucous glands of Ng pupae.