Jun-Ichiro Mukai

Action of silkworm endonuclease on oligoribonucleotides terminating in 3′-phosphate

Abstract Studies have been carried out on the action of purified silkworm endonuclease on oligonucleotides of various chain lengths separated from a pancreatic ribonuclease (EC 2.7.7.16) digest of RNA. 1. 1. Dinucleotides were totally resistant. 2. 2. Trinucleotides of the type PupPupPyp were slowly and only partially hydrolysed as follows: PupPupPyp → PupPu+pPyp 3. 3. Tetranucleotides of the type PupPupPupPyp were completely hydrolysed by 2 alternative mechanisms as follows: 4. 4. Pentanucleotides of the type PupPupPupPupPyp gave the same types of terminal fragments as above and pPupPupPyp from the 3′-phosphate end, and also pPupPu from inside the chain. 5. 5. On digestion with silkworm nuclease, the unfractionated pancreatic ribonuclease digestion products gave a pattern identical to that from the pentanucleotide fraction except that pPupPupPyp was missing. 6. 6. The original positions of all of these fragments in the corresponding substrates could be located from which they arose. 7. 7. On the basis of these results, the possible usefulness of silkworm endonuclease as a reagent in the base sequence analysis of polynucleotides and also for determination of the base specificity of nucleases of the 3′-phosphate-forming type is discussed.

NAD- and FAD-3′-pyrophosphates — enzymic synthesis and inertness

We have shown [ 1,2] that ATP:nucleotide pyrophosphotransferase (EC 2.7.6.4) purified from Streptomyces adephosphoZyticus [3,4] catalyses the transfer of the S’y several 5’-diphosphonucleosidic coenzymes such as NAD, FAD, GDP-choline and ADPglucose; short oligonucleotides; tRNA and G and a cap structure found at the 5’-termini of eucaryotic mRNA. These products of 3’-pyrophosphorylation may be supposed to have lost or altered their initial biological activity. In the present experiments, we isolated and identified the transfer prod-. ucts formed from NAD and FAD, i.e., their 3’-pyrophosphoryl derivatives (at the adenosine moiety), then found these products to be devoid of the respective cofactor and inhibitory activities.

l-Leucine, a natural inhibitor of silkworm larval nuclease

Abstract In a comparison of larval and pupal alkaline endonucleases of silkworm, extracts from pharate adults were found to inhibit specifically larval but not pharate adult nuclease activity with DNA but not RNA as the substrate. An inhibitor was purified from pharate adult extracts and identified as l -leucine. Kinetic analyses disclosed an uncompetitive inhibition mechanism and formation of a ternary complex of the larval nuclease, l -leucine, and DNA.

Synthesis of deoxynucleoside-5'-tri-3'-diphosphates by Streptomyces adephospholyticus ATP:nucleotide pyrophosphokinase.

Abstract ATP: nucleotide pyrophosphokinase (EC 2.7.6.4.) of Streptomyces adephospholyticus catalyzes an efficient transfer of the 5′-β,γ-pyrophosphoryl group of dATP to the four common deoxynucleoside-5′-triphosphates at their 3′-OH positions in the alkaline pH and in the presence of Co 2+ ions, giving the respective 3′-pyrophosphoryl derivatives. Deoxyadenosine-5′-tri-3′-diphosphate was chromatographically prepared and structurally characterized.

Effects of purine and pyrimidine nucleoside 5′-di(tri)phosphate-3′-diphosphates on the Escherichia coli cell-free transcription and translation activity

The pleiotropic roles of (p)ppCpp as a unique effector or a signal molecule in stringent control of bacteria have been well studied [l-3]. Occurrence and special function of its adenine analogues (p)ppApp were suspected in possible relation to the bacterial growth and sporulation [3-71. We have studied the effects of the 8, namely cytidine and uridine as well as the above 2 purine nucleoside S’-di(tri)phosphate3’-diphosphates on the wheat germ cell-free eukaryote mRNA translation system and found that each of them exerts various and specific effects, either positive or negative, depending on the mRNAs employed [8]. Here, we have tested the effects of these 5’,3’polyphosphonucleotides (p)ppNpp on the bulk RNA and protein synthesis by Escherichia coli cell-free coupled transcription-translation and uncoupled poly(U)-directed translation systems. (42 Ci/mmol), [U-14C]leucine (339 mCi/mmol) and [U-‘4C]phenylalanine (521 mCi/mmol) were purchased from the Radiochemical Centre (Amersham).

A biliprotein from the digestive juice of Bombyx Mori L. Its purification and partial structural study of the chromophore

Abstract 1. 1. A blue-colored, red-fluorescent protein was purified from the digestive juice of silkworm, Bombyx mori L. 2. 2. The chromoprotein and its chromophore show well-defined absorption peaks at 278 and 605 mμ, and 495 and 575 mμ respectively. 3. 3. The chromoprotein and chromophore were negative to Gmelin, Ehrlich, diazo- and Carr-Price tests but were positive to the chromium oxide-chlorine-benzidine test for pyrrole. 4. 4. Chemical and spectral results obtained, together with the previous findings mentioned in the text, suggest that this chromoprotein is a biliprotein, probably formed in the midgut of the silkworm from the chlorophyll lipoprotein complex of mulberry leaves.

Species-specificity of nucleotide composition of ribonucleic acids from different silkworms

Abstract Highly polymerized ribonucleic acids were prepared by a phenol method from the posterior silk glands of larvae of a domestic silkworm, Bombyx mori L., and four species of wild silkworms: Antheraea pernyi Guer., Dictyoploca japonica Moore, Philosamia cynthia ricini Donovan, and Philosamia cynthia pryeri Butler. Paper chromatographic analysis after hydrochloric acid hydrolysis revealed that the nucleotide composition of ribonucleic acid is species-specific and subspecies-nonspecific.

Several nucleoside-3' and/or 5'-polyphosphates stimulate β-galactosidase induction in escherichia coli

Streptomyces nucleotide 3’-pyrophosphokinase catalyzes the transfer of 5’+y-pyrophosphoryl group of either ATP, dATP or pppApp to 3’-OH of acceptor nucleotide’s leading to the formation of an unusual variety of nucleoside polyphosphates [ 1,2]. Among the unusual nucleotides, ppGpp has received much attention. It is thought to be a signal molecule in bacterial metabolism [3] and to act both as a positive and a negative effector of several specific operons [4-81. ppGpp inhibits the key enzymes in several metabolic pathways [9,10]. Moreover, (p)ppApp, @)ppGpp and some other highly phosphorylated nucleotides may be the regulators in bacterial differentiation or early sporulation [l l-131. pppdApp, GPP, PPGPP, PPCPP, PPUPP as web as CAMP and cGMP stimulate fl-galactosidase induction at the cellular level. We presuppose the natural occurrence of pppdApp, ppCpp and ppUpp in addition to the already known natural occurrence of (P)PPGPP [16J71, (P)PPAPP 1171 and PPGP P81. Guanosine-3’-diphosphate (Gpp) was included here as it is known to be a natural amplification factor of glucocorticoid-induced operon transcriptive action in rat liver system [ 191.

Comparison of larval and pupal alkaline endonucleases in silkworm, Bombyx mori L.

Abstract 1. 1. Larval and pupal nucleases are equally activated by Mg ions, inhibited at a high ionic strength and attack both DNA and RNA endolytically, producing 5′-phosphoryl oligonucleotides. 2. 2. Their optimal pHs are 10·3 and 9·0 respectively. 3. 3. The two nucleases are separable on polyacrylamide gel electrophoresis. 4. 4. The two nucleases are immunologically similar. 5. 5. Their approximate molecular weights are 22,000 and 86,000 respectively. 6. 6. Conversion or switching of larval to pupal nuclease during development is discussed.