Kohji Ueno

Activation of fat body by 20-hydroxyecdysone for the selective incorporation of storage protein in Sarcophaga peregrina larvae

Abstract When haemolymph of third instar larvae of Sarcophaga peregrina was radioiodinated in vitro, most of the radioactivity was incorporated into 75 k storage protein which constitutes about 70% of the total haemolymph protein at this stage. Using radioiodinated haemolyph, the fate of storage protein during metamorphosis was followed. It was found that this protein was selectively incorporated into the fat body in the early pupal stage and that 20-hydroxyecdysone was essential in this process to activate the fat body to incorporate storage protein. This was demonstrated in vitro by incubating larval fat body in the presence of 20-hydroxyecdysone. It was suggested that storage protein was accumulated in large granules in fat body cells that appeared in the cells responding to 20-hydroxyecdysone.

Antibody against a stimulatory factor of RNA polymerase II inhibits nuclear RNA synthesis

THE mechanisms by which eukaryotic genes are differentially activated are mostly unknown. However, the most fundamental process of gene expression is thought to be transcription by RNA polymerase II, and thus the regulation of RNA synthesis by this enzyme is one of the important points of study of eukaryotic gene expression. It is generally believed that structural modification of chromatin governs the transcriptability of the template1, and consequently studies have been carried out on the activity of chromatin2–4. Recently, another possibility has been considered, that various regulatory proteins may be involved in the process of transcription and regulate the gene expression5. However, although there are many reports of proteins stimulating the activity of RNA polymerase II in vitro6–11, no conclusive evidence has yet been obtained to show that these proteins are, in fact, functioning in the process of nuclear RNA synthesis. We have purified a protein from Ehrlich ascites tumour cells that specifically stimulates the activity of RNA polymerase II on homologous DNA12. Named S-II, this is a basic protein with a molecular weight of 40,500. We report here that an antibody against S-II markedly inhibits RNA synthesis in isolated nuclei, but that it does not affect the activity of purified RNA polymerase II. These findings suggest that the processes of RNA synthesis in isolated nuclei and with purified RNA polymerase II and deproteinised DNA are basically different, and that S-II has an essential role in nuclear RNA synthesis.

Identification and characterization of amino acid residues essential for the active site of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureus.

The enzymes essential for bacterial peptidoglycan biosynthesis are attractive targets for antimicrobial drug development. One of these is MurB, which contains FAD as a cofactor and catalyzes the NADPH-dependent reduction of UDP-N-acetylenolpyruvylglucosamine (UDP-GlcNAcEP) to UDP-N-acetylmuramic acid. This study examined the roles of the conserved amino acid residues of Staphylococcus aureus MurB, which are located near the active site in x-ray crystal structures. Seven of 11 site-directed mutated murB genes lost the ability to complement a temperature-sensitive S. aureus murB mutant. Biochemical characterization of the seven mutated MurB proteins revealed that they cannot carry out the reduction of UDP-GlcNAcEP, although they can all catalyze the intramolecular reduction of FAD via NADPH. Spectrometric analyses of the oxidized form of the mutated proteins in the presence and absence of NADP+ or UDP-GlcNAcEP revealed that these essential amino acid residues play four distinct roles in substrate interactions: Arg213 is essential for maintenance of the electronic state of FAD; Arg176 is required for interaction with UDP-GlcNAcEP; His259 is required for interaction with both UDP-GlcNAcEP and NADP+; and Asn71, Tyr175, Ser226, and Glu296 are not apparently required for interaction with either ligand. The results presented here identify for the first time the amino acid residues of MurB that are required for the interaction with UDP-Glc-NAcEP and NADP+.

Cloning and in vitro transcription of the Sarcophaga lectin gene.

A genomic clone for the Sarcophaga lectin gene was isolated. This gene was a compact single copy gene. Two transcription initiation sites were located by S1 nuclease mapping and primer extension. However, transcription from one of these initiation sites was much greater than that from the other site under all conditions in which this gene was expressed. This gene was found to be transcribed efficiently in a nuclear extract of NIH-Sape-4 cells, an embryonic cell line of Sarcophaga synthesizing Sarcophaga lectin constitutively, but not in that of Ehrlich ascites tumor cells. These results suggested that the former extract contains a specific transcription factor(s) for this gene that is not present in the nuclear extract of Ehrlich cells.

Counteraction by 20‐hydroxyecdysone of the effect of juvenile hormone on phosphorylation of ribosomal protein S6

Phosphorylation of ribosomal protein S6, induced when the fat body of Sarcophaga peregrina (flesh‐fly) larvae was incubated in vitro in the presence of 32P, was found to be suppressed by juvenile hormone. This suppressive effect of juvenile hormone was counteracted by a physiological concentration of 20‐hydroxyecdysone. Since 20‐hydroxyecdysone is known to induce phosphorylation of S6 in vivo, this reaction was suggested to be regulated by the balance of the effects of ecdysone and juvenile hormone.

SEPARATION OF A STIMULATORY FACTOR OF RNA POLYMERASE II FROM PROTEIN KINASE ACTIVITY OF EHRLICH ASCITES TUMOR CELLS

The regulation of eukaryotic gene expression is one of the most exciting problems in modern biology. Since Roeder and Rutter reported the multiple forms of eukaryotic RNA polymerase [l] , extensive studies have been done on the purification of these enzymes. Along with the purification of the enzymes, there have been many papers reporting the factors stimulating these enzymes, especially RNA polymerase II [2-61. These factors are expected to play important roles in the regulation of eukaryotic transcription. In order to elucidate the function of these factors, we purified one of these factors, named S-II, from Ehrlich ascites tumor cells [7] . This factor is a basic protein having molecular weight of 38 000 and stimulates only RNA polymerase II and distinctly insensitive to RNA polymerase I. The stimulation cannot be detected when poly [d(A-T) is used as template under the conditions where more than tenfold stimulation is obtained with homologous DNA as template. Recently, Dahmus showed that a stimulatory factor of RNA polymerase II from Novikoff hepatoma cells, named HLF2, contained protein kinase activity [8] . He proposed that phosphorylation of RNA polymerase II by this factor was required for the subsequent stimulation of RNA synthesis. In this paper we report results showing that S-II could not be copuritied with protein kinase and that protein kinase activity was not essential for the stimulation of RNA polymerase II. 2. Methods

Preferential deadenylation of Sarcophaga lectin mRNA during its acute phase expression

The sizes of mRNAs of various defense proteins of Sarcophaga peregrina (flesh fly) were shown to become progressively shorter. We studied the shortening of the Sarcophaga lectin mRNA, because this mRNA shows acute phase expression on injury of the larvae and programmed expression in the pupal stage. Results showed that this mRNA underwent deadenylation when expressed in response to body injury, but no appreciable change in size during programmed expression in the pupal stage. During acute phase expression, the size of the poly(A) tail of the Sarcophaga lectin mRNA decreased from 130 bases to about 30 bases in 6 h. The half-lives of the forms with different sizes of poly(A) tail were almost identical.