Yoshinobu Nakanishi

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.

Invitro transcription of a chromatin-like complex of major core protein VII and DNA of adenovirus serotype 2

Major core protein VII of adenovirus serotype 2 (Ad2) is thought to play a role as a histone octamer in eukaryotic cells. We compared the template activity of the VII-DNA complex formed in vitro with that of protein-free DNA. Hybridization assay of in vitro transcripts showed that transcription from regions located in the middle of Ad2 DNA decreased when Ad2 DNA formed a complex with VII. This suggests that the chromatin structure plays a role in regulation of transcription of the adenovirus genome.

Characterization of three proteins stimulating RNA polymerase II

We have purified three proteins (S-II, S-II’, S-I(b)) that specifically stimulate the activity of RNA polymerase II of Ehrlich ascites tumor cells [ 1,2]. Structural studies showed that S-II’ was a phosphorylated form of S-II [3] and that radioiodinated S-I(b) and S-II’ gave identical peptide maps, whereas the M, of S-I(b) (24 000) was significantly different from that of S-II’ (41 000) [2]. These proteins were found to share common antigenicity and to be located entirely in the nucleoplasm, not in the nucleoli [4]. Antibodies raised against S-II selectively inhibited a-amanitin-sensitive RNA synthesis in isolated nuclei, indicating that these proteins are essential for transcription by RNA polymerase II in vivo [5]. Moreover, when S-II was added exogenously to isolated nuclei from spleen cells of anemic mice, it significantly enhanced the synthesis of globin mRNA as well as cw-amanitin-sensitive RNA synthesis [6]. This paper describes studies on the optimum conditions for stimulation of RNA polymerase II using purified stimulatory proteins and enzyme. Under these conditions, RNA polymerase II and a stimulatory protein are suggested to interact in a molar ratio of 1: 1, and it was possible to obtain significant stimulation with a few nanograms of purified stimulatory proteins. These proteins did not seem to participate in the initiation of RNA synthesis from the promoter region of the adenovirus major late gene, under these conditions.

Stimulation of transcription from accurate initiation sites by purified S-II

The effects of transcription factors S‐II and S‐II′, a phosphorylated form of S‐II, on accurate transcription were compared in a reconstituted transcription system greatly depleted of S‐II. S‐II, but not S‐II′, stimulated the syntheses of run‐off products of various truncated class II genes in this system, suggesting that the activity of this factor is regulated by its phosphorylation and dephosphorylation.

Apparent difference in the way of RNA synthesis stimulation by two stimulatory factors of RNA polymerase II

There are 3 species of RNA polymerase in eukaryotic cells. Among them, RNA polymerase II is known to participate in the transcription of heterogeneous nuclear RNA [l]. However, it is unknown how the enzyme transcribes various genes differently. A hypothesis was proposed [2] that structural modifications of chromatin govern the transcribability of specific genes. Since some evidence has been obtained in support of this hypothesis, it is possible that the state of the template is important in regulation of eukaryotic transcription [3-S]. In addition, specific proteins affecting the activity of RNA polymerase II may regulate eukaryotic transcription. Such proteins have been found in various eukaryotic cells, although their functions are unknown [6-l 11. We have reported two protein factors that stimulate RNA polymerase II of Ehrlich ascites tumor cells [ 121. One of these factors, named S-II, has been purified [ 131 and shown to enhance the formation of the initiation complex with homologous RNA polymerase II and DNA in the presence of nucleoside triphosphates [ 141. This paper describes evidence that, like S-II, the other stimulatory factor, named S-I, specifically stimulates RNA polymerase II, but that the modes of action of the two factors are probably different.

Phosphorylation of S-II is not affected by inhibitors of RNA synthesis

S-II is an essential factor for RNA polymerase II-mediated transcription. A phosphorylated form of S-II, termed S-II has been shown to be present in cells at half the concentration of S-II. In studies on the role of phosphorylation and dephosphorylation of S-II in transcription, the possibility that phosphorylation of S-II is coupled with transcription in vivo was investigated. The phosphorylation of S-II was measured in mouse L cells cultured with two typical inhibitors of RNA synthesis. Neither of these inhibitors, 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and actinomycin D, affected the phosphorylation of S-II under conditions where 75 to 98% of RNA synthesis was inhibited at the initiation and elongation step, respectively. These results indicate that the phosphorylation of S-II and transcription are independent processes.

Robotic pitching by rolling ball on fingers for a randomly located target

This study considers a robotic pitching task with the goal of implementing dynamic manipulation. The strategy used to control the pitching direction of the ball is based on human pitching action, and involves rolling the fingers on the ball at release. The effect of varying the time of release in the pitching direction is analyzed based on ball dynamics. Experiments to test the proposed method featured a high-speed manipulator, composed of a robotic arm with four degrees of freedom (DoFs) and a hand with 10 DoFs, throwing a ball toward a randomly located target recognized by using high-speed vision.