| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1989: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1988: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
Normal barley strain (Nami Akashinnriki) and dwarf strain (uzu Akashinriki) were grown under the dark. The coleopitles were harvested on day 3, when the growth rate of the normal strain was ca. two times as fast as that dwarf strain. mRNA was extracted and purified from the coleoptile segments (1 cm in length, excised from 5 mm below the tip). mRNA thus obtained was introduced into the in vitro translation system using wheat germ extracts with radio labeled ^<35>S-methionine or ^3H-leucine. The transcripts were dissolved on two-dimensional gel electrophoresis. The comparison of the fluorogram of the normal strain with that of the dwarf strain revealed that there were 11 species of proteins distinctly appeared in the transcripts from mRNA of the nominal strain, but were not or little transcribed from mRNA of the dwarf strain.
IAA was added to the in vitro translation system to see the effect of exogenously applied IAA on the translation process. IAA did not affect the translation in stra
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in uzu either using methionine or leucine, while in stmin Nami, IAA substantially inhibited the incorporation of labeled leucine into the proteins but not the incorporation of methilnine. The unexpected results remained to be answered.
As reported previously, strain uzu produced less IAA than strain Nami. To see which step for IAA bioshynthesis was impeded in strain uzu, crude enzyme preparation was extracted from the coleoptile segments of uzu and Nami. IAA was produced from D-tryptophan in both strains. The production was inhibited by D-cycloserine (an inhibitor of D-transaminase), suggesting that IAA is produced through D-tryptophan in both strains. Next, racemase activity which converts L-trp to D-trp was assayed. pH optimum for the racemase of Nami was 7.6 while that of uzu was over 9.7. The difference of the optimum pH for the racemase between Nami and uzu strongly suggested that the amino acid composition of racemase of uzu was different from that of Nami, and DNA sequence for racemase of uzu was also different from that of Nami. The isolation of the racemase was now undertaken by ion- exchange and gel permeation chromatography. Purified enzyme and its amino acid composition will afford a great facility for the analysis of dwar gene of uzu. Less
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