| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1993: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
1.We have cloned three genes, RRN3, RRN6, and RRN7 involved in the transcription of 35S rRNA genes by RNA polymerase I in Saccharomyces cerevisiae, From deduced amino acid sequences, RRN3 appcars to encode a protein of 627 amino acids, RRN6 to encode a protein of 894 amino acids, and RRN7 to encode a protein of 514 amino acids. Standard gene disruption experiments showed that RRN3, RRN6, and RRN7 are essential genes. By use of immunoaffinity purification and biochemical fractionation, we obtained a highly purified preparation (Rrn6/Rrn7 complex), which consisted of Rrn6p, Rrn7p, and another protein with an apparet molecular weight of 66,000. In vitro transcription experiments showed the Rrn6/Rrn7 complex is required for the formation of a transcription -competent preinitiation complex. The role of Rrn3p in the transcription process ia still unclear. However, a overproduction of Rrn3p in S.cerevisiae inhibits cell growth, suggesting that Rrn3p traps some essential protein.
2.Schizosaccha
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romyces pombe cDNA bank in S.cerevisiae expression vector and Kluveroyces lactis genomic DNA bank, respectively, were used to complement S.cerevisiae rrn3, rrn6, and rrn7 mutants to isolate functional homologues. We have found SRN31 and SRN32 obtained from S.pombe cDNA bank can complement temperature-sensitive rrn3 mutation, determined the complete nucleotide sequence of SRN31 and found that SRN31 encodes a protein of 434 amino acids with a calculated molecular weight of 50,200. There is no apparent homology between Rrn3p and Srn31p. SRN33 derived from K.lactic can also complement temperature-sensitive rrn3. We have not analyzed SRN32 and SRN33 yet.
3.We attempted to transcribe the functional 35S ribosomalRNA from RNA polymerase II promoter in S.pombe, We fused nmt1 promoter to one unit of rDNA to construct a nmt1-35S rDNA fusion gene. The fusion gene on a multicopy vector was transformed into a temperature-sensitive RNA polymerase I mutant (nuc1) of S.pombe. However, the growth of transformants was not observed at a high temperature, indicating that the amount rRNA supplied from nmt1 promoter is not enough to complement nuc1 mutation. Less
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