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Nucleic Acids Research, 1981, Vol. 9, No. 22 5905-5916
© 1981

MOLECULAR BIOLOGY

Localization of putative transcription initiation site on the cloned rDNA fragment of Tetrahymena pyriformis

Torn Higashinakagawa*, Hidetoshi Saiga, Naoko Shintani, Masako Narushima-Iio and Takashi Mita

Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health 1-1 Iseigaoka, Yahata Nishi-Ku, Kitakyusyu 807, Japan

*To whom correspondence should be addressed.

Received September 3, 1981. A DNA fragment (1.4 Kb) which codes for 5' region of 35S ribosomal precursor RNA (pre-rRNA) in Tetrahymena pyriformis was cloned with pBR322. The fragment was cleaved from the central part of the palindromic rDNA with restriction endonuclease Kpnl and Hindlll, and ligated to the larger moiety of pBR322 DNA-Hindlll-BamHI fragment together with {lambda}DNA-KpnI-BamHI fragment through trimolecular ligation. The analysis of R-loop formed between Kpnllinearized recombinant plasmid and 35S pre-rRNA revealed a DNA:RNA hybrid region of 465±30 base pairs in length. Considering the contraction of DNA: RNA hybrids relative to DNA duplexes (Philippsen et al. , J. Mol. Biol., 123, 387–404, 1978), the size of the hybrid region was corrected to about 490 base pairs. Alternatively, the size of DNA which was protected againstnuclease SI due to hybrid formation with 35S pre-rRNA was estimated to be 490 nucleotides long. These data indicate that the transcription initiation site is localized at about 490 base pairs from the Hindlll site of the cloned rDNA fragment.


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