Cloning of the Escherichia coli gene for the stringent starvation protein |
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| Authors: | Ryuji Fukuda Ryoji Yano Toshikazu Fukui Toshiharu Hase Akira Ishihama Hiroshi Matsubara |
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| Affiliation: | (1) Department of Biochemistry, Institute for Virus Research, Kyoto University, Sakyo-ku, 606 Kyoto;(2) Faculty of Pharmaceutical Sciences, Osaka University, 565 Osaka;(3) Department of Biology, Faculty of Science, Osaka University, 560 Osaka, Japan;(4) Present address: Department of Molecular Genetics, National Institute of Genetics, 411 Mishima, Japan;(5) Present address: Department of Biochemistry, Research Laboratories, Dainippon Pharmaceutical Co, LTD, 564 Osaka, Japan |
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| Abstract: | Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with 32P at their 5 -termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP stringent starvation protein - PTH phenylthiohydantoin |
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