Information extracted from genomes of three hyper-thermophilic archaea
Yutaka Kawarabayashi
Biotechnology Center, National Institute of Technology and Evaluation, Shibuya, Tokyo 151-0066, JAPAN
In my Institute, for several years the genome projects of hyper-thermophilic archaea have been performed for identification of enzymes and thermostable proteins useful for industry, and for obtaining the information for mechanism of thermostability of proteins in thermophilic microbial. At present, sequencing of entire genomes of three hyper-thermophilic archaea were completed. The first target was anaerobic hyper-thermophilic euryarchaeon, P. horikoshii OT3 which was isolated from a hydrothermal vent at Okinawa Trough in 1992, possessing the 1,738,505 bp-long and 41.9% G+C containing genome. This archaeon had 2,061 ORFs, one 16S-23S rRNA cluster and 46 tRNA genes.1.) The second target was aerobic hyper-thermophilic crenarchaeon, A. pernix Kl which was isolated from solfotaric thermal vent in Kodakara-Jima Island in 1993, containing 1,696,695 bp-long and 56.3% G+C containing genome. On this genome 2,694 ORFs, one 16S-23S rRNA cluster and 47 tRNA genes were isolated.2.) An aerobic thermoacidophilic crenarchaeon, S. tokodaii strain7 which was isolated from Beppu hot springs, was the third target of our thermophilic genome project. This genome possessed the 2,694,756 bp-long and very low G+C content, 32.8%, genome. On this genome 2,911 ORFs, one 16S-23S rRNA cluster and 45 tRNA genes were identified.3.) Approximately 23-31% of all ORFS were assigned their functions, 20-29% had similarity to hypothetical protein assigned on the other genomes.
These information and additional information were already opened in the internet homepage of our Institute. (URL; http://www.bio.nite.go.jp/E-home/genome_list-e.html)
To extract specific features of these archaeal genomes, comparative analysis among these three archaea and against all archaea determined of the entire genomic sequences was performed. On the genomes of M. jannaschii and M. thermoautotrophicum, two 16S-23S rRNA clusters were identified, but on other species just one 16S-23S rRNA cluster was isolated. Although M. jannaschii and M. thermoautotrophicum had just less than 40 tRNA genes, other archaea contained over 45 tRNA genes. In addition to the result of comparison of organization of RNA genes, and the result of comparative analysis of protein-coding genes indicated that P. horikoshii OT3 and A. pernix K1 were more close than the positions in the phylogenic tree. The comparative analysis will be one of the big target of research when using entire genomic data.
For application of these genomic data, the libraries of plasmid clones containing 2kb-long genomic DNA fragment which cover each ORFs were constructed. These clones were used for expression of thermostable proteins in E. coli. With collaboration to many laboratories in University, several number of gene products were synthesized in E. coli, and the some 3D structure were already dissolved. I would like to summarize these expression analysis in this symposium.
Among 47 tRNA genes in A. pernix's genome and 45 tRNA genes in S. tokodaii's genome, the 11 and 21 tRNA genes were identified as intron-containing tRNA genes, respectively. To confirm these estimations, we have tried to clone these tRNA molecules present in living A. pernix K1 cells. The cDNA molecules, which were amplified from tRNA molecules by RT-PCR, were cloned and analyzed the sequences of inserts. From these data it was indicated that both of intron-containing and mature forms of predicted tRNA molecules were actually present in living A. pernix K1 cells.
1) DNA Res. Vol.5, No.2, 55-76 & 147-155 (1998)
2) DNA Res. Vol.6, No.2, 88-101 & 148-155 (1999)
3) in preparation
