Pyrococcus horikoshii OT3T(= NBRC 100139T), the first organism whose genome was analyzed at the Genome Analysis Center is a hyperthermophilic archaeon that was isolated from a hydrothermal fluid in Okinawa Prefecture in 1992 by the manned deep-sea investigation vessel "Shinkai 2000" in the DeepStar project that was carried out by the Japan Marine Science and Technology Center in collaboration with University of Maryland of U.S.
P. horikoshii OT3T is an obligate anaerobe, harboring such characteristics
as growing at temperatures between 88¡ëC and 104¡ëC with the optimal
temperature of 98¡ëC in the presence of sulfur, and possessing circular
genomic DNA. The proteins and enzymes produced by this hyper-thermophilic archaeon have outstanding heat resistance and may, therefore, be utilized in various industrial fields such as chemistry, food, medical supplies, etc.
Analysis of the 1.74 MB genome of this archaeon revealed the presence of only one set of genes for 16S and 23S rRNA and two genes for 5S rRNA, while it is likely to harbor 11 genes/ORFs encoding intein-containing proteins and two tRNA genes with introns. Most genes/ORFs were conserved in Pyrococcus abussi, Pyrococcus furiosus, and Thermococcus kodakaraensis, only 3% of the 1,883 genes/ORFs assigned appear to be organism-specific.
2007-04-06 ..... 1
Comparative genomic data of pyrococcal genomes was released
We analyzed the relationships among all genes/ORFs between Pyrococcus horikoshii OT3 and the following organisms and the results were incorporated into the DOGAN.
The nucleotide sequence of the P. horikoshii OT3 genome was determined as described below. This was the first attempt of genome analysis at NITE which was started in August 1996 and finished in February 1998.
The genome of P. horikoshii was completely re-annotated in March 2007. See the summary below.
The physical map of the P. horikoshii OT3 (= NBRC 100139) genome was constructed with three restriction enzymes, Srf I, Sse8387 I and Not I, to facilitate alignment of the fosmid clones used for sequencing.
Fosmid clone library
The genomic DNA was prepared in agarose, partially digested with Hind III and size-fractionated by electrophoresis to obtain fragments of approximately 40-kb in size, which were then inserted into the pBAC108L fosmid cloning vector. A total of 1,248 clones were isolated.
The purified DNA from each of the fosmid clones was sonicated to different extents, size-fractionated and cloned into the HincII site of pUC118. The clone library thus obtained was subjected to nucleotide sequencing with dye-primer or dye-terminator sequencing kits and a 373XL or 377XL DNA sequencer from ABI.
The raw sequence data were collected and assembled into contigs using Phred-Phrap programs and edited with Sequencher program. The remaining gaps between the resultant contigs were filled with end-to-end PCRs.
Validation of the nucleotide sequence data
The genomic DNA was used as the template for PCR amplification of 15-20 kb-long overlapping segments by designing primers based on the determined nucleotide sequence. The resultant PCR products were subjected to restriction enzyme digestion and the sizes of individual fragments obtained were compared with those of computer-generated fragments for comparison. The final genomic nucleotide sequence thus determined was 1,738,505 bp long.
ORF Assignment and Annotation (re-annotation)
ORFs were predicted by using gene-finding programs Glimmer ver. 2.0 and GeneHacker followed by extensive manual curation.
The deduced protein sequences were subjected to BLASTP (Altschul et al., 1997) search against Swiss-Prot/TrEMBL database and were analyzed by InterPro program (Mulder et al., 2003).
Signal peptides were predicted by SignalP program (Bendtsen et al., 2004), and transmembrane regions were predicted by TMHMM program (Krogh et al., 2001).
Ribosomal RNA sequences were predicted by Infernal software with Rfam database, then modified according to the homology among archaeal rRNA, especially in Pyrococcus abyssi, Pyrococcus furiosus and Thermococcus kodakaraensis.
Transfer RNAs were first predicted by using tRNAScan-SE and Infernal software with Rfam database.
Small RNAs were quoted from snoRNA database (Omer et al., 2000) including three sRNAs (Gaspin et al., 2000).
Other noncoding RNAs were predicted by using Infernal software with Rfam database.
Summary of the P. horikoshii Genomic Data
The genomic data of P. horikoshii was changed in this release (March 2007) as follows.
Genomic size: 1,738,505 bp -> 1,738,506 bp (single bp insertion at position 1,257,711)