Defining Genes in the Genome of the Hyperthermophilic Archaeon Pyrococcus furiosus : Implications for All Microbial Genomes

Abstract

The original genome annotation of the hyperthermophilic archaeon Pyrococcus furiosus contained 2,065 open reading frames (ORFs). The genome was subsequently automatically annotated in two public databases by the Institute for Genomic Research (TIGR) and the National Center for Biotechnology Information (NCBI). Remarkably, more than 500 of the originally annotated ORFs differ in size in the two databases, many very significantly. For example, more than 170 of the predicted proteins differ at their N termini by more than 25 amino acids. Similar discrepancies were observed in the TIGR and NCBI databases with the other archaeal and bacterial genomes examined. In addition, the two databases contain 60 (NCBI) and 221 (TIGR) ORFs not present in the original annotation of P. furiosus. In the present study we have experimentally assessed the validity of 88 previously unannotated ORFs. Transcriptional analyses showed that 11 of 61 ORFs examined were expressed in P. furiosus when grown at either 95 or 72°C. In addition, 7 of 54 ORFs examined yielded heat-stable recombinant proteins when they were expressed in Escherichia coli, although only one of the seven ORFs was expressed in P. furiosus under the growth conditions tested. It is concluded that the P. furiosus genome contains at least 17 ORFs not previously recognized in the original annotation. This study serves to highlight the discrepancies in the public databases and the problems of accurately defining the number and sizes of ORFs within any microbial genome.

Parallel evolution of transcriptome architecture during genome reorganization

“…Altogether, the revised genome annotation includes an additional 127 ORFs that were not reported in the original GenBank submission (Poole et al 2005). Our tiling array analysis has validated transcription of at least 58 of these ORFs and discovered an additional 12 novel transcripts, of which two are antisense to annotated ORFs (Fig.…”

“…This newly discovered gene is interspersed between and cotranscribed with MMP1635, a redoxactive disulfide protein, and MMP1637, a hypothetical protein. It should be noted that PF0736.1n was previously believed to be a bona fide gene based on RNA hybridization to a PCR-based double-stranded microarray (Poole et al 2005). This example showcases the value of strand-specific analysis.…”

“…As is the case for most genome annotations, the number of putative ORFs in the Pfu genome has been a subject of debate (Poole et al 2005); the current GenBank annotation (RefSeq) includes 60 additional ORFs, such as PF0736.1n. Altogether, the revised genome annotation includes an additional 127 ORFs that were not reported in the original GenBank submission (Poole et al 2005).…”

Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus : Characterization of a Coenzyme A- Dependent NAD(P)H Sulfur Oxidoreductase

“…Total RNA was extracted from cell extracts of P. furious by use of acid-phenol (46) and stored at Ϫ80°C until needed. The design and construction of DNA microarrays containing all of the predicted 2,192 open reading frames (ORFs) in the annotated genome of P. furiosus (37), preparation of cDNA from the RNA samples, and hybridization experiments were all performed as previously described (46). Fluorescently labeled cDNA was prepared using an ARES DNA labeling kit (Molecular Probes, Eugene, OR).…”

Structure determination of a new protein from backbone‐centered NMR data and NMR‐assisted structure prediction

“…50 The refinement procedures described should be applicable to a significant fraction of the 60% judged to be small enough for study by NMR (35% of the genes in the P. furiosus genone (767 of 2198) are predicted to encode proteins of less than 20 kDa). 51 Excluding membrane proteins (20%) and those that are too large for NMR, about 17% of the total genome should have conventionally identified homologs that can be refined using the methods described (60% 3 0.80 3 0.35 ¼ 17%). While the single application we have presented does not allow an estimate of the success rate in finding homologs for the remaining 40% of the genome, the large number of new protein structures that fall into existing fold families would suggest it to be high.…”