Archaeon Methanococcus Voltae JORDAN KONISKY,* SUZANNE M

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JOURNAL OF BACrERIOLOGY, Oct. 1994, p. 6402-6403 Vol. 176, No. 20 0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology The DNA Polymerase Gene from the Methanogenic Archaeon Methanococcus voltae JORDAN KONISKY,* SUZANNE M. PAULE, MARIA E. CARINATO, AND JANICE W. KANSY Department ofMicrobiology, University of Illinois, Urbana, Illinois 61801 Received 23 June 1994/Accepted 16 August 1994

Previous studies have identified intervening sequences that encode homing endonucleases within the genes encoding several archaeal DNA polymerases. We report the sequence of the gene encoding the DNA polymerase of Methanococcus voltae and describe evidence that it lacks analogous intervening sequences.

Recent studies have identified the presence of in-frame littoralis DNA polymerases (1,311 and 1,702 amino acids, insertions in the structural genes encoding the DNA poly- respectively), the lengths of the mature forms of these polymerases of the archaeon Thermococcus littoralis (7, 10) and merases are more similar (775 and 774 amino acids, respec- Pyrococcus species strain GB-D (17). In both cases, the pri- tively) to that of the M. voltae translation product. Multiple- mary translation product is processed to yield an internal alignment analysis of the amino acid sequences derived for the protein(s) (termed the intein[s] [11]) and the active mature mature forms of the Pyrococcus species strain GB-D and T. protein formed by the joining of the external sequences littoralis enzymes and the presumptive primary translation (termed the extein [11]). Similar protein-splicing events have products of the M. voltae, Sulfolobus solfataricus (882 amino been described for the production of the mature 69-kDa acids [12]), and P. furiosus (775 amino acids [14]) DNA subunit of the vacuolar ATPase of Saccharomyces cerevisiae (3, polymerases by using the University of Wisconsin Genetics 4, 6, 8) and Candida tropicalis (5) and the RecA protein from Computer Group PILEUP program led to the alignments Mycobactenium tuberculosis and Mycobacterium leprae (2). In shown in Fig. 1. In addition, there were many regions of each of these systems, the inteins have sequence similarity to sequence identity and similarity elsewhere in the proteins (data homing endonucleases, and in a few cases endonuclease activ- not shown). While the PILEUP program generated gaps ity has been directly established (13). interspersed throughout the full sequences, the gap lengths To explain the finding that alleles without inteins are found were small. For example, for the M. voltae sequence, 22 gaps in related species, it has been suggested that inteins may spread were generated and the average gap size was 3.5 residue through lateral transfer (13). Thus, the pattern of specific equivalents (range, 1 to 11 residues). In contrast, alignment of intein types among species can provide information on the the precursor forms of both the Pyroccocus species strain history of genetic exchange between species and, in addition, GB-D and T littoralis enzymes with the sequences derived by provide a perspective on the evolution of genome content and the direct translation of the M. voltae, P. furiosus, and S. organization. solfataricus genes yielded major gaps that corresponded exactly We report the DNA sequence of a DNA polymerase gene to the positions of the two inteins found in the T littoralis from a methanogen, a representative of the Euryarchaeota and the one intein identi- branch of the domain Archaea (16). We identified the Meth- primary translation product (7, 10) an analysis of fied in the Pyrococcus species strain GB-D precursor protein anococcus voltae DNA polymerase gene through (17). The results of our analysis are consistent with the an open reading frame immediately upstream of the methano- of P. gen S-layer structural gene (9). A search of protein sequence previous report that the DNA polymerase gene furiosus databases by the National Center of Biotechnology Informa- does not contain intervening sequences of the type observed in tion Blastp program (1) yielded highest sequence similarities to T. littoralis (14). the DNA polymerases of Pyrococcus furiosus, Pyrococcus spe- With the University of Wisconsin Genetics Computer Group cies strain GB-D, and T littoralis (identity to each, 45%). The BESTFIT program, a direct comparison of the amino acid complete sequence of the M. voltae DNA polymerase gene can sequence of each of the intein endonucleases derived from the be obtained from GenBank (accession no. L33366). intervening sequences found in the T littoralis and Pyrococcus The a-type DNA polymerases have characteristic inter- species strain GB-D DNA polymerase genes to the M. voltae, spersed regions that are thought to comprise functional do- S. solfataricus, and P. furiosus DNA polymerases did not mains (15). These motifs are easily identified in the M. voltae identify significant amino acid sequence similarity in any DNA polymerase and are present in the same order as that region of these proteins. While region I of the M. voltae observed in a-type enzymes (Fig. 1). For comparison, similar enzyme has a stretch of 22 amino acids that is absent in regions for the human a-type DNA polymerases are shown. the other archaeon enzymes, it is not known if this region is While the length of the presumptive primary translation present in the active DNA polymerase. However, the region product of the M. voltae gene (823 amino acids) differs lacks the motif that has been suggested to promote protein substantially from those of the primary translation products of splicing (7). the genes encoding the Pyrococcus species strain GB-D and T. Thus, neither the M voltae, S. solfataricus, nor P. furiosus DNA polymerase gene harbors intervening intein sequences of the type reported for the Pyrococcus species strain GB-D and * Corresponding author. T. littoralis DNA polymerase genes. Therefore, endonuclease- 6402 VOL. 176X1994 NOTES 6403

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