Molecular characteristics of spontaneous deletions in the hyperthermophilic archaeon Sulfolobus acidocaldarius

Abstract

Prokaryotic genomes acquire and eliminate blocks of DNA sequence by lateral gene transfer and spontaneous deletion, respectively. The basic parameters of spontaneous deletion, which are expected to influence the course of genome evolution, have not been determined for any hyperthermophilic archaeon. We therefore screened a number of independent pyrimidine auxotrophs of Sulfolobus acidocaldarius for deletions and sequenced those detected. Deletions accounted for only 0.4% of spontaneous pyrE mutations, corresponding to a frequency of about 10(-8) per cell. Nucleotide sequence analysis of five independent deletions showed no significant association of the endpoints with short direct repeats, despite the fact that several such repeats occur within the pyrE gene and that duplication mutations in pyrE reverted at high frequencies. Endpoints of the spontaneous deletions did not coincide with short inverted repeats or potential stem-loop structures. No consensus sequence common to all the deletions could be identified, although two deletions showed the potential of being stabilized by octanucleotide sequences elsewhere in pyrE, and another pair of deletions shared an octanucleotide at their 3' ends. The unusually low frequency and low sequence dependence of spontaneous deletions in the S. acidocaldarius pyrE gene compared to other genetic systems could not be explained in terms of possible constraints imposed by the 5-fluoroorotate selection.

FIG. 1.

Deletions and other sequence features of the S. acidocaldarius pyrE gene. Horizontal open bars on the genetic map depict coding sequences of the pyrB, pyrE, and pyrF genes of S. acidocaldarius; arrows show direction of transcription. Open bars above the map show the size and location of spontaneous deletions (designated in italics). All other numbers identify nucleotide positions in the pyrE coding sequence (GenBank accession number Y12822). Positions of the first and last nucleotides removed are given above the genetic map on either side of the open bars. Regions of potential stem-loop structures, numbered in order of decreasing thermodynamic stability, are indicated above the map. The first and last nucleotides of these regions (in roman numerals) are as follows: I, 200 to 217; II, 133 to 153; III, 67 to 88; IV, 528 to 541; V, 481 to 508, VI, 340 to 350. Regions which have been duplicated by various spontaneous mutations are represented by the trapezoidal symbols below the genetic map. Sites of frequent mutation are shown as partial sequences in parentheses below the map; numbers indicate the position of the first nucleotide in each region. More-detailed information on the gene sequence and mutations are provided in reference 15.

FIG. 2.

Octanucleotides corresponding to novel joints. As a convenient way to show the relative positions of the sequences, the upper strand of the wild-type pyrE gene is drawn annealed to the octanucleotide in the lower strand that represents the novel joint of the corresponding deletion. This method of depiction is that used in other deletion studies (11, 12, 16, 30) and is not a proposal of a specific mechanism. Sequences remaining after deletion are shown in bold type, as for Table. 1. Numbers with vertical lines indicate the nucleotide position in each strand with respect to the pyrE coding sequence; the remaining numbers indicate the sizes of the loops shown.