The halobacterial insertion element ISH28.

A characteristic feature of the halophilic archaeon Halobacterium halobium (now termed Halobacterium salinarium) is the high genetic variability due to frequent transposition events. The organism contains a number of insertion sequences (ISH1, ISH2, ISH11, ISH23/ISH50, ISH24, ISH26, ISH27, ISH28, ISH SI, and ISH1.8) most of which have been isolated from the bacterioopsin {bop) gene region of purple membrane mutants (1, 2, 5, 6, 7). Many of these elements are characterized at the DNA sequence level (1 -10). Their size varies from 520 bp determined for ISH2 up to 3000 bp found for ISH24 (Table 1). Copies of ISH1 and ISH2 characterized so far are identical to the respective ISH1 or ISH2 element. In contrast, copies of ISH26 or ISH27 constitute a family with related though not identical members (6, 8). The ISH28 insertion element described in this report was isolated from the bacterio-opsin {bop) gene region of the purple membrane mutant O6 (7). This mutant derives together with other Bop mutants from Bop revertant reIV-4, that occurred at a frequency of 10~ (7). The parental Bop mutant of reIV-4 contains an ISH24 insertion element in the 3' region of the brp gene which is located upstream of the bop gene and transcribed from the opposite DNA strand (Figure 1A). Both genes are involved in purple membrane synthesis (11). The genetic basis for the restoration of the purple membrane synthesis in the revertant reIV-4 is the insertion of the 588 bp pHH 1 sequence termed 'ISH25' adjacent to the ISH24 (see Figure 1A) (5, 7). Bop mutant 06 incurred an additional insertion element, ISH28, upstream of the bop gene (7). Here we report the isolation and DNA sequence determination of the ISH28 insertion element. A 1.5 kbp BgH fragment containing the ISH28 element together with 406 bp of bop gene sequence was used to determine the integration site of the element near the bop gene by dideoxy sequencing. The integration of ISH28 occurred 120 bp upstream of the bop mRNA start site (see Figure IB). The element is flanked by an 8 bp direct repeat of target DNA duplication. This region upstream of the bop gene incurred other ISH-elements in various Bop mutants: ISH2 in mutant SD19 (2), ISH23 in mutant IV-10 (5), and ISH27 in mutant 05 (7, 8). The target DNAs of inserts in these mutants are indicated in Figure IB. Subfragments of the 1.5 kbp BgK fragment were used to determine the entire ISH28 sequence. The element is 932 bp in size and contains a 16 bp perfect terminal inverted repeat with no similarity to inverted repeats of other halobacterial insertion elements (Table 1). The ISH28 sequence is available in the EMBL DNA data base. No similarity was found with previously characterized halobacterial insertion elements (1-10) . H.halobium contains several copies of ISH28; one is present on the 150 kbp plasmid pHHl (7). REFERENCES