Characteristics of w ^hd81b9  mutant demonstrate its M’ cytotype.

Khromykh, Yu. M., E.R. Varentsova, S.V. Sarantseva, and L.V. Kotlovanova.   Petersburg Nuclear Physics Institute, Russian Academy of Sciences, Gatchina 188300, Russia. E-mail: khromykh@omrb.pnpi.spb.ru.

      Induced by hybrid dysgenesis, the w ^hd81b9 mutation of Drosophila melanogaster (abbreviated w^hd) was derived from the Harwich P strain and selected by Rubin et al. (1982). It was characterized as a null allele with a 0.5-kb P-element insert in exon 6 of the white gene. The mutant was stable when kept in P cytotype but reverted at a high rate (4´10^-3) when kept in M cytotype, suggesting that an intact P element was present elsewhere in the genome of mutant strain.

      First, the number of P element insertions in the w^hd genome was determined by nonradioactive in situ hybridization technique (Ashburner, 1989) with the use of Vectastain ABC Kit (Vector Labs, USA). As illustrated by Figure 1, chromosomes of w^hd flies contain a big set of P elements.

      Second, germ line and somatic cells (larval imaginal discs and nervous ganglia) of w^hd strain were tested in the presence of  P repressor. For this purpose we applied the P-lacZ expression technique developed by Lemaitre and Coen (1991). P-lacZ constructs in which the Esherichia coli lacZ gene is fused in-frame with P element transposase gene was originally designed for the detection of genomic regulatory elements (O’Kane and Gehring, 1987) and they function as a reporter of P promoter activity. It was shown by Lemaitre and Coen (1991)  that P-lacZ insertions have their expression repressed by P cytotype.

      To construct the w^hd, P-lacZ strain, we used the ABOO strain obtained from Stephane Ronsseray. Chromosome 2 of ABOO flies contains an insertion of P-lacZ element, (P[lac, ry⁺]A)  that is expressed in germ line and soma (Lemaitre et al.,1993). One copy of this element was introduced into the w^hd genome by proper crosses of w^hd flies with flies of ABOO  genotype. Similar crosses were performed with the Lk-P(1A) strain obtained from Stephane Ronsseray, in order to construct a Lk-P(1A), P-lacZ strain. The latter was used as the strain with known characteristics: it was shown by Ronsseray et al. (1991), that Lk-P(1A) strain fully represses germ line P element activity but does not fully repress transposase activity produced by an in vitro modified P element called P[ry⁺ ∆2-3]99B (abbreviated ∆2-3). Ovaries of adult females and larval neural ganglia and imaginal discs were dissected and stained for ß-galactosidase activity as described by Lemaitre and Coen (1991). Results of staining are presented in Figure 2.

Table 1.  P element insertions are mainly distributed on the autosomes and show some cell-to-cell variations. chromosome number of P insertions X 2 2L 11-12 2R 9-11 3L 7-9 3R 10-12 4 0 Total number: 39-46

     Judging from the results of X-gal staining, Lk-P(1A), P-LacZ strain, indeed, fully represses ovarian P-element activity and permits it on the middle level in somatic cells. At the same time, P-lacZ strain demonstrates a high level of P element activity independently from the type of cells.  Because the w^hd strain does not contain P repressor despite the large number of P element insertions in the chromosomes, its cytotype may be classified as M’. Evidently, this strain contains defective P-elements and the role of intact P elements inserted elsewhere in the genome is unclear.

      Third, w^hd strain cytotype was revealed by a test on the appearance of P element induced somatic mosaics. To do it, females of the w^hd  genotype were crossed to males having the first chromosome with the yellow (nonessential to this report) and white markers and the third chromosome with the kinked marker and  ∆2-3 source of P-transposase (y¹ w¹; Ki¹ ∆2-3 strain was obtained from the Bloomington Stock Center, Bloomington, Indiana University). Females and males of w^hd strain itself and the F₁ generation of the mentioned cross were analysed in respect of red mosaic spots on the eyes. No mosaic eyes were registered among females and males of w^hd genotype. F₁ females showed the mosaic eyes at a rate 7% while the lack of mosaics was showed by males. This fact demonstrates that P element induced somatic mosaics arise as response of w^hd  M’ cytotype to the action of Δ2-3 P transposase. The differences between F₁ females and males are in good agreement with similar differences established by Engels et al. (1990) under analysis of w^hd germ line reversion and can be explained by the action of the same pairing dependent repair mechanism.

Figure 2.  Photomicrographs showing  X-gal staining of ovaries (above) and larval neural ganglia and imaginal discs (bottom) from the Lk-P(1A), P-lacZ and whd, P-lacZ strains.

      Acknowledgments: We thank Stephane Ronsseray for providing ABOO and Lk-P(1A) strains.  This work was supported by a grant from Russian Foundation of Basic Researches.

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