Khromykh, Yu. M., E.R. Varentsova, S.V. Sarantseva, and L.V. Kotlovanova. 2002. Characteristics of w hd81b9  mutant demonstrate its M’ cytotype. Dros. Inf. Serv. 85: 37-40.

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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:

      Induced by hybrid dysgenesis, the w hd81b9 mutation of Drosophila melanogaster (abbreviated whd) 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.


Figure 1.  Photomicrograph showing  total number of  P element hybridization signals on the polytene chromosomes of  whd strain.  Some of signals are indicated by arrowheads.

      The whd81b9 strain used by our laboratory was obtained from the Mid America Stock Center (Bowling Green, Ohio). Based on the results of the above-mentioned work we supposed that this whd strain has M’ cytotype and may be used as a source of nonautonomous P elements to induce the somatic dysgenesis in the experiments with the use of repair-defective mutants. To check this supposition some additional characteristics of whd strain were studied.

      First, the number of P element insertions in the whd 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 whd flies contain a big set of P elements.

      Second, germ line and somatic cells (larval imaginal discs and nervous ganglia) of whd 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 whd, 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 whd genome by proper crosses of whd 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.


number of P insertions













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 whd 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, whd strain cytotype was revealed by a test on the appearance of P element induced somatic mosaics. To do it, females of the whd  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 whd 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 whd 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 whd  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 whd 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.

       References:  Ashburner, M., 1989, Drosophila: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, USA;  Engels, W.R., D.M. Johnson-Schlitz, W.B. Eggleston, and J. Sved 1990, Cell 62: 515-525;  Lemaitre, B., and D. Coen 1991, Proc. Natl. Acad. Sci. USA 88: 4419-4423;  Lemaitre, B., S. Ronsseray, and D. Coen 1993, Genetics 135: 149-160;  O’Kane, C.J., and W.J. Gehring 1987, Proc. Natl. Acad. Sci. USA 84: 9123-9127;  Ronsseray, S., M. Lehmann, and D. Anxolabéhère 1991, Genetics 129: 501-512;  Rubin, G.M., M.G. Kidwell, and P.M. Bingham 1982, Cell 29: 987-994.