High genetic instability of heterochromatin after transposition of the LINE-like I factor in Drosophila melanogaster

Abstract

In the present work, we have asked whether a group of 13 essential genes mapping to the heterochromatin of Drosophila melanogaster chromosome 2 are mutable following transposition of the I factor during I-R hybrid dysgenesis. We found that the frequency of lethal events mapping to chromosome 2 heterochromatin is surprisingly high, despite the low density of genetic functions identified in this region compared with euchromatin. Cytogenetic and molecular analyses indicated that the recovered mutations correspond either to insertions or to rearrangements. Moreover, chromosomes bearing specific heterochromatic lethal mutations were generated by recombination in the heterochromatin. Together, these data indicate that I factors transpose with high frequency into pericentric regions of chromosome 2 and may play a role in the evolution of constitutive heterochromatin.

Figure 1

Cytogenetic map of chromosome 2 heterochromatin, showing the extent of the I-R-induced deficiencies along the heterochromatin of the right arm. Thirteen cytologically distinct regions, designated h35 to h46, can be resolved in chromosome 2 heterochromatin (24). Filled areas represent the Hoechst 33258-bright regions; the shaded boxes represent regions of intermediate fluorescence, and the open boxes are regions of dull fluorescence. Only the heterochromatic regions are shown. Above the diagram are bars indicating the mapping of 13 vital heterochromatic genes. The genes in the left arm of chromosome 2 (2Lh) are indicated from top to bottom according to their left to right mapping along the chromosome. The extent of the I-R-induced deficiencies was determined cytogenetically as described in detail elsewhere (24, 30) and is represented by thick lines (below). 2L, left arm; 2R, right arm; C, centromeric region.

Figure 2

(A) Southern hybridization of genomic DNA from the Charolles parental strain (Ch) carrying the rl⁺ allele, and from rl^IR1 homozygotes (rl^IR), digested with EcoRI and probed with the 1.4-kb rl cDNA probe (30); black arrows indicate the bands whose mobility varies in the rl^IR1 mutant and in the Charolles strain. (B) Filters were stripped and reprobed with I sequences; the open arrow indicates I-homologous sequences in the dysgenic mutants that comigrate with rl homologous sequences. (C) Southern hybridization of genomic DNA from Canton-S (C) and Charolles (Ch) parental stains carrying the rl⁺ allele, and from rl^IR1 homozygotes (rl^IR), digested with EcoRV and probed with the HindIII–DraI fragment. (D) PCR amplification reaction from genomic DNA of Canton-S (C), Charolles (Ch), and rl^IR1 strains. A 982-bp fragment was specifically amplified from genomic DNA of rl^IR1 homozygotes. M, molecular weight marker. (E) Northern blot assays of second-instar larvae total RNA from rl⁺, rl^IR1, and Df(2Rh)Rspⁱ¹ homozygotes lacking the rl gene. The 1.4-kb rl cDNA probe reveals two major transcripts of 2.6 kb and 1.9 kb, both absent in larvae homozygous for Df(2Rh)Rspⁱ¹. The same filter was reprobed with the rp49 ribosomal protein gene probe as a loading control.

Figure 3

Schematic representation of the I factor insertion in the rl gene. The white and shaded lines represent 125 bp of the rl exon 2, and 394 bp of the rl intron 2 respectively; the filled line represents the 5′ end of the I factor. Dashed lines represent rl^IR1 genomic sequences and I factor sequences that were not included in the 982-bp PCR fragment. The I factor insert is not in scale.