Reduced DNA polytenization of a minichromosome region undergoing position-effect variegation in Drosophila

Abstract

Molecular analysis of a Drosophila minichromosome, Dp(1;f)1187, revealed a relationship between position-effect variegation and the copy number reductions of heterochromatic sequences that occur in polytene cells. Heterochromatin adjacent to a defined junction with euchromatin underpolytenized at least 60-fold. Lesser reductions were observed in euchromatic sequences up to 103 kb from the breakpoint. The copy number changes behaved in all respects like the expression of yellow, a gene located within the affected region. Both copy number and yellow expression displayed a cell-by-cell mosaic pattern of reduction, and adding a Y chromosome, a known suppressor of variegation, increased both substantially. We discuss the possibility that changes in replication alter copy number locally and also propose an alternative model of position-effect variegation based on the somatic elimination of heterochromatic sequences.

Figure 1. Origin and Genetic Structure of Dp1187

The origin of the ln(1)sc⁸ progenitor chromosome from a normal X chromosome and its subsequent derivitization to produce Dp1187 is diagrammed schematically (not drawn to scale). See text and Lindsley and Grell (1968) for further details. A genetic map showing the ten known complementation groups from the X tip that remain on Dp1187 is drawn below. Heterochromatin is shown as a stippled box and euchromatin as a solid line.

Figure 2. Molecular Structure of Dp1187

(A) The restriction map of Dp1187 was compiled using maps and cloned DNAs from the first 130 kb of euchromatin and by Southern blot analysis similar to that shown in (B). The solid line is euchromatin and the stippled box is heterochromatin. Sites of the rare-cutting enzymes Notl (N) and Sfil (S) are indicated near the top. Additional sites may be present if the chromosome extends beyond position −320 (dashed line). Only the restriction sites flanking the sc⁸ breakpoint (coordinate 0) are shown for the following enzymes: X (XhoI), H (HindIII), E (EagI), B (BamHI), R (EcoRI), V (EcoRV), NC (NcoI), G (BgIII), and Sa (SaII). The locations of the erect wing (ewg, coordinates −92 to −100 kb), yellow (y, −20 to −25 kb), and achaete (ac, −10 to −13 kb) genes are indicated (Campuzano et al., 1985; Fleming et al., 1989). (B) DNA inserts were prepared from imaginal discs and brains of sibling third instar larvae (X/O males) either lacking (Dp−) or containing (Dp+) one copy of Dp1187. Inserts were subjected to pulse-oriented gel electrophoresis either uncut (U) or following digestion with XhoI (X) or Notl (N), and the DNA was transferred and hybridized with a probe from the scute region (pBSscXR3.7). Electrophoresis conditions were 120 V, 104 s pulse at 8.5°C for 3.5 days.

Figure 3. Position-Effect Variegation of yellow⁺ in Wing Triple-Row Bristles

A segment of the triple row at the anterior margin of the wing is shown for the following males. (a) Wild type. (b) yellow mutant (y¹). (c) Dp1187 without Y chromosome (X,y/O; Dp1187 y⁺; ry⁵⁰⁶). Arrows indicate y⁺ bristles. (d) Dp1187 with Y chromosome (X,y/Y; Dp1187 y⁺; ry⁵⁰⁶). Arrow points to a y bristle. Magnification, ×738.

Figure 4. Underpolytenization of Dp1187 at Sites Near the sc⁸ Breakpoint

A map of the region of Dp1187 flanking the sc⁸ breakpoint is shown at the top using the kb coordinates of Figure 2. Four regions where probes were used to determine DNA copy numbers are shown, along with local restriction site polymorphisms that were used to distinguish bands derived from the normal X(X) and Dp1187 (Dp). Below each map are corresponding lanes from Southern blots containing DNA from the predominantly diploid imaginal discs and brain cells (DB) or from highly polytene salivary gland cells (SG) isolated from the same larvae. The probe used for each blot is indicated by a heavy line on the map above along with the enzyme used; the origin and size in kb of the labeled fragments are also given (arrows at the right). The rightmost blot was from a pulse-oriented electrophoresis experiment; electrophoresis conditions were 120 V, SO s pulse at 8.5°C for 2 days. The other blots utilized standard electrophoresis. All DNAs were isolated from X/O;Dp1187 male larvae.

Figure 5. Underpolytenization Responds to a Modifier of Position-Effect Variegation

A Southern blot similar to those in Figure 4 is shown. DNA was prepared from disc and brains (DB) or salivary glands (SG) of male larvae containing Dp1187 and either a normal Y chromosome (XY) or lacking a Y chromosome (XO). DNAs were digested with BgIII and probed with the −1.9 kb region probe shown in Figure 4. The location and size of fragments deriving from the normal X and from Dp1187 are indicated.

Figure 6. Cell-to-Cell Variation of Dp1187 Labeling In Situ

In situ hybridization was carried out to salivary gland polytene chromosomes from Dp1187-bearing male larvae containing or lacking a Y chromosome. A sample of the results is shown from a preparation hybridized with the −54 kb probe shown in Figure 4. Labeling of Dp1187 (large arrow) was not detected in (a), was intermediate in (b), and was approximately equal in (c) to labeling of the X tip (small arrow). All three nuclei were from a single individual of the genotype X/O;Dp1187 Magnification, ×467.

Figure 7. Dp1187 Labeling In Situ Depends on Probe Location and the Presence of a Y Chromosome

Data from grain counts of polytene chromosomes labeled by one of the three euchromatic probes was tabulated. Each of the three probes was hybridized to chromosomes from Dp1187-bearing male larvae containing (X/Y) or lacking (X/O) a Y chromosome. In each case the ratio of grains associated with Dp1187 to those associated with the normal X was calculated after subtracting for background. The ratios (r) were grouped into four classes: 1.0>r>0.7, 0.7>r>0.3, 0.3>r>0.1, and 0.1>r. The percent of nuclei falling in each class is plotted for the six combinations and displayed at the top of the bars. N, the total number of nuclei.