A comparative genetic analysis of fourth chromosome vs. pericentric PEV. (A) Photographs of male progeny from crosses between females carrying a PEV reporter (as in Figure 1) and the following males: 1, y w^67c23 (wild type); 2, Su(var)3-9⁰⁶/Su(var)3-9⁰⁶ [SU(VAR)3-9 null (Reuter et al. 1986)]; 3, Dp(2;2)P90/CyO [two doses of HP1 (Wustmann et al. 1989), provided by J. C. Eissenberg]; 4, l(3)810/TM6 Tb [haplo-deficient H2Av (van Daal and Elgin 1992)]; 5, Su(var)3-7^P43/TM6 [G416E point mutation in Zn finger 4 of SU(VAR)3-7 (Bushey and Locke 2004)]; 6, Su(var)3-7^P12/TM6 [C-terminal deletion of SU(VAR)3-7 (Bushey and Locke 2004)]. (B) Eye pigment measurements and photographs of progeny without the balancer chromosome were carried out as described in the Figure 1 legend. Mean values that are significantly different from those of wild type (P < 0.05) are highlighted in yellow. Responses to a loss of SU(VAR)3-9 distinguish the fourth chromosome distal arm from the pericentric heterochromatin.

Increased copies of chromosome 4 suppress PEV of chromosome 4 distal arm P-insert reporters. Variegating P[hsp26-plant, hsp70-white] reporters within the distal portion of chromosome 4 (39C-12, 39C-33, and 39C-42 at polytene divisions 102B, 102D, and 102F, respectively), pericentric heterochromatin (118E-10 and 39C-3, bases of chromosomes 4 and 2L, respectively), or telomeric heterochromatin (39C-27, chromosome 2) (described in Wallrath and Elgin 1995; Sun et al. 2000, 2004) were introduced into backgrounds with varying chromosome 4 dosage. Males were photographed 3–5 days postelcosion. (A) Columns (1–4) from left to right: 1, diplo-4 (+/+); 2, haplo-4 (+/0); 3, triplo-4 [C(4)ey^R/+]; and 4, fourth chromosome deficiency [+/Df(4)B2-7AT]. Genotypes and graphic depictions of chromosome 4 karyotypes are shown below each column of photographs. (B) Quantitative measurements of eye pigment from the progeny shown in the photographs (same order from left to right) were carried out as described (Sun et al. 2004). Bars indicate means of three to five samples (five adult males each) with the standard error shown as a vertical goalpost. Mean values that are significantly different from diplo-4 (P < 0.05) are highlighted in yellow. Enhancement of PEV in flies carrying distal fourth chromosome reporters in the presence of a distal chromosome 4 deletion demonstrates that the haplo-enhancer effect can be attributed to the banded distal portion of chromosome 4. Drosophila cultures were raised on cornmeal sucrose-based medium at 25° as described (Shaffer et al. 1994). Stock BL1785 [C(4)RM, ci¹ ey^R/0] (Bloomington Drosophila Stock Center) is the source of the attached fourth chromosome for the line y w^67c23; C(4)RM, ci¹ ey^R/0 used in our analysis. Stock y; Df(4)B2-7AT/ci^D spa^pol was provided by R. Sousa-Neves (described in Sousa-Neves et al. 2005). In each experiment, PEV reporter lines were crossed to host stock y w^67c23 as a wild-type control. To produce haplo- and triplo-4 flies, virgin females carrying the PEV reporter were crossed with males carrying an attached fourth chromosome [y w^67c23; C(4)RM, ci¹ ey^R/0]. The fourth chromosome homologs segregate together, resulting in haplo-4 “minute” +/0 and triplo-4 C(4)RM, ci¹ ey^R/+ siblings.