Copyright 0 1989 by the Genetics Society of America

Position Effect Variegation inDrosophila melanogaster: Relationship Between Suppression Effect and the Amount ofY

Patrizio Dimitri* and Claudio Pisanot *Dipartimento di Genetica e Biologia Molecolare and TCentro di Genetica Evoluzionistica del CNR, Universita di Roma, Piazzale A. Moro 5, 00185 Roma, Italy Manuscript received August 23, 1988 Accepted for publication March 13, 1989

ABSTRACT Position effect variegation results from chromosome rearrangements which translocate euchromatic close to the . The euchromatin-heterochromatin association is responsible for the inactivation of these genes in some cell clones. In Drosophila melanogaster the Y chromosome, which is entirely heterochromatic, is known to suppress variegation of euchromatic genes. In the present work we have investigated the genetic nature of the variegation suppressing propertyof the D. melanogaster Y chromosome. Wehave determined the extent to which different cytologically characterized Y chromosome deficiencies andY fragments suppressthree V-type position effects: the Y-suppressed lethality, the white mottled and the brown dominant variegated phenotypes.We find that: (1) which are cytologically different andyet retain similar amounts of heterochromatin are equally effective suppressors, and (2) suppression effect is positively related to the size of the Y chromosome deficiencies and fragments that we tested. It increases with increasing amounts of Y heterochromatin up to 6040% of the entire Y, after which the effect reaches a plateau. These findings suggest suppressionis a function of the amount of Y heterochromatin presentin the genome and is not attributable to any discrete Y region.

ARIEGATION due to thechromosomal position Although the molecular basis of position effect var- V of a was first described in Drosophila mel- iegation remainsas yet unknown, differentsets of data anogaster by MULLER(1930) and has since been ob- suggest that histone proteins play an important role served in several species. The phenomenon is the in this phenomenon. In fact both reduction of histone consequence of the inactivation of euchromatic genes gene multiplicity (MOOREet al. 1979; MOORE,SIN- which are placed into a disruptedblock of heterochro- CLAIR and GRIGLIATTI1983) and histone protein matin by a chromosome rearrangement. The hetero- modifications result in the suppressionof position chromatin-directed cis-inactivation only occurs in a effect variegation (MOTTUS,REEVES and GRICLIATTI proportion of cells duringdevelopment. Thus the 1980).Furthermore, the dominant mutation Su- individual carrying the chromosome rearrangement uar(2)IO', which suppresses position effect variegation is phenotypically a mosaic of mutant and wild-type and shows a lethal interactionwith the Y chromosome, clones. (For review, see SPOFFORD1976.) correlates with histone H4 hyperacetylation and with Cytogenetic analysis of D. melanogaster polytene a significantly increased chromatin accessibility to en- chromosomes has shown that segmentscontaining dogenous nucleases (DORN et al. 1986).Altogether variegating genes lose their typical banded morphol- thesefindings indicate that changes in chromatin ogy and show irregular or diffuse banding (SHULTZ structure and organization play an important role in and CASPERSON1934; PROKOFYEVA-BELGOVSKAYAmodifying variegating . 1939; HARTMAN-GOLDSTEIN1967; KORNER and In the last few years nonhistone chromosomal pro- KAUFFMAN1986). This suggests thatthe adjacent teins (NHC proteins) were isolated which bind pre- heterochromatin causes some modification(s) in the dominantly to the heterochromatic sequences of D. chromatin structure of the variegating gene, which melanogaster and may be involved in the acquisition possibly results in the inhibition of its normal expres- and/or the maintenance of a compact heterochroma- sion. A recent molecular analysis of the rosy locus has tin structure (HSIEHand BRUTLAC1979; LEVINGER shown in fact that the rosy-variegated phenotype is and VARSHAVSKY1982; JAMESand ELGIN1986) and relatedto adecrease in transcription (RUSHLOW, thus in position effect variegation. BENDERand CHOVNICK1984). In D. melanogaster position effect variegation can be enhanced or suppressed by a variety of physical, chem- The publication costs of this article were partly defrayed by the payment of page charges. This article must therefore be hereby marked "advertisement" ical and geneticfactors (reviewed by SPOFFORD1976). in accordance with 18 U.S.C. $1734 solely to indicate this fact. In particular, addition of heterochromatin to the ge-

Genetics 122 793-800 (August, 1989) 794 P. Dimitri and C. Pisano nome suppresses, while subtraction enhances, typical is due tospecific Y regions. To this end we have tested variegation. The Y chromosome, which is entirely the effectiveness of nine different Y chromosome de- heterochromatic, suppresses position effect variega- ficiencies and five Y fragments in suppressing three tion (GOWENand GAY 1934)in a cell autonomous way different V-type position effects: the Y-suppressed le- (BECKERandJANNINC 1977). Attempts were made to thality, the white mottled and the brown dominant phe- identify specific Y region(s) involved in suppression of notypes, associated with the In( I)lv23I, In( 1)~”~~ position effect variegation. Early studies on the white WmSlbR and Zn(2R)bwvDe2rearrangements, respectively. mottled position effect indicated that the amount of We have found that the suppression of all three var- Y chromosome added to the genomewas unrelated to iegated phenotypes depends on the amount of the Y the efficiency of suppression (BAKERand SPOFFORD chromosome present in the genome and is not attrib- 1959). In a furtheranalysis, the BSvariegated pheno- utable to any discrete Y region. type was investigated and two suppressing regions, one close to the kl-2 fertility factor and anotherprox- MATERIALS AND METHODS imal to ks-I, were identified (BROSSEAU1964). It has recently been suggested that the Y chromo- For the description of FA47 and Zn(1)~~~~wM5lbRre- some suppresses position effect variegation by com- arrangements see APPELSand HILLIKER(1982). Other chro- peting for free histone or NHC protein(s) responsible mosomes and genetic markers used in this work are de- scribed in LINDSLEYand GRELL (1968). for the “heterochromatinization” of the variegating Culture conditions: Flies were maintained on a standard gene (ZUCKERKANDL1974; MOOREet al., 1979). That Drosophila medium containing cornmeal,yeast, sucrose agar would result in a general dilution of these proteins at with nipagin added as a mold inhibitor. All crosses were the variegating sites, thusreducing the probability grown at 25”. Male-sterile Y chromosomes: The genetics and cytology that these sites become inactive (MOTTUS,REEVES and of the y+Y deficiencies have been previously described GRICLIATTI1980). (GATTIand PIMPINELLI1983) except for Df(Y)G22, Bsy+, In the lastfew years the Y functions have been which was characterized genetically by KENNISON (1981) analyzed cytogenetically. The Y chromosome hetero- and cytologically by C. PISANO,S. BONACCORSIand M. chromatin has been subdivided into 25 differentially GATTI(personal communication). Y chromosome fragments:The Y fragments employed in stainedregions by combiningQuinacrine, Hoechst this work are Y distal-X proximal elements (X‘ Y”) derived 33258 and N-band staining techniques. Geneticcom- from five different fertilereciprocal X-Y translocations (V36, plementation tests coupled with the cytological analy- V24, W19, P7 and W28) that involve a y w f X chromosome sis of several rearranged Y chromosomes have led to and the BsY y+ chromosome (see Figure 2). The X-Y trans- locations were induced and genetically characterized by mapping of the Y functions to these cytological regions KENNISON (198 1). The X chromosome breakpoints are lo- (GATTIand PIMPINELLI1983). The loci defined in cated in the heterochromatin (Xh) proximal to the bb locus. these studies are physically very large. The kl-5, kl-3 (Forthe cytological map of the X heterochromatin, see and ks-1 fertility factors are 3 to 4 Mb long,for PIMPINELLIet al. 1985.) TheBSYy+chromosome breakpoint example. Different types of genetic organization are of V24,P7 and W28 is located in the long arm (YL), while the Y chromosome breakpoint of W19 and V36 in the short evident among the component of the Y chromosome: arm (YS). Therefore, besides the different portions of Y (1) the six fertility factors require structural integrity chromosome heterochromatin, the X‘ Y” elements carry for functioning; (2) the collochore (COOPER1964) and either y+Xh (Xp YSDV36, X‘ YS” W19)or B‘Xh (X‘ YLDV24, the Cry locus (HARDY andKENNISON 1980; HARDYet X‘ YL” P7 and X‘-YL” W28) blocks. Both y+Xh and BSXh blocks are derived from the proximal Xh region and are al. 1984), like the bb locus (reviewed by RITOSSA comparable in size. (For the nomenclature of the hetero- 1976), are inactivated only by deletions (rather than chromatic blocks of y+Y and BSYy+, see GATTIand PIMPI- by breakpoints) and therefore possibly consist of a NELLI 1983.) These elements X‘Y” were isolated from the large number of repeated subunits (reviewed by PIM- parental translocations by crossing females homozygous for YSX.YL, l)EN,y (=XY, y) to T(X;Y)y w BSy+ males and PINELLI et al. 1986); and (3) the AB0 factors, which In( f by recovering the XYy/X‘ Y”, BSor y+ F1 males. Except for map to specific heterochromatic sites of different P7, cytologically described in the paper by HARDYet ul. chromosomes and may correspond to anotherkind of (1984), the cytological analysis using Hoechst and N-band- repeatedgenetic element (PIMPINELLI et al. 1985). ing techniques of the X-Y translocations was carried out by Furthermore, the Y chromosome heterochromatinin- S. BONACCORSI(personal communication). Calculation of the Y deficiency and fragment sizes:The cludes several classes of highly-repeated and middle cytological size of Y deficiencies and X‘ Y” elements, includ- repetitive DNAs (PEACOCKet al. 1977;SPRADLINC ing the y+ or B’Xh blocks (Figures 1 and 2) is expressed in and RUBIN 198 1) which may be functional parts of percent according to the cytological map of the standard the genetic loci (GATTIand PIMPINELLI1983; LIVAK fertile y+Y (Figure 1) elaborated by GATTIand PIMPINELLI 1984). (1983). Eye pigment measurement. Heads were collected 2 days In thepresent work we have asked whetherthe after esclosion by freezing the adults in Eppendorf tubes suppression of position effect variegation is a general and vortexing for a few seconds. The red pigment was feature of the heterochromatic Y-DNA or whether it extracted according to EPHRUSSIand HEROLD (1944).Spec- Position Effect Variegation 795

FIGURE1 .-Hoechst staining banding pattern of the standard y+Y chromosome according to GATTI and PIMPINELLI (1983). The darkareas correspond to OfI V)S7 DIIyIss Dt Wf34 bright regions; the hatched areas, to dull DIIVtSlO 0fIV)SB DIIVtSlO Dt1Y)SlZ regions and theopen areas, to nonfluores- OflVtG22 cent regions. The bars below indicate the physical sizeand the chromosome location Df(VtS9 of the deficiencies employed in our analy- DtlVtSll sis. trophotometric analysis was performed at 480 nm. For each the Y chromosome deficiencies assayed in this experi- chromosome samples consistingof five heads were analyzed. ment. In Figure 3 the absolute viability of each class of1231 males bearingdifferent Y chromosomes is RESULTS plotted us. their physical size. Deficiency mapping shows that chromosomes still Suppression of three different variegated position retaining 60 to 95% of the total heterochromatin do effect was analyzed: (1) the Y-suppressed lethality not drastically differ from the control y+Y in their (LINDSLEY,EDINCTON and VONHALLE 1960), (2)the effectiveness of suppression. In particular the suppres- white mottled (wm), and (3) the brownvariegated sion effectiveness is the same for Df(Y)s SIO, SII, S7, phenotypes (bw'), which are respectively associated S6 and S5. Since the deficiencies used altogether cover with the following rearrangements: (1) In( I )v231 an the entire Y chromosome length except for the cen- inversion with breakpoints lB/C; 20F on the cytoge- tromeric region (Figure l), these results suggest that netic map, (2) I~(I)w"~~wm51bR, and (3) Zn(2R)bwVDe2 the suppression effect doesnot map to specific Y (41A-B; 59 D6-El). In both Zn(I)v231 and In(l)wm4 Wm51bR the variegation-inducing regions are derived region. The possibility might still remain that the Y cen- from the X chromosome heterochromatin, whereas tromere orits neighboring regions,which are retained the heterochromatin of the right arm of the second inall deficiencies, are responsible for the observed chromosomeinduces the bw' phenotype. All three suppression. This possibility was tested using five dif- rearrangements induce a strong variegated position ferent fragments (Figure2). These were recovered as effect, which can be efficiently suppressed by the addition of a Y chromosome to the genome. half translocations (X' YD elements) from the fertile Y chromosome deletions (Figure 1) and X' YD ele- Xh-Y translocations V36, V24, WIY, P7 and W28 (KEN- ments (Figure 2) recovered from fertile T(X;Y)s were NISON 1981), which lack the Y centromere. The XpYD tested for their ability to suppress these V-type posi- elements retain, in addition to they+ or BSXh blocks tion effects. It is worth noting here that the Df(Y)s present in the BS Y y+ chromosome from which they are from the same parent Y. As can be seen in Figures originated, different amounts of the Xh proximal to 1 and 2, the rearrangements used in this work alto- the bb locus. In particular, both V36 and V24 contain gether cover the entire Y length. Thus, no Y regions roughly the whole of the proximal Xh region, while were left genetically unexplored in our experiments. P7, W28 and W1Y retain only smaller Xh portions next The Y suppression effect on Z(2)v 232: In a first to the centromere. The heterochromatic content of set of experiments, the suppression of the lethal phe- X' YD elements, including y+ or BS Xh blocks, is ex- notype of I( I)v231, associated with In( l)Z v231, was pressed as a percent of the cytological size of the analyzed. control y+Y. In this calculation the Xh proximal to the YL .X. Y S/Y* males (where Y* are eitherY deficien- bb locus is not included, since its cytological content cies or XpYDelements marked with y+ or BS) were does vary among the elements. Furthermore, since crossed with FM7, y wa v B/I(I)v231 y females. We the y+Y chromosome heterochromatin retainedin the have suppressed the absolute viability of the F, males X' YD elements represent 12-55% of the control y+Y as the ratio of surviving I( I)v231 males to the total size, that also enabled us to investigate the pattern of female offspring whose viability was assumed to be suppression obtained with Y portions representingless constant. The ratio of I(I)v231/Y* male absolute via- than 60% of the y+Y. The results are shown in Table bility to that of E(I)v231/y+Y males, which carry a 1 and are graphically expressed in Figure 3. cytologically normal and genetically fertile y+Y, gave The maximum background of suppression attrib- arelative viability which measured theextent of utable to the proximal Xh present in the X' YD ele- suppression exerted by each rearranged Y chromo- ments does not go beyond 26%, observed with V36 some relative to that caused by y+Y. which carries the largest Xh amount (i.e., the Xh region Table 1 shows the absolute and relative viability proximal to bb plus the y+Xh block derived from the values calculated for differentmale genotypes bearing original BSYy+).Therefore theYh present in V24, WIY, 796 P. Dimitri and C. Pisano 8 PSVLC FIGURE2.-Hoechst staining band- [ ing pattern of the standard BSYy+chro- " mosome according to GATTIand PIM- Xp- Yo V36 PINELLI (1983). The bars below indicate the physical size and the chromosome XC-YD P7 location of five different deficienciesre- XP-VD wl9 covered as X' Y" elements from recip- Xp- Yo V24 rocal T(X;Y)s (see MATERIALS AND METHODS). Xp- Yo W28

0.6 ' TABLE 1

Suppression effect of different Y chromosomes on the Z(I)v231 0.5 phenotype E W28 3 p7 Progeny 0.4 t s' l(I)u231 Total females V24 Zm w19 Chromosome Y* @E and YE') a.m.v." ? SE r.m.v.b ('70) d E 0 101 2663 0.038 0.001 7.3 0.3t V3h 23 1 1409 0.164 0.004 31.5 V24 1223 386 0.31 0.008 59.6 I , I CV19 2820 910 0.32 0.005 61.5 :f P7 957 370 0.386 0.01 74 0.0 CV28 443 1020 0.43 0.01 82.7 0 20 40 60 80 100 SI0 589 1151 0.51 0.013 98 sz2 1879 929 0.494 0.009 95 % of y*Y chromosome G22 1349 660 0.49 0.01 94.2 FIGURE 3.-The suppression-effect exerted by different Y chro- SZI 1164 596 0.51 0.012 98 mosome amounts onthe lethality associated to the In 1( Z)u231. The s7 1190 2261 0.526 0.009 101 absolute viability of each class of 1(Z)u231 males bearing different S6 1780 940 0.528 0.01 101 Y chromosomes is plotted us. their physical size. The physical size 84 637 1291 0.493 0.01 1 95 of Y deficiencies and XD-YDelements, including the y+ or Bs Xh s5 899 469 0.521 0.014 100 blocks, is expressed as a percent of the cytological size of the control y+Y, according to the cytological map of the standard fertile y+Y Y+Y 2769 1449 0.52 0.009 100 stained with Hoechst 33258 elaborated by GATTIand PIMPINELLI a The absolute male viability is expressed by the ratio of vital (1983). In that calculation the Xh proximal to the bb locus present l(I)u2?l males to the total of females. .I in the X' YDelements is not included. l(l)u23Z/Y* (a.m.v.) ' Relative male viability (%) = x 100. l(l)u231/y+Y (a.m.v.) ' Y * = Y chromosome deficiencies andfragments. A critical threshold (60% of the y+Y) is apparent, at which the maximum suppression occurs. Therefore P7 and W28 fragments is indeed responsible for the the possibility that any specific Y region is responsible increased suppression exerted by those Xp YD frag- for the suppression of that position effect variegation ments in comparison to V36. can be ruled out. It is apparent from theplot in Figure 3 that suppres- The white mottled phenotype: The deletions used sion increases as a function of the amount of the Y in the test with 1( 1)231 were also used to test w"' and added to the genome. Small amounts of Y chromo- bw" position effect variegation. Only the yf-marked some are able to induce a relevant suppression effect. and X-Y rearrangements could be used since the BS In fact, it appears that the Yh present in V24 and W19 marker in the others drastically reduces the number (about 10% and 15% of the control y+Y, respectively) of ommatidia. is responsible at least for the 30% suppression differ- The effect exerted by the Y chromosome in sup- ence observed between these elements and V36. These pressing zn(1)wm4 wm51bR (w")was tested by crossing data show thatthe Y centromeredoes not play a single wm/Ymales by y w fly w fly* females (where Y* specific effect in suppressing 1( 1 )v231,since the tested were either Y chromosome deficiencies or XpYD ele- XpYD elements lack the Y centromere and all of them ments from T(X;Y)marked with y+). In each cross, show a suppression effect related to the physical size. the red pigment levels were measured in both they w Furthermore the similarly sized V24 and W19 show fly W" and they w fly w"/Y* F, female offspring taken similar effects, although they contain opposite ends of as control and experimental values, respectively. The the Y chromosome (see Figure 2). results are summarized in Table 2 and thesuppression Takentogether, these findings indicate that values are shown in Figure 4. suppression of 1( 1 )v231 is a quantitative phenomenon, Suppression values obtained with W19 (in which the independent from the genetic constitution of the Y 2 1 to 25 Yh regions represent in size 15% of the y+Y) chromosome heterochromatin presentin the genome. element confirms that small fragments of y+Y hetero- Position Effect Variegation 797

TABLE 2 0.20 - Suppression effect of different Y chromosome amounton the white mottled phenotype

0.15 No. of - obser- OD vations" Percent of - Chromosome C E C E Amb ? SE suppression 2 0.10 a zw19 V36 11 11 0.0360.087 0.0510,005 26.7 WI9 7 10 0.046 0.131 0.085 0.0047 44.5 SI0 13 18 0.035 0.198 0.1630.004 85.3 0.05 - lV36 s12 15 180.039 0.197 0.158 0.0045 82.7 SI1 9 0.04712 0.208 0.161 0.004 84.4 s9 9 9 0.0360.201 0.165 0.006 84.8 0.00 ' s7 10 8 0.035 0.2200.185 0.005 97.4 0 20 40 60 80 100 S6 13 21 0.039 0.2200.181 0.003 94.7 X of Y+r chromosome 84 7 11 0.0470.222 0.1750.005 92 FIGURE 4.-The suppression effect exerted by different Y chro- s5 7 10 0.043 0.219 0.176 0.00592.1 mosome amounts on the Zn(l)wm41 w'"'''~ variegated phenotype. Y+Y 18 14 0.041 0.2320.1910.005 100 The percent of the Y chromosome added to the genome is plotted " - Optical density (OD480nm)levels were measured in pf/p"/Y*(E) us. the AOD(OD(E)OD(C)) values. The physical size of Y deficiencies and rwflrw"(C) siblings females (see text). and X' YDelements, including the y+ or BSXh blocks, is expressed Ea&obs%vation based on a pigment extractedfrom five heads. as a percent of the cytological size of the control y+Y, according to AOD = OD(E) - OD(C). - the cytological map of the standard fertile y+Y stained with Hoechst Percent of suppression = (AOD)Y*/(A~)Y+Y. 33258 elaborated by GATTI and PIMPINELLI(1983). In that calcu- lation the Xh proximal to the bb locus present in the X' YDelements chromatin are highly effective in suppressing the var- is not included. iegated phenotype. The presence of the y+Xh block retained in both V36 and W19 is not crucial per se, as ments. That suggests either that small amounts of Y can be seen by comparing Df(Y)SlO (deleted in the heterochromatin-regardless of genetic content-are y+Xh block) with Bf(Y)S12 which is of a comparable sufficient for effective suppression of bwV,or that W19 size and yet retains the y+Xh block (Figure 4). Both fragment includes a specific bwVsuppressor located in SI0 and S12, which remove the opposite ends of the the h21-hZ5 regions of the Y. The effect of these Y,leave about 60% of the chromosome and bothshow regionspresent in W19 can beevaluated from a suppression effect approaching 85% of the y+Y con- Df(Y)B4 which is deleted for thesame regions (Figure trol.Suppression remains constant for deficiencies 1). Suppression efficiency observedfor Df(Y)B4 is retaining 60 to 80% of heterochromatin, after which identical to that shown by the control y+Y chromo- the maximum suppression is reached. some. Thus the effect of the W19 element seems to Taken together these results show that suppression be only attributable to its quantitative rather than to of the wmphenotype, as previously found forI( l)v231, its qualitativeheterochromatic content. The bwv is a function of the amountof Y chromosome present suppression with heterochromatic portions represent- in the genome. ing 60% or more of the y+Y is very similar to that The brown dominant variegated phenotype: The observed for suppression of the w"phenotype (Figures effect of the Y chromosomeheterochromatin on 4 and 5). However, this is an exception to the other- bw VDP2 expression was tested by crossing single y/Y; wise generally observedquantitative correlation. bwVD"/+ males to pf/pf/Y* females. The amount of Df(Y)S6 is similar in size to S7 and B4 chromosomes, red pigment was measured in the F1 female offspring. but its suppression value is closer to that observed for The F1 females from each cross are either y w f/y; SIO, S12, Slland S9 than to the control. We are bw VDe2/+ (control group) or y wf/y/Y*; bwVDP2/+ (ex- unable to analyze in any further detail the region perimentalgroup). The results fromthese experi- removed in Df(Y)S6 (M-I-), which appears to be ments are summarized in Table 3 and plotted in the responsible fora 9% decrease in suppression com- graph in Figure 5. pared to the y+Y control. Thus S6 chromosome may The V36 suppression effecton bw variegation is identify a cytological region relatively more efficient similar tothat observed onboth previously tested than the rest of the Y in suppressing the bwVpheno- phenotypes. The W19 fragment is more effective in tYPee bw" suppression (70%) than in either Z(l)v231 or wm Besides the weak suppressor region identified by suppression. The W19 and V36 fragments differ in the S6 chromosome, it is apparent from the graph that W19 carries the 21 to 25 regions of the Y short that, on the whole, bw'suppression does not substan- arm, which represent15% of the y+Y insize and tially differ from that reported for either 1(1)231 or appear to be responsible at least for the 50% differ- wmin that no discrete region of the Y chromosome is ence between the suppression values of those frag- responsible for a strong suppression effect. 798 P. Dimitri and C. Pisano

TABLE 3 Suppression effect of different Y chromosome amounton the brown variegated dominant phenotype ::j No. of - obser- OD € w19 vations" 0 0.15o.m[ Percent of Chromosome C E C E Am' SE suDpression W6 9 80.018 0.064 0.046 0.012 17.4 W19 9 12 0.0220.206 0.184 0.005 69.7 SI0 11 10 0.0220.264 0.242 0.007 91.7 SI2 22 14 0.0210.268 0.247 0.0045 93.5 SI 1 15 14 0.0190.257 0.238 0.006 90 s9 9 80.018 0.256 0.242 0.007 91.7 0.w I s7 14 16 0.0210.282 0.261 0.0035 98.8 0 20 40 60 80 100 S6 9 12 0.0330.272 0.239 0.0076 90.5 B4 6 6 0.0190.285 0.266 0.0084 100.7 % of Y+Y chromosome s5 9 40.029 0.293 0.264 0.0085 100 FIGURE 5.-The suppression effect exerted by different Y chro- Y+Y 11 11 0.0140.278 0.264 0.007 100 mosome amounts on the In(2R)bwVDeZvariegated phenotype. The

~ ~~ ~ ~ ~~ ~~ ~~ percent" of the Y chromosome added to the isgenome plotted versus 0 tical density (OD48onm)levels were measured in pf/y/Y*; bwvD"/+ (E) and ywf/y;bwVDC2/+(C) siblings females (see text). the AOD(OD(E)-OD(C)) values. The cytological size of Y deficien- or a &&obs=&n based on a'pigmencextractedfrom five heads. cies andXpYD elements, including they+ BSXhblocks, is expressed * AOD = OD (E) - OD (C). - as a percent of the cytological size of the control y+Y, according to Percent of suppression = (AOD) Y*/(A~)Y+Y. the cytological map of the standard fertile y+Y stained with Hoechst 33258 elaborated by GATTIand PIMPINELLI(1983). In that calcu- DISCUSSION lation the Xh proximal to the bb locus present in the X' YDelements The Y chromosome has been known for a long time is not included. to be an efficient suppressor of position effect varie- mosome rearrangementsthat were tested. In fact gation (GOWENand GAY 1934). Early investigations Df(Y)SIO and Df(Y)SI2chromosomes, which are cy- favored the view thatdiscrete suppressor regions togenetically different and yet retain similar amounts could be mapped to specific Y sites (BAKERand SPOF- of heterochromatin are equally effective suppressors FORD 1959; BROSSEAU1964). Chromosome banding of all three variegated phenotypes. The same applies techniques were not available at thattime. Therefore, to V24 and W19 X' YDelements as suppressorsof the both the qualitative and quantitative content of the Y lethal variegated phenotype. chromosomes assayedin those studies were poorly The suppression effect appears to be related to the resolved. Some of the Y fragments used in those size of the Y chromosome deficiencies and fragments. studies may have in fact included euchromatin from Suppression increases with increasing amounts of Y X or autosomes thatwas not identifiedas euchromatic, heterochromatin up to 60-80% of the entire Y, after as well as heterochromatin from other sources that which the effect reaches a plateau. In particular, for could have the same generalized effect as the Y het- I)v23I a critical threshold (60%of the y+Y) is appar- erochromatin. I( ent, above which the maximum suppression occurs. In thepresent work, we have found thatsuppression Small Y chromosome fragments (V24 and W19 ele- of the variegating state is a function of the quantity ments) were found to be already highly effective in of Y heterochromatin present in the genome at least for three different V-type position effects: the Y-sup- suppressing the variegatedphenotypes. This effect pressed lethality, the white mottled and the brown var- reflect again the quantitative feature of the suppres- iegated phenotypes. We wish to point out the consist- sion phenomenon, suggesting thatin the absence of a ency with which this phenomenon was observed. In Y chromosome, the repressed chromosomal state of fact similar suppression patterns were found in males the variegating genes is particularly sensitive to the (Z(I)v231)and females (wmand bw") using unrelated addition of small amounts of Y heterochromatin. Since analytical methods: recovery of viable male offspring the variegation-inducing heterochromatic regionsare and eye pigment measurement. different for the three position effects examined in Our studies were based on a detailed cytological this study (Xhand 2Rh),it appears that thesuppression analysis of the Y chromosome deficiencies and frag- effect exerted by the Y is also largely independent ments that were employed. Since the different dele- fromthe genetic constitution of theinducer sites, tions and fragments together cover the entire length being only related to their common heterochromatic of the Y,we are confident thatin our tests no Y regions organization. were left genetically unexplored. These results indicate that the variegation-suppres- Several points are worth stressing. Suppression is sion property is a general feature of the Y heterochro- unrelated to the cytogenetic content of the Y chro- matin, in that it is homogeneously spread along the Position Effect Variegation 799 entire length of this chromosome rather than being 1984 Cytogenetic analysis of a segment of the Y chromosome associated with a specific mappable element.That may of Drosophila melanogaster. Genetics 107: 59 1-610. HARTMAN-GOLDSTEIN,I. J., 1967 Onthe relationship between be explained postulating that proteins involved in the heterochromatization and variegation in Drosophila with special “heterochromatinization” of the variegating genesare reference to temperature-sensitive periods. Genet. Res. 10 actually structural components of all the heterochro- 143-159. matic regions of the Y chromosome and are present HSIEH,T., and D. BRUTLAG,1979 Sequence and sequence varia- in limiting amount in the cell. According to this tion within the 1.688 g/cm3 satellite DNA of Drosophila mela- model, thegreater the amount of Y chromosome nogaster. J. Mol. Biol. 135: 465-481. JAMES, T. C., and S. C.R. ELGIN,1986 Identification of non- heterochromatin added to a variegating genome, the histone chromosomal protein associated with heterochromatin better the chance that heterochromatic proteins (his- in Drosophilamelanogaster and its gene. Mol. Cell. Biol. 6 tones or NHC proteins), would be “sequestered” by 3862-3872. the Y DNA. 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