Copyright 0 1994 by the Genetics Society of America

Modification of the Drosophila Heterochromatic Mutation brownDominant by Linkage Alterations

Paul B. Talbert*91, Cosette D. S . LeCiel*'* and Steven HenikofP".? *Fred Hutchinson Cancer Research Center and THoward Hughes Medical Institute, Seattle, Washington 98104

Manuscript received August 13, 1993 Accepted for publication October 8, 1993

ABSTRACT The variegating mutation brownDmniMnf (bp) of Drosophila melanogaster is associated with an insertion

of heterochromatin into chromosome arm 2R at 59E, the site of the bw gene. Mutagenesis produced 150 dominant suppressors of bwD variegation. These fall into two classes: unlinked suppressors, which also suppress other variegating mutations; and linked chromosome rearrangements, which suppress only b p . Some rearrangements are broken at 59E, and so might directly interfere with variegation caused by the heterochromatic insertion at that site. However, most rearrangements are translocations broken proximal to bw within the 52D-57D region of 2R. Translocation breakpoints on the X chromosome are scattered throughout the X euchromatin, while those on chromosome 3 are confined to the tips. This suggests that a special property of the X chromosome suppresses b d variegation, as does a distal autosomal location. Conversely, two enhancers of bp are caused by translocations from the same part of 2R to proximal heterochromatin, bringing the b d heterochromatic insertion close to the chromocenter with which it strongly associates. These results support the notion that hetero- chromatin formation at a genetic locus depends on its location within the nucleus.

C HROMOSOMES of Drosophila melanogaster and many other eukaryotes are composed of two distinct kinds of chromatin: the pericentric regions consist of heterochromatin, which remains condensed throughout the cell cycle, and the chromosome arms consist of euchromatin, which decondenses during interphase. A large body of work has shown that DNA sequences in heterochromatin are largely repetitive and contain relatively few identifiable genes; the bulk of functional genes reside in euchromatin (for a re- view, see GATTI and PIMPINELLI 1992). In Drosophila embryos, centromeric regions coalesce at the apex of blastoderm nuclei, and heterochromatinization of these regions appears to occur around the time of cellularization (RABINOWITZ 1941). In salivary gland nuclei, the pericentric sequences are underrepre- sented relative to the polytenized euchromatic arms and the coalesced centromeric regions form a heter- ochromatic chromocenter (RUDKIN 1969; LAIRD et al. 1973). The functional significance of this nuclear compartmentalization of heterochromatin is not well understood.

Evidence for incompatibility of euchromatin and heterochromatin comes from chromosome re- arrangements that juxtapose the two. Such chromo- some rearrangements typically exhibit a variable cis- inactivation of adjacent euchromatic genes, known as

15, Seattle, Washington 98195.

50, Seattle, Washington 98195.

Genetics 136: 559-571 (February, 1994)

' Present address: University of Washington, Department of Botany KB- * Present address: University of Washington, Department of Genetics SK-

position-effect variegation (PEV). For example, when a rearrangement places heterochromatin next to the wild-type white (w+) gene, which is necessary for pig- mentation of the eye, the inactivation can be visualized as a variable pattern of pigmented and nonpigmented ommatidia in the eye (for reviews, see SPOFFORD 1976; HENIKOFF 1990; SPRADLING and KARPEN 1990; REU- TER and SPIERER 1992). Studies of salivary gland chromosomes show that cis-inactivation of euchro- matic genes correlates with a change in cytological appearance from euchromatic to heterochromatic (HARTMANN-GOLDSTEIN 1966; HENIKOFF 198 1 ; HAY- ASHI et al. 1990; KARPEN and SPRADLING 1990; UM- BETOVA et al. 1991). This has been interpreted as a spreading of heterochromatin formation across the rearrangement breakpoint into normally euchromatic adjacent genes, causing their inactivation. Since PEV results from gene inactivation, PEV alleles are nor- mally recessive to their wild-type homologs.

PEV can be suppressed by dominant mutations in a large number of euchromatic loci, as well as by an extra Y chromosome (reviewed by EISSENBERG 1989; GRIGLIATTI 1991; REUTER and SPIERER 1992). Many of the suppressor loci, known as Su(uar)s, show dosage effects such that a single dose of a Su(var)+ gene will suppress PEV relative to the normal two doses; a third dose will enhance PEV. Conversely, some dominant E(uar)s enhance when wild type in one dose and sup- press when in three doses. T o account for these ob- servations, a mass-action model has been proposed in which dosage-sensitive suppressor loci encode proteins

560 P. B. Talbert, C. D. S. LeCiel and S. Henikoff

that complex with DNA to form heterochromatin (LOCKE et al. 1988). Conversely, enhancer loci might encode proteins involved in limiting heterochromatin formation. The process of heterochromatin formation is then dependent on the concentrations of each of these component proteins. Other Su(var) and E(var) genes that do not show dose effects may encode en- zymatic functions that are necessary to assemble chro- matin.

A form of PEV complementary to the cis-inactiva- tion of euchromatic genes occurs for genes that nor- mally reside in heterochromatin (BAKER 1968). The light ( I t ) gene at the base of chromosome arm 2L apparently requires a heterochromatic environment to function since it variegates when rearrangements move it to distal euchromatin (HESSLER 1958; HILLI- KER and HOLM 1975; WAKIMOTO and HEARN 1990). Interestingly, some suppressors of PEV of euchro- matic genes act as enhancers of It variegation, as would be expected if they limit heterochromatin formation (HEARN et al. 1991). The fact that no variegating rearrangements have been observed that move It to proximal euchromatin has led to suggestions that proximity to a centromeric compartment in the nu- cleus may also be an important determinant of heter- ochromatin formation (WAKIMOTO and HEARN 1990). A study of the heterochromatic rolled ( r l ) gene has shown that it variegates when it is present in a small block of heterochromatin surrounded by euchroma- tin, but regains more normal function when it is moved near a large block of heterochromatin, regard- less of its distance from the centromere per se (EBERL et al. 1993). These studies argue that distance from other heterochromatic elements is an important de- terminant of PEV for heterochromatic genes (BAKER 1968).

In contrast to the situation for PEV of heterochro- matic genes, involvement of nuclear localization in PEV of euchromatic genes is less clear. MULLER (cited by EPHRUSSI and SUTTON 1944) was the first to predict that differences in chromosomal positioning in so- matic cells might account for PEV of euchromatic genes. Indeed, numerous early studies showed that PEV mutations can often be reverted by re- arrangements in the heterochromatin adjoining a var- iegating gene, usually relocating that gene to a eu- chromatic site (DUBININ 1936; PANSHIN 1938; GRIF- FEN and STONE 1940; KAUFMANN 1942; HINTON and GOODSMITH 1950). Molecular studies with the chro- mosomal inversion Zn(l)w"', which moves the w+ gene proximally next to a disrupted block of pericentric heterochromatin, have indicated that reinversions placing thew+ gene back to a distal location revert the variegating phenotype, even though some heterochro- matic repeat sequences remain adjacent to the w+ gene (TARTOF et al. 1984). Similarly, in reversions of

T(1;2)dorUar7, at least 20 kb of heterochromatin re- mains adjacent to the deep orange gene (POKHOLKOVA et al. 1993). In all of these cases, however, it is difficult to evaluate whether the reversions resulted from the change in position relative to a chromocentral com- partment, the change in the amount or kind of adja- cent heterochromatin, or all of these. Only DUBININ (1936) reported revertant chromosomes in which a block of heterochromatin and the adjacent breakpoint were moved to new locations by euchromatic re- arrangements that left the heterochromatic block in- tact.

Here we provide evidence that altering the chro- mosomal location of euchromatic genes subject to PEV can have striking phenotypic consequences, sim- ilar to what is seen for PEV of heterochromatic genes. These findings were made as a result of attempts to obtain dominant suppressors of PEV of the brown (bw) gene, which controls production of the pteridine pigments of the eye, using the variegating allele

being a spontaneous insertion of pericentric hetero- chromatin, rather than an X-ray induced chromosome rearrangement (SLATIS 1955). Insertion of hetero- chromatin occurred distally in chromosome arm 2R, which otherwise retains its normal gene order. There- fore, euchromatic rearrangements of 2R can change the position of b d relative to its centromere without simultaneously altering the block of heterochromatin that induces PEV.

Variegating bw alleles not only inactivate the bw gene on the rearranged chromosome (cis-inactiva- tion), they also inactivate the bw gene on the homol- ogous chromosome (trans-inactivation; reviewed by HENIKOFF et al. 1993). We screened for suppressors of b d [Su(bd)s] in an effort to isolate mutations that specifically suppress trans-inactivation. The majority of suppressors recovered were Su(var)s similar to those previously found to suppress cis-inactivation of wm4. Surprisingly, nearly a fifth of our Su(bd) lines carried rearrangements that modified PEV of bw by changing the chromosomal position of the b d allele, suggesting that this PEV phenotype depends on nuclear position.

brOwnDominanl (bd). This PEV mutation is unique in

MATERIALS AND METHODS

Fly stocks and culture conditions: Fly stocks were grown on standard corn meal-molasses medium in shell vials or on instant food (Carolina Biological Supply) in plastic specimen bottles at room temperature except as noted. No difference in phenotype was observed between genotypically identical flies grown on the two kinds of food.

The b d allele has an insertion of heterochromatin which is visible as an extra polytene band just proximal to 59E (SLATIS 1955). As it presently exists, b d has the bw gene interrupted by sequences that lack tested restriction sites (HENIKOFF et al. 1993) and that are likely to be simple sequence repeats characteristic of heterochromatin. The gene has no detectable bw+ activity (SLATIS 1955; DREESEN

56 1

FIGURE 1.-Eye pigmentation in wild-type and variegating bw geno- types. Clockwise from upper left:

bup/bw'; st, E(b4)144 b4/+ bw+; st, h+/bw+; st, Szl(b4)20 b4/+ bw+; st,

bWDlb4; st.

et al. 1988). The allele appears to have changed since its isolation, as HINTON and GOODSMITH (1 950) were able to revert it to wild type, whereas the current allele appears to be nonrevertable (K. LOUCHNEY, unpublished data).

The P[bw+]92C strain carries a bw+ transposon inserted 92C (DREESEN et al. 1991). Its variegating derivative T(2:3)V21e, P[bw+ /" ("V218") was previously described (HEN- IKOFF et al. 1993). Other mutations not reported here are described by LINDSLEY and ZI" (1 992).

Mutagenesis and screen: We screened for dominant sup- pressors of the variegated eye pigment phenotype of bP/+ flies (Figure 1). The production of pteridine eye pigments by the activity of the bw locus is more easily monitored in

the absence of ommochrome pigments. The screen and subsequent analyses were therefore conducted in a scarlet (st) background, since st+ activity is necessary to produce ommochromes in the eye. The marker speck (SF) is closely linked to bw (1 -2 cM) and was sometimes used to distinguish bw+ from bu?' regardless of eye phenotype (see below).

White-eyed b d sp+; st males were mutagenized with ethyl methane sul honate (EMS) (GRIGLIATTI 1986) and mass- mated to bw P s t ; st virgin females (Figure 2). Nearly all of the b d +/+ sp; st progeny had eyes that were white with a quite uniform scattering of individual reddish spots within ommatidia (Figure 1, lower right). Offspring with increased pigmentation ("suppressed flies"; Figure 1, upper right)

562 P. B. Talbert, C. D. S. LeCiel and S. Henikoff

white I scarlet F1 b w D + sf [F] b w D + ; st FIGURE 2.-Screen for suppressors of b d / + ; st.

" x ' s p . s ' + sp Sf + sp Sf + sp Sf

pale yellow variegated orange variegated scarlet

Eye phenotypes are indicated. The nonsuppressed phenotype is pale yellow variegated (Figure 1, lower right). Individuals with orange variegated pigmenta-

of about 0.5%. Less than a third of these proved to be fertile and heritable. The large brackets indicate that the linkage of the suppressor is unknown.

PmmEl [ -500 (0.5%) I tion (Figure 1, upper right) were recovered at a rate

t F2 " b w D + , sf

+ sp Sf

pale yellow variegated

[F] -,- b w D + ,sf + sp Sf

orange variegated scarlet scarlet m

99 x T(2;3) V21: bwD Sp; sf Sb P[bw 7 " I bwD sp; ln(3R) P, st +

orange variegated eyes axillary speck; stubbly bristles; white eyes with very few pigmented spots

T(2:3) V2 1: bwD sp; sf Sb P[bw +] " T(2;3) V21: bwD sp:sfSb P[bw+]" FIGURE 3.-Test for suppression of cis-inacti- vation of V21'. The dominant markers s@+ and Sb

+ - +

su - .L

t si

bwD sp'/n(3R)P, sf

stubbly bristles; white eyes with very eyes

white axillary speck;

few pigmented spots stubbly bristles; pale yellow variegated eyes

r - - - - . - - - - : orange , variegated eyes? I test class) L _ I " " " _ J variegated eyes

stubbly bristles; 1 white axillary speck; eyes stubbly bristles;

orange

were used to establish selection stocks by backcrossing them to bw+ sf; st flies and selecting the suppressed b d +/+ sp; st progeny. In addition to the suppressed flies, two flies with phenotypic enhancement (Figure 1, lower left) were also recovered from the screen and used to make analogous selection stocks.

In the course of propagating the selection stocks, many of the suppressors and both enhancers showed linkage to bw", as determined by the recovery of only a few or no nonsuppressed or nonenhanced b d +/+ sp; st progeny. These linked suppressors and enhancers were subsequently balanced over Zn(2LR) SMI, Cy in a st background unless they were sterile or lethal in one sex. As the Cy-bearing chromosomc? enhances PEV (SPOFFORD 1976 and our un- published data), the variegation phenotype of these balanced flies was always checked by crossing to the bw+ sf chromo- some.

Test for suppression of &-inactivation: Since the b d allele is null, it cannot be used to test for cis-inactivation. Therefore, suppressors were tested for their ability to sup- press the cis-inactivation of the P[bw+] gene at 92C in V21'. Suppressor-bearing b d / + females were crossed to V21'- bearing males as described in Figure 3. Some of the sup-

-.- + SP ' sf are closely linked to b4 and V21', respectively, bwD sp ' / n ( 3 ~ ) p , st and can be used to identify the latter mutations

independently of their eye pigmentation pheno- axillary speck; types. pale yellow variegated eyes

axillary speck;

variegated eyes orange

pressors were subsequently tested for suppression of cis- inactivation using wm4. Suppressor-bearing +/Y; b d / + ; st males were crossed to wm4; +; + virgin females, and progeny were examined for the expected class of males suppressed for wm4. Although w n 4 / K b P / + ; st/+ male progeny could not easily be distinguished from the wm4/Y; +/+; st/+ male progeny, suppression of wm4 could be observed in either genotype.

Test for suppression of telomeric variegation: Some suppressors were tested for their effect on w""; P((w,ry)A] 4-4, a stock in which a w+ gene inserted at lOOF is subjected to variegation induced by proximity to the right telomere of the third chromosome (HAZELRIGG et al. 1984; LEVIS et al. 1985). Suppressed +/Y; b d / + ; st males were crossed to

"s t male progeny (with second chromosomes either b d / + or +/+) were evaluated for suppression.

Test for suppression of trans-inactivation: P[bw+]92C is trans-inactivated by its variegating derivative V21" (DREESEN et al. 1991). Some suppressors of b d / + were tested for their ability to suppress this trans-inactivation at 92C. Tests were conducted at 18 a , since this trans-inactivation is more pronounced at that temperature. Two different crossing

w J 1 1 8 . , P[(w,ry)A]4-4 virgin females. The wJJ18/Y; + P[(w,ry)A]

Modifiers of bp 563

bwD +; st + E] T(2:3) V2 1 bw sp; st Sb P[bw 'I " bwD Sp , st P[bwf]92C st P[bw+]92C * x % stubbly bristles; orange variegated eyes scarle7leyes

axilla speck;

1 bwD Sp; st + P[bw']92C I E] T(2;3) V21e, bwD f; st Sb P[bw']'

+ - bwDSp; St + P[bw']92C + T(2:3) VZC, bwD f; st Sb P[bw+lV

l - - - - - - - - - - 1 stubbly bristles; yellow variegated eyes

(control)

Su bwD+, St $q X + + w st

orange variegated eyes

/ Su bwD+ st

+ bwDsp' st P[bwf]92C ? ? X

dull scarlet eyes

t

I stubbly bristles; I I red variegated eyes I

I (suppression) I I """"" A

bwD sp . st P[bw'192C bwD sp ' st P[bwf]92C bb

T(23) V27' bwD sp;stSb P[bw+]'

bwD sp: st +

white eyes wlth very axillary speck; stubbly bristles;

few pigmented spots

T(2;3) V21: + bwD sp; st Sb P[bw+]" r - - - - - - - - - - I no axillary speck; I

1

I stubbly bristles; I yellow variegated eyes I

I

I (no suppression) ' L """"" J FIGURE 4.-Tests for suppression of trans-inactivation with V21e/

P [ h + ] 9 2 C . The strategies for testing suppressors unlinked to b4 (A) and for testing suppressors linked to b4 (B) differ to avoid crossing linked suppressors (which are rearrangements) onto T(2;3)V21'. The hypomorphic bw+ phenotype resulting from a single dose of P [ h + ] (DREESEN et al. 1991) is described as ''dull scarlet." See MATERIALS AND METHODS for additional details.

strategies were required depending on whether or not the suppressor was linked to bwD (Figure 4). Unlinked suppres- sors yielded a class of red-variegated (suppressed) V21'/ P[bw+]92C progeny, whereas linked suppressors yielded only yellow-variegated (nonsuppressed) V2lU/P[bw+]92C progeny. To verify that the Su(bd) was indeed present in these nonsuppressed V21e/P[bw+]92C flies, individuals were crossed to bw+ sp; st and their progeny backcrossed to bw+ sp; st to remove the P[bw+]-bearing chromosomes (not shown). Suppressed b d +/+ sp; st flies reappeared, indicat-

ing that the Su(bp) was present in the non-suppressed V21'/ P[bw+]92C grandparent.

A single suppressor, Su(bd)44, which was not linked to b p , gave inconsistent results in the test for suppression of cis-inactivation, apparently because the suppression was very weak. The test for suppression of trans-inactivation with this suppressor followed the procedure outlined above for linked suppressors, except that the V21e/P[bw+]92C flies clearly fell into suppressed and nonsuppressed classes. Individuals in both classes were tested as described above to verify the presence of the suppressor in individuals of the former but not the latter class.

Complementation tests: A subset of suppressors, en- hancers, recombinants and deficiencies were tested for com- mon lethal mutations in crosses that generally took the form lethal#l/SMl X lethal#2/SM1. Between 100 and 200 prog- eny were scored for survival of the lethal#l/lethal#2 progeny except in a few crosses where it was clear that these progeny were viable after scoring 50-70 progeny. The combinations were classified as lethal if no lethal#l/lethal#2 progeny were recovered, as semilethal if fewer than 10% of the expected progeny (as estimated from the sibling classes) were re- covered, and as viable otherwise.

Pairing tests: Since a large fraction of the suppressors were rearrangements of chromosome arm 2R that might potentially have affected pairing between homologs, several 2R chromosome rearrangements (with bw+) were tested for effects on the eye phenotype of b d as follows: crosses were performed using appropriate markers to make b d / + ; st flies carrying the rearrangements on the bw+ chromosome. In four cases, these were compared with MI+; st sibs to determine any suppressive effects of the rearrangements on the eye phenotype. In the remaining two cases, they were compared with standard b d +/+ sp; st flies.

Cytology: Suppressors and enhancers showing linkage to b d were examined for rearrangements on the chromosome arm carrying b d (2R). Suppressor-bearing or enhancer- bearing W/+; st flies were crossed to bw; st flies, and wandering third-instar larvae were identified as being b d / bw; st by their colorless (rather than yellow) Malpighian tubules. These larvae would carry any rearrangements linked to b d . Salivary glands were dissected in 45% acetic acid and then transferred to a drop of 45% acetic acid 1% orcein on a siliconized coverslip. The coverslip and glands were transferred to a clean slide and examined on an in- verted microscope while gently tapping the coverslip with a pencil. When chromosomes were well spread, they were examined by phase contrast microscopy.

RESULTS

Trans-inactivation at the bw locus is dependent on pairing between a heterochromatin-associated bw al- lele and its homolog, but specific sequences from the bw gene region are not required adjacent to the het- erochromatic breakpoint (HENIKOFF et al. 1993). To explain this, a model for trans-inactivation was pro- posed in which a protein essential for bw transcription becomes inactivated when it binds at the bw locus and is brought into contact with heterochromatin-binding proteins by somatic pairing of adjacent homologous sequences. Although it mediates truns-inactivation, such a protein might be expected to have no role in cis-inactivation. We reasoned that a mutation in this hypothetical factor which rendered it insensitive to

564 P. B. Talbert, C. D. S. LeCiel and S. Henikoff

contact with heterochromatin, but still permitted bw transcription, might act as a suppressor specific for trans-inactivation and be genetically identifiable on this basis. Although it is not possible to predict whether such a mutation would be dominant or re- cessive, a dominant mutation would be much easier to identify both in an initial screen for phenotypic suppression and in subsequent genetic tests.

Isolation of modifiers: We chose to look for sup- pressors of the dominant variegating allele bd. This allele is the strongest of all trans-inactivating bw alleles and is fully viable. This allele is also null (see MATE- RIALS AND METHODS) and results in a completely un- pigmented eye in a mutant scarlet background (in which ommochrome pigments are absent). As a con- sequence, cis-inactivation of brown in b d is undetect- able and the level of pteridine production in M/+; st flies directly reflects trans-inactivation of the bw+ allele. Flies of this genotype produce pigment in a number of individual ommatidia scattered throughout the otherwise very pale eye (Figure 1 , lower right). The consistency of this variegated phenotype among flies allows sensitive identification of suppressors, al- though the overall lack of pigment makes enhancers more difficult to detect. We treated b d ; st males with EMS and crossed them to bw+; st females (Figure 2). Approximately 100,000 b d / + offspring were screened to yield 150 dominant suppressor mutations [Su(bd)s] with more eye pigment than their b d / + ; st siblings (Figure 1, upper right). With one exception (discussed below), all of the suppressed lines still had variegated eyes. Thirty-nine of the suppressors either proved to be too weak to reliably identify in crosses or were lost prior to analysis. Two dominant enhancer mutations [ E ( b d ) s ] were also recovered in which flies had fewer pigmented ommatidia than their b d / + ; st siblings (Figure 1 , lower left).

Suppression of cis-inactivation: The suppressors were tested for their ability to suppress cis-inactivation of the bw+ gene on the V21" translocation (Figure 3). This translocation juxtaposes heterochromatin from the base of 2R to a bw+ gene present within a P element transposon at 92C on the third chromosome (HENIKOFF et al. 1993). The bw gene at 92C on the V21' chromosome is strongly cis-inactivated, yielding an essentially white eye with only a few pigmented ommatidia in a homozygous b d ; st background. Since this gene has no paired homolog on a normal (st) third chromosome, trans-inactivation cannot occur and any phenotypic suppression observed must be due to the suppression of cis-inactivation.

Of 1 1 1 suppressors tested, 87 suppressed the cis- inactivation of bw+ on V21", and 24 did not. One of the 87, designated Su(bd)44, suppressed V21" only weakly and inconsistently and was initially classified as failing to suppress. It will be discussed further below.

None of the tested suppressors of cis-inactivation showed tight linkage to b d , and so are second-site suppressors. Although no systematic effort was made to map them, in the course of further testing it became apparent that suppressors were recovered on both of the large autosomes. A single recessive suppressor, su(bd)62, is X-linked. Since this suppressor was (nec- essarily) recovered from a heterozygous female, this female may have been selected because of a second dominant suppressor in the same fly that subsequently segregated away and was lost.

To determine whether these suppressors of cis- inactivation were typical Su(var) mutations or might represent a distinct class of suppressors of bw varie- gation, 37 of these 87 Su(@)s were tested for their effect on wm4. All but four suppressed wm4. We there- fore concluded that about 90% of the suppressors of cis-inactivation were likely to be typical Su(var)s.

Failure to suppress telomeric variegation: Genes transposed to Drosophila telomeres are also subject to variegated position effects (HAZELRIGG et al. 1984). The stock w1'18; P[(w,ry)A]4-4 carries the w+ gene inserted next to the 3R telomere which causes the gene to variegate. Thirty-four of the 87 Su(bd)s were crossed to P[(w,ry)A]4-4. All failed to suppress the telomeric variegation, consistent with tests of other modifiers of wm4 (R. LEVIS, unpublished data). This suggests that while telomeric variegation appears phe- notypically similar to PEV caused by pericentric het- erochromatin, different genes probably are involved in these two phenomena.

Suppression of trans-inactivation: The 24 Su(bw")s that do not suppress cis-inactivation in V21" might be expected to include any suppressors that specifically affect trans- but not cis-inactivation, as well as any that are allele-specific suppressors or revertants of b d . To determine if any of the Su(bd)s are specific suppres- sors of trans-inactivation, candidates were tested for their ability to increase the amount of eye pigmenta- tion in V21*/P[bw+]92C flies (see MATERIALS AND METHODS). P[bw+]92C is the parent allele of V21', and the bw+ genes on these chromosomes can pair so that V21" acts as a moderately strong trans-inactivator of P[bw+]92C at 18 O .

As a preliminary positive control, six of the 87 Su(bd)s that suppress cis-inactivation were tested for suppression of V2lC/P[bw+]92C flies (Figure 4A); all showed increases in eye pigmentation consistent with suppression of trans-inactivation. This was especially clear for Su(bd)44, an unlinked suppressor that was originally scored as having no effect on cis-inactiva- tion. Repeated assays revealed a weak effect of Su(bd)44 on cis-inactivation and a pronounced effect on trans-inactivation. The stronger effect of Su(bd)44 on expression from the trans copy of bw than the cis copy suggests that it might be a heterochromatic com-

Modifiers of bp 565

TABLE 1

Suppressors linked to b d

Separable from Suppresses

Suppressor0 b9, V21~lP[bW+]92C? Cytologyb Viable over Dx2R)aJ?

Su(bwD)5 No T(2;3)55B; 1 OOD Viable Su(bwD)20 Yes No T(1;2)1 lC;52D Su(MY2 Yes No T(1;2)2B;57D Su(bwD)55 No T(1;2)15A;54E Su(hD)59 No T(2;3)54C;62A Viable Su(bw")73 Yes T(1;2)15E;53F Viable Su(bwD)87 No No T(Y;2)59E Lethal Su(bwD)98 No ln(2R)59E;60E Viable Su(bwD)125 No No T(1;2;3)5A;8D;59E;74Cc Su(bwD)126 Yes No T(2;3)55F;lOOD Su(bwD)131 No In(2LR)26F;59E Viable Su(bwD)133 Yes No T(2;3)53A;lOOB9 Su(bwD)151 No No T(1;2)17E;57A Viable Su(bwD)l 58 Yes No T(1;2)8E;56E Su(bwD)l 69 No Unrearranged Viable Su(bwD)l 89 No Dj(2R)59E;6OC Viable

a Phenotypes of all suppressors revealed no consistent differences, except for Su(bwD)169, which is weak, and Su(bwD)189, which showed

* All suppressors except Su(bwD)189 have a doublet band at 59E associated with bd'. no variegation. New ;;der uncertain.

. .

ponent that is more directly involved in mediating trans-inactivation than the other suppressors of cis- inactivation.

The 24 Su(bwD)s that did not suppress cis-inactiva- tion in the test with V21a all showed linkage to b d in crosses. Six were clearly separable from the bw locus, but others were apparently inseparable, since no un- suppressed b d / + flies were recovered during stock maintenance (Table 1). Five of the six separable linked suppressors and three apparently inseparable suppres- sors were tested for their effects on the phenotype of V21g/P[bw+]92C flies (Figure 4B). None of these eight S u ( b d ) s suppressed either cis- or trans-inactivation of the bw+ insert at 92C (Table 1). These results sug- gested that many or all of the Su(bd)s linked to b d are specific suppressors of the b d allele, since they do not affect cis- or trans-inactivation when present with other variegating alleles.

Cytology of suppressors linked to bz8: The exist- ence of at least 5 Su(bd)s that were linked to but separable from b d and that were apparently specific suppressors of b d raised the possibility of an unusual interaction between bwD and a neighboring locus or cluster of loci. Most of the 24 linked Su(6d)s behaved genetically like translocations: for example, 11 Su(bwD)s were either male-lethal or male-sterile, indi- cating that they were probably translocations between the X and second chromosomes (LINDSLEY 1982). Cytological examination was undertaken to determine whether breakpoints common to different re- arrangements would identify a unique suppressor lo- cus. The cytology for the surviving 16 of the original

24 linked Su(M)s is presented in Table 1. Of the 16 suppressors, five have breakpoints at 59E,

the site of the bw gene: one of these is a deficiency, two are inversions, one is a translocation to the Y chromosome, and one is a complex rearrangement involving three chromosome arms. Su(bwD)I89 deletes the b d doublet at 59E, which explains why this chro- mosome alone of all the Su(bd)s does not cause de- tectable trans-inactivation. Su(M)87 segregates ster- ile D m Dp(2R)59E;60F, bp +/bw+ sp/bw+ sp; st males as well as fertile T(y;2)59E, b d +/bw+ sp; st males. The former are distinguished by their more deeply pigmented red-variegated eyes, presumably a result of having two bw+ genes compared with only one in their less pigmented fertile brothers. Su(bd)87 and the other three lesions at 59E might be expected to disrupt pairing at bw, which could account for the observed phenotypic suppression.

The remaining 1 1 linked suppressors do not show alterations at 59E: one is a weak suppressor that is cytologically indistinguishable from its b d parent chromosome, six are translocations to the X chromo- some, and four are translocations to the third chro- mosome. The distribution of breakpoints in the latter 10 rearrangements appears to be very nonrandom (Figure 5). The breakpoints on the second chromo- some are all between 52D and 57D, although no two are in the same place. The six breakpoints on the X chromosome are scattered from 2B to 17E. In con- trast, the four breakpoints on the third chromosome are at the distal tips. Given the clustered but noncoin- cident distribution of breakpoints on 2R, it seems

566 P. B. Talbert, C. D. S. LeCiel and S. Henikoff

Suppressors

2L I

Enhancers 2L 1

II I II

4 + FIGURE 5.-Distribution of translocation breakpoints of suppres-

sors and enhancers. T(J;2) and T(2;3) suppressors as well as T(2;het) enhancers all have breakpoints on chromosome arm 2R clustered between 52D and 57D, proximal to bup at 59E. The breakpoints on the third chromosome of T(2;3) suppressors are confined to the distal tips, while the breakpoints on the X chromosome of T(1;2) suppressors are widely distributed. The enhancers have breakpoints in heterochromatin: E(b4)40 = T(2;4)54F;JOIhet and E(bup)144 = T(2;3)54B;8Ohet.

unlikely that any breakpoint represents the position of a gene that acts as an allele-specific suppressor of M/+. It appears instead that translocations in a por- tion of chromosome arm 2R proximal to bp can affect its ability to trans-inactivate its homolog, and that all such translocations to the X chromosome affect trans-inactivation while only those at the tips of a large autosome are able to do so.

One possible way in which a rearrangement might affect trans-inactivation is by affecting the probability of synapsis of homologs. If a rearrangement in one member of a chromosome pair decreases the proba- bility of somatic pairing, we would expect that such rearrangements would suppress trans-inactivation. We tested six rearrangements of 2R isolated in other studies for their possible effects on trans-inactivation by bwD and found none (Table 2). One notable differ- ence between the rearrangements in these tests and the Su(bp)s, other than breakpoint position, is that the rearrangements listed in Table 2 are all on the bw+ chromosome, while the Su(bp)s are all re- arrangements on the bwD chromosome. While this difference in linkage should not affect pairing, it might affect other processes such as nuclear position- ing or heterochromatin formation at bp.

Enhancers: The distribution of rearrangement breakpoints at the distal tips of chromosome arms 3L and 3R in Su(bwD)5, Su(bwD)59, Su(bp)I26 and Su(bp)133 suggests that the placement of the bwD heterochromatic block distally, i e . , farther from the pericentric heterochromatin, may reduce the effi- ciency of trans-inactivation. This leads to the expec-

TABLE 2

2R rearrangements tested for suppression of trans-inactivation

Suppresses Rearrangement Cytology bg/+; Sf?

In(2R)AA21 + Dfl2R)AAZJ In(2R)56E;58E + No DfT2R)56F;57D

w2R)vgu In(2R)49C;5OC No Tp(2;y)bw+ Tp(2;y)58F- Noa

T(2;3)Antpd T(2;3)4 1 F;84B No T(2;3)C287 T(2;3)56D;89F No T(2;3)Ta’ T(2;3)5lE;84B No

a Data from (HENIKOFT and DREESEN 1989).

59A;60EF

A.

FIGURE 6.-Association of bup with the chromocenter in a 7‘(2;3)54B;80het. E(b4)144 b4/+bw; st salivary gland nucleus. The entire synapsed right arms of the second chromosomes are visible. The bw chromosome arm is continuous while the E(bup)J44 bup arm is interrupted by the translocation at 54B and is associated with the heterochromatic chromocenter at 59E, the location of b4.

tation that, conversely, enhanced trans-inactivation will be observed when the bp heterochromatic block is moved proximally, i e . , closer to the pericentric heterochromatin. This is precisely what was seen for the two E ( b p ) s isolated in the screen for mutations.

Cytological examination showed that both E(bp)s were associated with translocations of 2R to het- erochromatin: E ( b p ) 4 0 is T(2;4)54F;IOlhet and E(bwD)144 is T(2;3)54B;80het. It is interesting that both E(bup) breakpoints on 2R are in the same region as the 2R breakpoints of the Su(bp)s. Enhancement cannot be explained by extreme heterochromatic spreading, since the distance from the breakpoint to bwD is several-fold greater than has been previously observed (SPOFTORD 1976), and since the banding pattern of the intervening euchromatin is unaffected (Figure 6). An alternative explanation for enhance- ment is suggested by a striking feature of the polytene chromosome cytology of these enhancers: the bwD heterochromatic block at 59E is frequently associated with the chromocenter in both stocks (Figure 6), al- though it is almost never associated with the chrom- ocenter in bp/+ (Table 3). Since the two enhancers have different translocation breakpoints, the associa- tion of 59E with the chromocenter is evidently

Modifiers of b P 567

TABLE 3

Association of a4 with heterochromatin of salivary chromosomesa

Distance of b9 from

heterochromatin Nuclei with Nuclei with (no. of lettered b9 at b9 not at

Genotype subdivisions) chromocenter chromocenter

bwD/+ 113 1 (2%) 50 T(2;4) E(bwD)40/bw 29 29 (78%) 8 E ( b ~ ~ ) 4 ~ ~ / b w 113 0 (0%) 42 T(2;3) E(bwD)144/bw 33 37 (86%) 6 E(b4)144REc/bw 113 4 (8%) 45 T(2;3) Su(bwD)5/bw 143 0 (0%) 74 T(1;2) Scl(bwD)73/bw 67 10 (21%) 38 T(1;2) Su(b4)151/bw 35 23b (66%) 12

,I Based on combined data for scorable nuclei from two squash preparations per genotype.

For all nine nuclei in which X-heterochromatin had separated from the bulk of the chromocenter, b w D was associated with X, not autosomal heterochromatin.

brought about by the proximity of b d to the hetero- chromatic breakpoints, and not to disruption of a gene on 2R euchromatin.

Evidence that the association of b d with the chrom- ocenter of salivary nuclei correlates with enhancement of PEV is provided by recombinant derivatives of the translocations. The recombinant E(bd)144REC was re- covered from our E(bd)144 b d +/+ + sp selection stock by virtue of its nonenhanced phenotype, which was indistinguishable from that of b d / + . A sim- ilar recombinant, E ( b d ) 4 P c , was obtained from E(bwD)40 b d +/+ + sp. E ( b d ) 4 P c is semilethal as a homozygote and shares its semilethality with E ( b d ) 4 0 , verifying its derivation from this enhancer. Cytologi- cal examination revealed that in both recombinants the b d heterochromatic block had recombined away from the translocation onto the normal second chro- mosome, and that 59E is no longer associated with the chromocenter (Table 3). Furthermore, both E ( b d ) 4 0 and E(bd)144 are semilethal over the small deficiency, Dfl2R)bw'. Semilethality in each case can be explained as enhanced cis-inactivation of a vital gene uncovered by the deficiency. This enhancement of cis-inactivation does not occur in the viable recom- binant E(bd)144REC, nor in E ( b d ) 4 p c , which is via- ble over Df12R)bw5, even though the bw region should be identical in these E ( b d ) s and the recombinants derived from them. Therefore enhancement of both cis-inactivation of a vital locus and trans-inactivation of bw' is relieved by separating b d from a transloca- tion breakpoint that causes it to associate with the chromocenter in salivary nuclei.

An example of a suppressor with a distal breakpoint, Su(bd)5 , showed no association between b d and the chromocenter of salivary gland chromosomes (Table 3). However, three of the bd-specific suppressors that translocate b d proximally did exhibit an association.

Su(bd)87 places band 59E1-2 adjacent to the hetero- chromatic Y chromosome. It is possible that the le- thality of Su(bd)87 over Df12R)bw5 (Table 1) is due to enhancement of cis-inactivation in this stock: the jux- taposition to pericentric heterochromatin may cause spreading to occur into a neighboring essential gene. However, the pairing of b d with its homolog is visibly disrupted in most Su(bd)87 salivary gland nuclei (P. B. TALBERT, unpublished data). A similar pairing disruption in the pigment cell nuclei would lead to suppression of trans-inactivation. Su(bp)73 and Su(bd)151 translocate bwD very proximally onto the X chromosome (15E and 17E, respectively), leading to association of b d with the base of the X chromo- some in salivary gland nuclei (Table 3). In many of these nuclei, the base of the X (and b d ) is separated from the rest of the chromocenter. If this feature parallels a difference in the position of the X chro- mosome in pigment cells of the eye, it might account for the lack of enhancement in these rearrangements.

DISCUSSION

A screen for modifiers of b d position-effect varie- gation led to the isolation of an unexpected class of mutations: translocations that alter the linkage of the b d heterochromatic element without affecting the element itself. These translocations all have one breakpoint within a region that is proximal to but separable from b d , and have the other breakpoint at a position that depends upon whether the chromo- some involved is the X or an autosome. Recovery of this unexpected class is accounted for by differences between our screen, designed to obtain specific dom- inant suppressors of trans-inactivation, and earlier studies.

Search for specific suppressors of trans-inactiva- tion: We sought mutations in a hypothetical hetero- chromatin-sensitive transcription factor that specifi- cally suppress trans-inactivation. The desired muta- tions would retain bw transcriptional activity while exerting dominant insensitivity to the presence of heterochromatin. None of the suppressors strictly ful- filled the criteria we imposed to identify such a mu- tation: that it have no effect on cis-inactivation but be able to dominantly suppress trans-inactivation at an ectopic site (92C) as well as at the bw locus. It may be that this combination of characteristics is very improb- able for mutations in the hypothetical transcription factor. For example, suppressors specific for trans- inactivation may only be recoverable as recessives. Alternatively, it may be that the hypothetical tran- scription factor does not exist and trans-inactivation is a direct interaction between the primary sequence or secondary structure of DNA in or near the bw gene and heterochromatic proteins brought near by pairing between homologs. Sequence analysis of the bw gene

568 P. B. Talbert, C . D. S . LeCiel and S. Henikoff

has not revealed any repetitive or unusual sequences that would make obvious targets for heterochromatin- forming proteins (MARTIN-MORRIS et al. 1993). The resolution of this question may be helped by further characterization of the bw sequences necessary to me- diate trans-inactivation.

Unlinked suppressors: The Su(bd)s can be grouped into two large classes based on their linkage to the bw locus and their ability to suppress cis-inacti- vation of V 2 P . The majority of suppressors are clas- sical Su(uar)s, typically unlinked to b w , which generally suppress &inactivating PEV mutations. Thus, despite its especially strong dominant effect caused by an interstitial insertion of heterochromatin, it is clear that b d is in many respects a typical PEV mutation. Pre- vious screens for Su(uur)s and E[var)s utilizing wm4 have failed to identify any Su(uar)s on the X chromosome. While our results are generally consistent, since we have not identified any dominant suppressors on the X chromosome, we did recover an X-linked recessive suppressor of variegation. It has previously been noted (GRIGLIATTI 1991) that a minority of Su(uar)s show some allele-specificity and this also may be true of about 10% of the Su(bd)s. None of the 34 tested Su(bd)s had any effect on telomeric variegation, in- dicating that different components are responsible for variegation caused by telomeric and centromeric re- gions.

Linked suppressors: The second large class of Su(bd)s consists of 24 suppressors linked to 6 2 8 itself. All of these failed to suppress the cis-inactivation of V21', and eight that were tested also failed to suppress trans-inactivation by this rearrangement, indicating that they are allele-specific suppressors of b d . All but one of the 16 examined cytologically proved to have rearrangements on 2R. Since EMS is thought to in- duce mutations primarily by base substitution (COTE et al. 1986), the almost perfect correlation between rearrangements linked to b d and allele-specific suppression is compelling evidence that it is the struc- tural alterations of the chromosome arm carrying b d rather than particular loci disrupted by the re- arrangements that lead to allele-specific suppression.

These bd-linked rearrangements can be further subdivided into three groups: rearrangements at 59E, translocations to the X chromosome, and transloca- tions to the distal tips of the third chromosome. The first type of rearrangement was recovered by HINTON and GOODSMITH (1 950) in their reversion study of b d using X-irradiation. Rearrangements at 59E would be expected to disrupt pairing locally, preventing trans- inactivation in some cells. They might also dissect the b d heterochromatic block into two pieces, altering its ability to induce variegation.

The other two classes of rearrangements were not recovered by HINTON and GOODSMITH. The de-

creased sensitivity to phenotypic changes afforded by using a st+ background may account for their failure to recover them. In addition, the b d allele appears to have changed structurally since 1950 (see MATE- RIALS AND METHODS), and its phenotype may have been less sensitive to this type of rearrangement at that time.

Breakpoint distribution: Translocation break- points associated with b d / + suppressors and en- hancers are strikingly nonrandom (Figure 5). All 12 breaks proximal to b d are clustered within a region consisting of about 20% of chromosome arm 2R. If the entire arm proximal to b d is considered to be a potential target, then the chance probability of such clustering is about (0.2)'' lo-'. The autosomal breakpoints associated with suppressors are also non- randomly distributed along 3L and 3R, the two au- tosomal arms represented. All four breakpoints lie within the distalmost 5% or so of each arm, a distri- bution expected by chance to occur at P = (0.05)4 =

The rarity of these autosomal breakpoints rela- tive to the six scattered breakpoints on the X chro- mosome is consistent with the assumption that the large majority of possible autosomal translocations failed to lead to detectable phenotypic change and so were not selected in the screen. This assumption seems reasonable, since we are unaware of evidence for restricted occurrence of chromosomal rear- rangements following mutagenesis (ASHBURNER

The clustering of Su(bd) and E ( b d ) translocation breakpoints between 52D and 57D might suggest that this region is important for initiating synapsis of the 2R chromosome arms, since trans-inactivation is a somatic pairing-dependent phenomenon (HENIKOFF et al. 1993). However, trans-inactivation is not the only bd-associated phenotype affected by linkage altera- tions: each of the E ( b d ) s is also enhanced for cis- inactivation of a nearby essential gene, which is not expected to be sensitive to somatic pairing disruptions. In addition, it is difficult to explain why translocations that move the trans copy of bw, such as T(Y;Z)bw+Y and T(2;3)C287 (Table 2) have no effect. Other explana- tions for this puzzling clustering of breakpoints must be considered, such as a special position within the nucleus. In this regard, we note that the only signifi- cant point of association between the nuclear envelope and salivary chromosome arm 2R proximal to b d is within the 52D-57D region (HOCHSTRASSER et al. 1986; MATHOG and SEDAT 1989).

The distal translocation breakpoints on the third chromosomes of the T(2;3) suppressors together with the heterochromatic breakpoints in the enhancers suggest that distance from the chromocenter is an important determinant of heterochromatin formation on the autosomes. The T(2;3) suppressors increase the

1990).

Modifiers of b d 569

length of the chromosome arm bearing b p , while the enhancers greatly decrease it. The proximity of 67.8 to the centromere in the enhancers leads to its asso- ciation with the chromocenter of salivary nuclei (Fig- ure 6). Although we do not know the nuclear location of b d in pigment cells, the correlation between its chromocentral location in salivary nuclei and the en- hancement of both cis-inactivation and truns-inactiva- tion in both E ( b p ) s is consistent with a similar local- ization in pigment cells. This localization may facilitate heterochromatin formation in the b d heterochro- matic block, leading to the observed enhancement. The T(2;3) suppressors may make heterochromatin formation at the b d heterochromatic block more difficult by moving it farther than normal from the chromocentral compartment. It should be noted, however, that the linear distance along the chromo- some arm need not accurately reflect the three-dimen- sional spatial relationship between b d and a chro- mocentral compartment in pigment cells. For exam- ple, it is possible that the existence of Su(bwD) break- points throughout the X euchromatin is accounted for by a special compartmentalization or loose association of the X chromosome, which places it effectively “far- ther’’ from the chromocentral compartment.

The abundance of X-linked suppressors perhaps reflects a large target size for translocation breaks in X euchromatin relative to the autosomal tips. Of the 24 linked suppressors recovered, 11 showed male lethality or male sterility indicative of a T(2;2) trans- location, and this conclusion was confirmed for all seven of those that survived long enough for cytolog- ical examination. These results are reminiscent of the findings of KHVOSTOVA (1939) for the cubitus inter- ruptus (ci) locus, which is located in the vicinity of the heterochromatin-euchromatin junction of chromo- some 4. Normally, ci+ is dominant over the ci’ allele. However, translocations of ci+ show reduced domi- nance when the locus is moved to distal but not to proximal regions of the autosomal arms. In addition, translocations of ci+ to all euchromatic portions of the X chromosome similarly showed reduced dominance. So like the reduced dominance of b7.8 (over bw+) observed for suppressors obtained in our screen, re- duced dominance of ci+ (over ci’) was seen for trans- locations both to distal autosomal sites and to any- where on the euchromatic X. We suggest that both phenomena have a similar causal basis. Furthermore, since breaks proximal to ci+ are likely to be in heter- ochromatin, the repositioning of ci+ with adjacent heterochromatin to euchromatic regions resembles the repositioning of the b d heterochromatic element described here.

The results of KHVOSTOVA (1939) and those of PANSHIN (1938) also indicate that the distal hetero- chromatin of the sex chromosomes differs from other

heterochromatin. In particular, KHVOSTOVA observed that X heterochromatin distal to the bobbed locus, the site of the nucleolus organizer, behaved similarly to X or distal autosomal euchromatin in its ability to reduce the dominance of ci+, but X heterochromatin proximal to the bobbed locus behaved like autosomal hetero- chromatin. This suggests that the nucleolus might form a boundary between two distinct kinds of het- erochromatin, the more distal of which resembles euchromatin in some properties. This may be relevant to the failure of Su(b7.8)s broken in proximal X eu- chromatin to enhance variegation: the observed asso- ciation of b7.8 with the (sometimes displaced) base of the X chromosome in these suppressors may involve only the distal “euchromatic-like” heterochromatin.

Relationship to models for position-effect varie- gation: Early studies of position effects on genes lo- cated in proximal regions led to the notion that blocks of heterochromatin are important for the functioning of heterochromatic genes (KHVOSTOVA 1939; LEWIS 1950; BAKER 1953; HESSLER 1958). Variegation of heterochromatic genes has been hypothesized to re- flect their association with a chromocentral compart- ment in some cells but not others (WAKIMOTO and HEARN 1990). These position effects depend on the distance of heterochromatic genes from large heter- ochromatic blocks, not the centromere itself (PANSHIN 1938; EBERL et al. 1993). Furthermore, heterochro- matic regions may need to aggregate to function (WAKIMOTO and HEARN 1990; EBERL et al. 1993).

Our results provide complementary support for these ideas, in that the ability of a block of hetero- chromatin to inactivate eushromatic genes also ap- pears to depend on its proximity to other heterochro- matin. This proximity might be necessary for access to heterochromatin-binding proteins localized in a chromocenter (WAKIMOTO and HEARN 1990). Al- though no association of b d with other heterochro- matic blocks is observed in b d / + polytene salivary glands, we suggest that this association occurs in the thinner and presumably more flexible diploid chro- mosomes of the pigment cells. The probability of this association, and therefore of cis- and truns-inactiva- tion, may be strongly dependent on the distance of bwD from the heterochromatic pericentric region as is observed in salivary nuclei of the E(bwD)s. The prob- ability of association might be decreased in the X- linked Su(bd)s because of the presence in X-hetero- chromatin of the nucleolus, which is suggested to impede access of b d to the chromocenter (A. HILLI- KER, personal communication). In the same manner, the nucleolus might interfere with the positioning of ci+ in X 4 translocations, thus accounting for the ability of all breakpoints distal to the nucleolus to reduce the dominance of ci+ over ci’ (KHVOSTOVA 1939).

Chromosome looping into a heterochromatic com-

570 P. B. Talbert, C. D. S. LeCiel and S. Henikoff

partment on a smaller scale could underlie the phe- nomenon of heterochromatic “spreading.” Faculta- tively susceptible euchromatic sequences might be recruited into the heterochromatic compartment by their close proximity. Such a model of spreading could explain the unusual cases of “discontinuous compac- tion” observed by BELYAEVA and ZHIMULEV (1991), in which polytene chromosome bands adopted a het- erochromatin-like appearance even though separated from heterochromatin by normally appearing bands. Perhaps in these cases, the heterochromatin-like bands are present in the heterochromatic compartment but are displaced when squashed preparations are made for cytological analysis.

This model might also account for an apparent discrepancy between our results (as well as those of R. LEVIS, unpublished data), which show that Su(var)s fail to suppress variegation of a w+ gene inserted into the 3R telomere, and those of KARPEN and SPRADLING (1992), which show that Y chromosomes indeed sup- press variegation of a rosy gene inserted into the telomere of a mini-chromosome, Dp(l;f l l l87. Since the telomere of Dp(l;f l l l87 is only about 250 kb from the pericentric heterochromatin (KARPEN and SPRA- DLING 1992), we suggest that the observed rosy varie- gation results from frequent looping into the chrom- ocenter where the gene becomes sensitive to PEV modifiers. In contrast, the 3R telomere is expected to be located at the opposite side of the nucleus (HIR- AOKA et al. 1993), where it would be unable to make contact with the chromocenter, and this difference would account for the insensitivity of the w+ gene to PEV modifiers. As is the case for the linkage altera- tions of b d described here, differences in variegating phenotypes would thus depend upon the relative lo- cations of the chromocenter and smaller genetic ele- ments (telomeres) which are rich in repetitive se- quences (KARPEN and SPRADLING 1992; R. LEVIS, unpublished data).

We thank JEFF JACKSON and ADRIAN QUINTANILLA for technical assistance and stock maintenance, and our many colleagues who provided critical comments on the manuscript. S. H. thanks GARY KARPEN and ALLAN SPRADLINC for discussions of the effects of Su(var)s on telomeric variegation and ART HILLIKER for pointing out that the nucleolus might impede associations between hetero- chromatic elements located on either side. This work was supported by grants to S. H. from the National Science Foundation (DCB8717937) and the National Institutes of Health (GM29009).

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Communicating editor: A. CHOVNICK