Modifiers of position-effect variegation in the region from 86C to 88B of the <i>Drosophila melanogaster</i> third chromosome

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Article

Reference

Modifiers of position-effect variegation in the region from 86C to 88B of the Drosophila melanogaster third chromosome

REUTER, Günther, et al.

Abstract

Four dominant suppressor and one enhancer of variegation loci were mapped in the polytene chromosome region extending from section 86C to section 88B of the Drosophila melanogaster third chromosome using a set of deficiencies. The suppressor locus Su-var(3)14 maps in 86CD, Su-var(3)13 in 86F4-7, Su-var(3)6 in 87B4-7 and Su-var(3)7 in 87E4-5. The enhancer locus E-var(3)3 maps in 87E12-F11. Su-var(3)13, Su-var(3)6 and Su-var(3)7 are also defined by point mutant alleles originally identified by other criteria (Reuter et al. 1986). Duplications covering the suppressor loci Su-var(3)14, Su-var(3)13, Su-var(3)6 and Su-var(3)7 were found to reduce considerably the haplo-abnormal effect of heterozygous point mutants of the corresponding loci. One suppressor locus, Su-var(3)7, maps within a region which has previously been cloned. The positions of deficiency breakpoints delimiting the suppressor locus indicate that all the necessary sequences for its function are located within 10 kb of cloned DNA.

REUTER, Günther, et al . Modifiers of position-effect variegation in the region from 86C to 88B of the Drosophila melanogaster third chromosome. Molecular and General Genetics , 1987, vol. 210, no. 3, p. 429-36

PMID : 3123888

DOI : 10.1007/BF00327193

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http://archive-ouverte.unige.ch/unige:9864

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Modifiers of position-effect variegation in the region

from 86C to 88B of the Drosophila melanogaster third chromosome

G. Reuter 1, j. Gausz 2, H. Gyurkovics

2,

B. Friede 1, R. Bang 1, A. Spierer 3, L.M.C. Hall 3, and P. Spierer 3 1 Department of Genetics, Martin Luther University, DDR-402 Halle, German Democratic Republic

2 Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, POB 521, Hungary 3 Department of Molecular Biology, University of Geneva, 30 quai E.-Ansermet, CH-1211 Geneva 4, Switzerland

Summary. Four dominant suppressor and one enhancer of variegation loci were mapped in the polytene chromosome region extending from section 86C to section 88B of the Drosophila melanogaster third chromosome using a set of deficiencies. The suppressor locus Su-var(3)14 maps in 86CD, Su-var(3)13 in 86F4-7, Su-var(3)6 in 87B4-7 and Su-var(3) 7 in 87E4-5. The enhancer locus E-vat(3)3 maps in 87E12-F11. Su-var(3) 13, Su-var(3)6 and Su-var(3) 7 are also defined by point mutant alleles originally identified by other criteria (Reuter et al. 1986). Duplications covering the suppressor loci Su-var(3) 14, Su-var(3) 13, Su-var(3)6 and Su-var(3)7 were found to reduce considerably the haplo-abnormal effect of heterozygous point mutants of the corresponding loci. One suppressor locus, Su-var(3)7, maps within a region which has previously been cloned.

The positions of deficiency breakpoints delimiting the suppressor locus indicate that all the necessary sequences for its function are located within 10 kb of cloned DNA.

Key words: Drosophila melanogaster - Position-effect variegation Cytogenetic mapping

Introduction

Euchromatic genes transposed by a chromosomal rear- rangement next to heterochromatic regions often show var- iegated expression of the mutant and wild-type phenotype when heterozygous with a mutant allele (reviewed by Lewis 1950; Baker 1968; Spofford 1976). Mutations affecting the extent of position-effect variegation are potentially valuable tools for studying the molecular mechanisms of gene inacti- vation in these instances, as these loci may be responsible for aspects of chromosome organization which are impor- tant for gene expression. Dominant suppressors as well as enhancers of position-effect variegation have been isolated and their genetic characteristics established (Henikoff 1979;

Reuter and Wolff 1981; Sinclair et al. 1983; Reuter et al.

1986).

Several dominant suppressors of variegation have been identified in the region from 86 to 88 of chromosome 3R (Henikoff 1979; Reuter et al. 1986). We have undertaken a further cytogenetic mapping of the corresponding genes.

This is facilitated by the fact that this region has already Offprint requests to." P. Spierer

been subjected to extensive genetic and cytogenetic analysis (Gausz et al. 1979; Hilliker et al. 1980; Gausz et al. 1981, 1986).

In this study, the modifiers of position-effect variegation with haplo-abnormal behaviour were mapped by segmental aneuploidy. The genetic characteristics of these loci were further established by studying the effects of duplications on mutant alleles. Some loci corresponded to known mutations or mutations described here. Finally, we made the first step towards molecular analysis of a suppressor locus by mapping the Su-var(3)7 locus to within a 10 kb region of DNA that we had cloned previously.

Materials and methods

Stocks and mutations. Flies were raised at 25 ° C under un- crowded conditions on standard Drosophila medium containing Tegosept M as a mould inhibitor. Description of chromosomes and mutations used in this study can be found in Lindsley and Grell (1968), and in other references cited below, or are described in this paper. Dominant suppressor mutations of position-effect variegation were isolated on the basis of their effect on white variegation in a w m4h background, using a selective system (Reuter and Wolff 1981). Those mutations studied here are described in Table 1.

Deficiencies uncovering different portions of the 86- 88 region are described in Results (Table 3) with their breakpoints and origin. The P and GE series of deficiencies were induced by X rays on chromosomes carrying the TE98 (w + rst +) or TE39 (w + rst +) transposons of Ising and Block (1981), respectively. The effects of the deficiencies on white variegation in wm4h/Y males were determined after outcrossing homozygote w m4h; + / + females to + / Y; Deficiency/

Bal males. The different balancer chromosomes (TM3, MKRS=Tp(3)MKRS, M(3)$34 kar ry2Sb and MRS which is like MKRS except for being kar +) do not affect position-effect variegation in w m4k flies. The wm4h/Y; Ball + genotype in each cross was used as a reference. The white variegation was quantified by measurements of the relative content of the red eye pigment (Reuter and Wolff 1981).

Lethals isolated independently within polytene sections 86 and 87 are described in Hilliker et al. (1980) and Gausz et al. (1981). These mutations were used for complementation analysis with the dominant modifiers of position-effect variegation. The eye pigment content of allelic mutations was also determined in a w m4h background.

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Table 1. Dominant suppressor mutations of position-effect variegation

Locus/mutation Source Cytology Viability Su-var(3) 6

Su-var(3)6 °1 EMS Normal Homozygotes strongly semi-lethal Df(3R)Su-var6 X rays Df(3R)87BI,2- Recessive lethal

5,6; 87D9-11

T(Y;3)15 X rays T(Y;3)Y; Lethal over a 87B5-7 deficiency of 87B Su-var(3) 7

T(Y;3)409 X rays T(Y;3)Y; Lethal over a 87E2-4 deficiency of 87E Su-var (3) 13

Su-var(3)13 °1 EMS Normal Recessive lethal No mutations have been identified for the Su-var(3)14 locus uncovered by deficiency Df(3R)cu 4°. All mutations except Su- var(3)13 °1 are described by Reuter et al. (1986)

Complementation analysis. The dominant suppressors of position-effect variegation used in the present study are homozygous lethal, semi-lethal or represent rearrangements with one breakpoint in the region analysed. The mutations were tested for non-complementation with the deficiencies of region 86C to 88B. Bal/Df females were mated to Su/TM3 and T(Y;3)se ss e ro males respectively and the viability of Su/Df transheterozygotes was recorded. These transheterozygotes are lethal if the deficiency uncovers the corresponding suppressor locus. Independently isolated lethals in the region from 86 to 87 (Hilliker et al. 1980; Gausz et al. 1981) were also tested for alMism with the suppressor mutations. The mutations were taken as allelic if transheterozygotes were lethal or significantly reduced in viability (semi-lethal).

Isolation of tandem duplications. Tandem duplications for the region from 86 to 87 were isolated as suppressors of Minute MS31. After irradiating w m4h; + / + females (4,000 tad) and mating to +/Y; Df(3R)cu4°Sb/TM6 males in the offspring, Df(3R)cu4°Sb/+ flies were inspected for the M + phenotype. Among 43,000 progeny, 54 M + flies (1.3 x 10 -3) were found with 5 clusters, and 7 proved to represent tandem duplications. Six duplications were used in our studies and are described in Table 2. Their effect on suppressor functions in the region from 86 to 87 was quantified by measurements of the relative content of red eye pigments after outcrossing with suppressor strains.

Results

Deficiency mapping and genetic characterization of position-effect variegation modifying loci in the region from 86C to 88B

Henikoff (1979) identified by deficiencies two haplo-abnor- real loci suppressing position-effect variegation in polytene chromosome section 87. One was mapped within polytene subsection 87B and the other within 87E. In another genetic analysis Reuter et al. (1986) found 12 loci on the third chromosome showing a dominant suppressor effect. For a pre-

Table 2. Tandem duplications

Duplication Breakpoints Viability"

Dp(3; 3)D1 86A1 ; 87C3-5 -

Dp(3; 3)D2 86AI-5; 86E9-Ft -

Dp(3; 3)D7 85F8-16; 87F2-7 -- Dp(3; 3)D10 86C2-7; 86E5-7 sl Dp(3; 3)D26 86B4-C1 ; 86D5-8 sl Dp(3; 3)D45 86C2-7; 86D8-E1 + a Viability of homozygotes: -, lethal; sl, semi-lethal; +, viable

cise cytogenetic mapping of suppressors as well as enhancers of position-effect variegation in the chromosomal region from 86C to 88B, we used a set of deficiencies and analysed them in a w "*h background. Suppressor mutations were tested for alMism with appropriate deficiencies (Ta- ble 3 and Fig. 1). Within the 86C to 88B interval we mapped four distinct suppressor loci and one enhancer of position- effect variegation.

Su-var (3) 14

Deficiency Df(3R)cu 4°, but not the smaller deficiency Df(3R)MS-31, shows a dominant suppressor effect in the w m4h background. Therefore, at least one suppressor locus is located either between 86C1-2 and 86D1-2 or between 86D4 and 86D8. The ratio of the relative content of red eye pigment in w m4h males of the sibling genotypes Df/Dp and Dp/TM6 generated by a cross of w m4h ;Dp/TM3 females with +/Y; Df(3R)cu4°Sb/TM6 males is 0.8 with Dp(3;3)DIO (Table 2) and 1.3 with Dp(3;3)D45 (Table 2) whereas Df/+ compared with TM6/+ gives a ratio of 3.2 (Table3). Therefore, since in transheterozygotes of Df(3R)cu 4° with duplications Dp(3 ;3)DIO and Dp (3 ;3)D45 the suppressor effect is abolished, the existence of a second site mutation on the deficiency bearing chromosome can be ruled out. Deficiency Df(3R)Kx-1 extends from 86C1 to 87BI-5 and shows an elevated suppressor effect (Table 3). This deficiency includes Su-var(3)14 and another suppressor locus, located more distally, Su- var(3)13. The balancer chromosome TM6 (Lindsley and Grell 1968) shows no effect on position-effect variegation.

No point mutant allele has been found for the Su-var(3)14 locus.

Su-var (3) 13

A second suppressor was found to map between the distal breakpoint of the deficiency Df(3R) TO7 and the proximal breakpoint of Df(3R)E-229, delineating the chromosome interval 86F4-5 to 86F6-7 (Fig. 1). The weak suppressor allele, Su-var(3)13 °1 is homozygous lethal and transheterozygotes with deficiencies uncovering the interval from 86F4- 5 to 86F6-7 are also lethal. Gausz et al. (1981) identified two lethal groups in this region. Allelism was found on the basis of lethality with the members of the ck3 complementation group. The two ck3 lethals tested (e277 and e297), however, do not show a suppressor effect. Duplica- tion mapping, on the other hand, proved that the dominant suppressor function has to be localized to the region (cf.

Table 6). One possible explanation might be that only amorphic or strongly hypomorphic mutations of the locus result in a detectable suppressor effect. Otherwise, Su-

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Table 3. Deficiency mapping and complementation analysis of position-effect variegation modifying functions in the region from 86C to 88B

Deficiencies Breakpoints Source" Effect on w m~h variegation b Complementation with Su-var(3) mutations c R modifying effect 13 °1 6 °1 T(Y; 3)15 T(Y; 3)409

Df(3R) cu 4° 86CI-2;86D8 (1) 3.2 Su + + 0 0

Df(3R)MS-31 86D1-2;86D4 (1) 1.2 0 0 0 0

Df(3R)Kx-1 86C1 ;87B1-5 (11) 19.0 Su - + + +

Df(3R) P-79 86EI-2;87B1-2 (5) 3.5 Su - + + 0

Df(3R) T-45 86F1-2;87B5-6 (4) 5.8 Su - + + 0

Df(3R) T-32 86E2-4 ;87C6-7 (2) 6.5 Su -- - -- +

Df(3R) P-IO 86E6-9;87A9-B1 (5) 4.2 Su -- 0 0 0

Df(3R)P-21 86E19-FI ;87Bl1-15 (5) 10.5 Su 0 - 0 0

Df(3R) T-07 86F1-2;86F4-5 (4) 1.4 - + 0 0 0

Df(3R)E-079 86F1-2;87BS-10 (4) 17.6 Su -- - -- 0

Df(3R) T-IO 86F1-2 ;87C5-7 (2) 5.2 Su - - - +

Df(3R) P-35 86F3-4;87B5-8 (5) 4.1 Su -- + + 0

Df(3R)E-229 86F6-7;87BI-2 (4) 1.0 - + + + 0

Df(3R) P-29 87A4-5;87B4-5 (5) 1.3 - 0 + 0 0

Df(3R)kar 3Q 87B2-4;87Dt-2 (8) 4.4 Su + 0 0 0

Df(3R)kar m 87A6-8 ;87D3-4 (6) 8.2 Su + 0 0 0

Df(3R)kar lw 87A6-7;87DI2-13 (8) 24.8 Su + - 0 0

Df(3R)kar sz12 87B1-3 ;87C8-9 (3) 8.7 Su 0 0 0 0

Df(3R)kar sz3° 87B2-4;87D2-3 (3) 8.0 Su 0 - 0 0

Df(3R)kar 3s 87Bt 5-C1 ;87C9-D1-2 (8) 1.0 - 0 0 0 0

Df(3R)kar sz8 87C1-3;87D14-15 (3) 1.0 - 0 0 0 +

Df(3R) kar 3z 87C2-3 ;87C9-D1 (6) 1.0 - 0 0 0 0

Df(3R)kar sz37 87C5-6;87D14-E1 (3) 1.3 0 + 0 +

Df(3R)kar szll 87C7-8;87E5-6 (3) 4.1 Su 0 + 0 --

Df(3R)kar szz8 87C7-8 ;87E9-10 (3) 3.9 Su + + + --

Df(3R)ry 614 87D2-4;87Dll-14 (7) 0.7 - 0 0 0 0

Df(3R) ry 14°2 87D2-4;87D14-E2 (7) 1.0 0 0 0 +

Df(3R)ry 16°7 87D4-6;87E1-2 (7) 1.0 - 0 0 0 +

Df(3R)126d 87D11-13;87E3-5 (7) 4.7 Su 0 0 0 --

Df(3R) lc4a 87E5-7;87E1 I-FI (7) 1.0 0 0 0 +

Df(3R)red 3L 87F12-14;88C1-3 (7) 1.2 - 0 0 0 +

Df(3R)GE41 87E3-4;88A6-8 (9) 0.3 E 0 0 0 +

Df(3R)N78 ND ;87E3-4 (9) 9.5 Su 0 0 0 -

Df(3R)AceHDI Not visible (10) 7.3 Su 0 0 0 -

Df(3R)N63 ND;87E4-6 (9) 3.5 Su 0 0 0 -

Df(3R)N42 ND;87E3-4 (9) 2.5 Su 0 0 0 0

ND, not determined

a Source of the deletions: (1) Ashburner et al. (1981), (2) Gausz et al. (1979), (3) Gausz et al. (1980), (4) Gausz et al. (1981), (5) J.

Gausz et al. (unpublished), (6) Scalenghe and Ritossa (1976), (7) Hilliker et al. (1980), (8) Ish-Horowicz et al. (1977), (9) Gausz et al.

(1986), (10) W. Gelbart (unpublished), (11) H. V/issin (unpublished)

b R is the ratio of the relative red pigment content of Df(3R)/+ to Balancer/+ males generated after outcrossing of wm4h;+/+

females with +/Y;Df(3)/Balancer males. Null expectation of 1.0. Su, the deficiency shows a dominant suppressor effect; E, enhancer effect; -, no effect

° In the complementation studies, the lethality of the mutations with the deficiencies was tested. Virgin females of Df/Bal stocks were crossed to Su/TM3 and T(Y;3)se ss e ro males respectively. -, Df/Su transheterozygotes are lethal; +, Df/Su flies are viable and their number is about one-third of the total number of flies. In the crosses with the translocations, only the male progeny can be analysed. 0, not determined

var(3)13 °1 could be a very small deficiency also including ck3 or the chromosome carries two independent mutations tightly linked to each other. Further cytogenetic fine structure studies are needed to characterize this suppressor locus in more detail.

Su-var ( 3 ) 6

The presence of a third suppressor locus was indicated by the suppressor effect of the deficiency Df(3R)kar 3Q which does not overlap deficiencies Df(3R)P-79 and Df(3R)P-IO.

This locus, Su-var(3)6, is separated from Su-var(3)13 by the deficiencies Df(3R)E-229 and Df(3R)P-29 neither of which shows any effect on position-effect variegation (Ta- ble 3 and Fig. 1). The localization of this locus is confirmed by complementation analysis with the dominant suppressor alleles Su-var(3)6 °1 and T(Y;3)15. Su-var(3)6 °a is semi- lethal as a homozygote and T(Y;3)15 has a breakpoint in 87B5-8. Both mutations are fully viable over Df(3R) T-45 but lethal over Df(3R)E-079. The locus is hence placed between the distal break of Df(3R)T-45 at 87B5-6 and the distal break of Df(3R)E-079 at 87B8-10 (Fig. 1). Deficien-

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432

Dp(3;3)D26 Op( 3;3) OtO r/e;

Dp(3;3)D2

Op (3,'3)D7

//////h~l

Op(3,'3)OI r/,~ ,,,.4 Dp ( 3,'3 / DZ,5

I II It ! I I I t t I'

I II II | | I I I I i

F

I Df(31?)cu z'° Df(31?)P-79

Su-var(3)14 Su-var(3)13 Su-var(3)6 Su-var(3) 7 E-var[3)3

' , * , I' , It !Ill'It I, ,i l" u ,,;J t , s, al,ll I

11 A B

86-87 II I 187-88 .

Of(3R)k~f )L2.6'd IDft3R}GEZ!

I I ~ I ... "11

Df(3R)MS3/ Df(3R)T-45 { Df(3R)kar sza Df(3R)lc4a I vJ | Df(3R)red 3/ rt~ J Df(3R) T-32

Df(317]t q I0 Df(3R)k°r3Li i Df (3 R ) P-21 Df(3R ) kor sz37

~.(/i//a I

Df(31?)T-07 Df(3R)kar 30

~ Df(3R)E-079 Df(3R)ker 5z!

Df(31?]T-/O Df(3R} sz2°

Of (3t?) P-35 ... Df(3R)ry 61v....//. ~

o~131?jE-p9 o(r31?J,/'o~

O,( ,1?Jry"o'

• Of(31?)k°rO!

D f(31?)kar I w Df(31?)ker szl2

Df(3R)Kx-I Df(31?)karSZ30

/(a

Fig. 1. Deficiency mapping of modifiers of position-effect variegation (see also Fig. 2). Deficiencies described in Table 3 are aligned with the salivary gland chromosome map (Bridges 1941). Deficiencies for which cytology was not determined at both ends were not drawn on the figure. Bars represent the extent of the deficiencies, and the hatched region represents uncertainty on the position of the breakpoint. Filled bars indicate deficiencies with a suppressor effect on variegation. Empty bars indicate deficiencies without effect on variegation, and the deficiency illustrated by a bar containing a dashed line has an enhancer effect. We have also drawn, above the chromosomes, the tandem duplications listed in Table 2

cies uncovering this region are lethal in transheterozygotes with both suppressor mutations (Table 3). Deficiencies including both the Su-var(3)13 and Su-var(3)6 loci do not always express elevated suppressor effects. This is probably due to genetic background effects rather than to other modifying functions in the region. Complementation studies based on lethality revealed that the EMS-induced suppressor mutation Su-var(3)6 °1 is allelic to the lethals of the complementation group ck19 (Table 4). One of them,

hs 16,

however does complement Su-var(3)6 °1 (Table 4). We also found that the viability of Su-var(3)6°l/l(3)e 7s transheterozygotes is three times higher when Su-var(3)6 °~ is ma- ternally inherited. The mutant alleles isolated previously as recessive lethals also show a dominant suppressor effect, but it is significantly weaker in ^{e 168}and hs ~6 (Table 4).

The extent of dominant suppressor effect and recessive lethality in transheterozygotes with Su-var(3)6 °1 does coincide, thus suggesting that Su-var(3)6 may be a complex locus or simply that Su-var(3)6 °1 represents a hypomorphic mutation only. This is also supported by the complementation pattern of the translocation T(Y;3)15 (Ta- ble 4). The translocation, which has a dominant suppressor

effect, complements the e t68,

hs 16,

and Su-var(3)6 °t alleles of the Su-var(3)6 locus, but only partially complements e 78. Full complementation of the translocation is obtained with representative alleles of the adjacent lethal complementation groups ck17 and ckl8 also excluding position effects on other genes immediately adjacent. The translocation is however lethal over deletions uncovering the Su-var(3)6 locus.

Su-var (3) 7

The fourth suppressor of position-effect variegation maps between 87E1-2 and 87E3-4, namely between the breakpoints of the two deficiencies Df(3R)ry t6°v and Df(3R)N78 (Table 3, Fig. J). Translocation T(Y;3)409, with a breakpoint just to the right of band 87EI,2 has been isolated as a dominant suppressor allele (Reuter et al.

1986). It uncovers a lethal function which can be localized to the same interval between the lethal complementation groups Ace and G7. T(Y;3)409 complements all lethals in the region except Ace J4°. Ace i4° and another recessive lethal from the Ace locus, Aee 126, were found to have a

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Table 4. Su-var(3)6 alleles, complementation analysis and suppressor effects on white variegation in wm4h/Y males

Paternal Maternal genotype a Su-var(3)6 °* Allele

genotype

e78 e 168 hs 16

Relative concentration of red eye pigments in w "4h

w =4h ; + / + Su/+ Balb/+ R ~

e 78 13/288 e 78 52.8_+1.1 1.5-t-0.2 35.2

(0.05)

e 168 - - - 63/289 e 168 10.3+0.4 2.2_+0.2 4.7

(0.22)

hs 16 - - - 115/362 hs 16 14.2_+0.4 5.0+0.4 2.8

(0.32)

Su-var(3)6 °1 61/378 6 4 / 2 9 3 93/253 67/457 Su-var(3)6 °1 78.2+2.0 9.2_+0.4 8.5

(0.16) (0.22) (0.27) (0.15)

T(Y;3)I5 64/170 8 1 / 1 7 6 67/148 79/169 T(Y;3)15 d 34.1_+5.7 9.2+2.6 3.7 (0.38) (0.46) (0.45) (0.47)

The lethals eva, e ~68, and ks 16 w e r e independently isolated and belong to the ck 19 complementation group which is localized in the region from 87B5-6 to 87B9-10 (Gausz et al. 1981). In the complementation crosses, the viability of mutant transheterozygotes was studied. The data indicate the number and the ratio of transheterozygotes per total number of progeny with the null expectation of 0.33. In crosses with the translocation T(Y;3)15 the null expectation is 0.5. -, transheterozygotes are completely lethal

b The third chromosome balancers TM3 and MKRS were used. These chromosomes do not show any effect on position-effect variegation in w m4h

c R is as in Table 3

d Translocation males wm4a/T(Y;3) 15 were compared with wm*h/Y; + / + males

Table 5. Complementation analysis of T(Y;3)409 the Ace region and their effect on position-effect wmgh/Y flies

with lethals of variegation in

Lethal mutation Effect on white variegation Comple-

in wm4h/Y mentation

with R" modifier effect T(Y;3)409 b

l(3)B16-4(B16-1) -- -- +

l(3)B26-2(C9a) 0.9 No effect +

l(3)Ace 19 1.1 No effect +

l(3)Ace 126 2.7 Very weak suppressor +

l(3)Ace jz9 1.4 No effect +

l(3)Aee j4° 2.2 Very weak suppressor --

l(3)Ace i5° 1.3 No effect +

l(3)G7 1.1 No effect +

l(3)m32 -- -- +

l(3)Ace T M -- Very weak suppressor c + T(Y;3) 409 4.4 a Suppressor effect

" R is the same as in Table t

b Viability of T(Y;3)409/ lethal mutation was analysed in males;

+, viable; - , lethal

c Suppressor effect only detected in the presence of an enhancer mutation

a wm4h/y males used as control for R calculation

very weak suppressor effect (Table 5) which was confirmed by repeated pigment measurements carried out in the different laboratories (not shown). Ace 7°1 was isolated as a weak suppressor of position-effect variegation. It later proved also to be allelic to the Ace locus. This suggests that Ace j4°

and Ace 126, together with Ace 7°1, have only a very weak effect on the 87E suppressor locus. The location of Su- var(3)7 between Ace and G7 is also strongly supported by the results obtained with a series of deficiencies with a breakpoint between Ace and G7. Df(3R)ry 16°7 uncovers

Ace but not the suppressor locus. Two other deficiencies, Df(3R)N42 and Df(3R)N78, also with a distal breakpoint between Ace and G7, display the same dominant suppressor effect as deficiency Df(3R)AceHD1 which uncovers both Ace and G7.

E-var(3)3

Deficiency Df(3R)GE41 with a breakpoint between the Ace and the G7 complementation groups has a strong enhancer effect. Deficiencies Df(3R)red 3L and Df(3R)lc4a do not uncover the haplo-abnormal enhancer function, which maps therefore between 87Ell-F1 and 87F12-14. The enhancer effect of deficiency Df(3R)GE41 is more pro- nounced in females as found in genotypes also carrying a strong second chromosomal suppressor Su-var(2)l °~

(Reuter et al. 1982). Females of the genotype w"4h; Su/+ ; Df/+ show strong variegation whereas sibling males express the suppressor phenotype (red eyes). Deficiency Df(3R)GE41 is viable in transheterozygote combination with T(Y;3)409 which might indicate that Su-var(3)7 is not included.

Analysis of tandem duplications

A set of tandem duplications was generated to study the effects of two wild-type genes on a single dominant allele of each of three different variegation suppressor loci. Four overlapping duplications were studied (Table 6). The suppressor mutations analysed are different with respect to the strength of their effect on position-effect variegation.

Some of the mutations display parental effects (G. Reuter, unpublished) and the pigment values, therefore, can only be compared between the different sibling genotypes of the same cross. The Bal/Su sibling flies were used as controls (Table 6). The suppressor effect of Su-var(3)13 °1, Su- var(3)6 °a and Df(3R)Su-var6 is significantly reduced in transheterozygotes with duplications Dp(3;3)D1 and

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434

Table 6. Duplication mapping of suppressor loci in the region from 86 to 87 Suppressor locus/ Genotype

mutations measured Duplications and cytology

Dp (3 ;3) D 1 Dp (3 ;3) D2 Dp (3 ;3) D 7 Dp (3 ;3) D26 86A1 ; 87C3-5 86A1-5; 86E9-FI 85F8-16; 87F2-7 86B4-C1 ; 86D5-8 Su-var(3)6

Su-var(3)6 °1 Df(3R) Su-var6

Su-var(3)7 Df(3R)AceHD1

T(Y;3)409

Su-var(3)13 Su-var(3)13 °I

e/Dp a 26.4±5.8 69.7±2.7 37.0±3.7 93.9±3.9

e/TM3 67.1±2.7 97.6±1.5 84.3±5.1 93.9±10.0

R e 2.5 1.4 2.3 1.0

Df/Dp b 17.7±2.4 ND 12.4±2.1 42.4±4.4

Df/TM2 52.4±3.5 ND 22.4±1.5 35.3±4.1

R ° 3.0 - 1.8 0.8

Df/Dp ° 33.5±4.1 ND 7.1±1.5 41.5±1.8

Df/TM3 16.5±1.2 ND 17.1±1.5 17.1±1.2

R e 0.5 2.4 0.4

~Dp a 41.8±6.5 39.7±5.9 17.7±0.9 75.3±2.7

~TM3 62.4±0.9 58.2±2.1 69.1±4.4 81.2±7.7

R e 1.5 1.5 3.9 1.1

~ / O p b 18.5±2.4 32.5±3.5 14.7±2.7 62.4±1.4

~/TM2 41.2±1.8 43.5±4.4 43.5±6.8 62.9±5.3

R e 2.2 1.3 3.0 1.0

ND, not determined

a Females of the genotype wm4h; Su-var(3)6 °1 e/TM3 were crossed to WinCh~ Y;Dp/TM3 males

b Females wm4h;Su/TM3 were crossed to wm4h/y;Dp/TM2 males. In the offspring the relative concentration of red pigments was determined in females

wm4h;Dp/TM3 females were crossed to +/Y; Df(3R)AceHD1/MKRS males and white variegation was measured in males only d Translocation males wm4h/T(Y;3)409/se ss e ro were crossed to w'gh; Dp/TM3 females and the offspring males studied

Ratio of the relative pigment content of Su/Bal to Su/Dp. If R is significantly higher than 1.0 the duplication covers the effect of the dominant suppressor function

Dp(3;3)D7. Both duplications cover the corresponding suppressor loci. The same experiments were performed with duplications Dp(3;3)D2 and Dp(3;3)D26, which do not cover the Su-var(3)13 and Su-var(3)6 loci. In these crosses, the two offspring genotypes Su/Dp and Su/Bal are not significantly different. Df(3R)AeeHD1/TM3 flies show a weaker suppressor effect than those carrying the same deficiency over duplication Dp(3;3)D1 or Dp(3;3)D26. This effect is specific to Df(3R)AceHD1, since it was not found for other suppressor mutations. Duplication Dp(3;3)D7 which also covers the 87E suppressor locus reduces significantly the dominant suppressor effect of Df(3R)AceHD1 and T(Y;3)409 (Table 6). Therefore, duplications of the suppressor loci detected in the region from 86 to 87 reduce considerably the dominant effect of the corresponding mutations or rearrangements in heterozygotes. These findings support the results of the deficiency mapping as well as the haplo-abnormal function of these suppressor loci.

Molecular mapping of the 87E suppressor locus

The suppressor of position-effect variegation Su-var(3)7 maps within a region which has been the subject of detailed genetic and molecular analysis. It was saturated with recessive lethal mutations by Hilliker et al. (1980), who also established a genetic map using a set of overlapping deficiencies. The interval from 87D5-6 to 87E5-6 was cloned by chromosomal walking as a series of overlapping recombi- nants by Bender et al. (1983). The deficiency breakpoints

Df ( 3R ) Ace Hol re, "~

Ace- Su- G7- l Df(3tP)N42 Ace-Su-G7 +

E w, I

Df(3tP)ry1607 Ace-Su+O7 + Df(3R)GE41 Ace+Su+ O7 -

Df(3R)N78 Ace-Su-G7 + J Df(3tP)N63 Ace-Su-GT-

J Df(3R) kor szll Ace- Su-GT-

!~i~i~i~ii~i~i ~1 11 Transcripts

Ace 0.8 1.6

+ 310 +/,10 + 510 + Jo + 710 +80 kb

Fig. 2. Molecular localization of Su-var(3)7. Deficiencies are marked as solid bars. The hatched regions indicate uncertainty in the position of the breakpoints (mapping from Gausz et al. 1986).

Transcript sizes and positions are from Hall et al. (1983), Bossy et al. (1984) and Hall and Spierer (1986). DNA map coordinates are as in Bender et al. (1983). Df(3R)AeeHD1 and pertinent unpublished information was kindly provided by W. Gelbart

used to establish the genetic map were mapped on the D N A by Spierer et al. (1983). Recently, new deficiencies were isolated and mapped in the same interval by Gausz et al.

(1986). These studies allowed us to map the 87E suppressor locus on the genomic D N A sequence (Fig. 2). The locus

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is delimited on its proximal end by the breakpoint of the deficiency Df(3R)AceHD1 and distally by the breakpoint of deficiency Df(3R)N78. This mapping, illustrated in Fig. 2, shows that the suppressor locus lies in an interval of about 10 kb. Three transcripts were detected in this interval by Hall et al. (1983), Bossy et al. (1984) and Hall and Spierer (1986). A transcription unit corresponding to the Ace locus lies within the proximal half of the interval, whereas 0.8 kb and 1.6 kb transcripts were detected at the distal extremity. These two transcripts are our candidates for the suppressor of position-effect variegation locus. Their small sizes and the availability of genomic DNA has led us to initiate work toward molecular identification by P element-mediated transformation (Rubin and Spradling 1982).

Discussion

Changes in chromosome configuration are generally in- voked as the cause of position-effect variegation. The genetic phenomena of position-effect variegation are well established; however biochemical studies have just begun. Tartof et al. (1984) have identified the DNA sequences at the euchromatin-heterochromatin junction of several rearrangements variegating for the white locus which is an im- portant starting point for molecular analysis of the mecha- nism. It has been speculated that mutations of genes con- trolling euchromatic or heterochromatic (or both) chromosome packaging may suppress or enhance position-effect variegation (Reuter and Wolff 1981; Sinclair et al. 1983;

Reuter et al. 1986). Indeed, a butyrate-sensitive suppressor of position-effect variegation which affects histone deacety- lation and chromosome condensation has been described (Reuter et al. 1982; Dorn et al. 1986).

As deficiencies for various regions had been found to have a dominant effect (haplo-abnormal) on the extent of variegation (Henikoff 1979; Reuter and Szidonya 1983) we used this feature to map modifiers of position-effect variegation between the proximal breakpoint of Df(3R)cu 4° in 86CI-2 and the distal breakpoint of Df(3R)red 3L in 88CI-3 and identified four suppressor loci and one enhancer locus in this interval of about 155-160 bands (Bridges 1941). That this chromosomal interval does not contain an exceptional cluster of such genes is strongly suggested from the results of comparable studies in other autosomal regions (Reuter and Szidonya 1983; J. Szidonya and G. Reuter, in preparation; G. Wustmann et al., in preparation). We thus estimate by extrapolation from the results described herein a total of about 135 loci modifying position-effect variegation in the Drosophila melanogaster autosomal complement. This estimate is higher than those deduced from studies of EMS or X ray induced mutations (Reuter et al. 1986, and in preparation). For some loci only amorphic or strongly hypomorphic mutations might result in a detectable dominant suppressor and enhancer effect, respectively. Other loci might be repeated genes detectable only by deficiencies.

Therefore, cytogenetic fine structure studies are needed to estimate the total number of such loci more exactly as well as to characterize their genetic properties in more detail.

At least three of the modifiers described in this report do not appear to be redundant. Su-var(3)6 is allelic to point mutations and Su-var(3)7 maps in a cloned region where no repeated DNA has been found (Bender et al.

1983 ; Hall et al. 1983). For the enhancer locus, a P element- induced mutation has recently been isolated (G. Wustmann and G. Reuter, unpublished). In our studies one suppressor locus (Su-var(3)6) was found clearly to represent an essential genetic function. On the other hand, we did not find EMS-induced mutations for the suppressor locus uncovered by Df(3R)cu 4° and for Su-var(3)13 only one mutant allele is available up to now. Further cytogenetic fine structure studies in the region are needed to characterize these loci more exactly.

Su-var(3) 7 is within the region which was saturated for recessive lethals by Hilliker et al. (1980). By deficiency mapping the suppressor locus was placed between the two lethal complementation groups Ace and G7. The break of T(Y;3)409 is lethal and maps to the same interval. A duplication covering the breakpoint of T(Y;3)409 corrects the dominant suppressor effect, also arguing that the lethal function and the dominant suppressor effect coincide. In the screen of Hilliker et al. (1980), some complementation groups were represented by very few alleles. It is therefore possible that an essential function with a small target size escaped detection. The two transcripts which we found to be candidates for the suppressor function are indeed short.

We also found that three of the five studied Ace alleles

(Ace j4°, Ace 126,

and

Ace 7°1)

show a very weak suppressor effect. Only one allele (Ace j4°) is lethal over T(Y;3)409 indicating that these alleles are mutations which affect both Ace (strongly) and the neighbouring variegation suppressor (weakly). Our studies in this region and in other parts of the genome (Reuter et al. 1986, and in preparation) show that several of the position-effect modifying loci encode essential functions.

Duplications of the Su + function correct the dominant suppressor phenotype, thus providing further evidence that these loci are dosage sensitive (haplo-abnormal). Therefore, experiments using P element-mediated transformation should be useful for a functional test of cloned DNA sequences from position-effect modifying loci.

Finally, the Su-var(3)7 locus was mapped to within a cloned interval (Fig. 2). Only three transcripts have been identified within the about 10 kb of genomic DNA. The Ace locus extends over the proximal half of this segment (Hall and Spierer 1986) and two transcripts have been identified in the distal half: a 0.8 kb polyadenylated transcript and a 1.6 kb polyadenylated transcript. The latter has its 5' extremity within the same restriction fragment as the breakpoints of the deficiencies Df(3R)N78 and Df(3R) GE41. It is likely but not proven that the breaks interfere with this transcription unit. Because of the small size of these transcripts, they are well suited for experiments using P element-mediated transformation, which would also demonstrate which of the two correspond to Su-var(3)7.

The 87E suppressor locus is so far the first modifier of position effect cloned. Work is now being undertaken to analyse its product and its expression.

Acknowledgements. We thank W. Gelbart for giving us the deficiency Df(3R)AceHD1 (and pertinent unpublished information) and A. Chovnick for providing many mutant stocks from the envi- rons of rosy. This work was supported by a bilateral agreement between the Academies of Science of Hungary and the German Democratic Republic, and by the Swiss National Science Founda- tion. We are grateful to R. Hagemann and A. Tissi6res for continu- ous support.

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Communicated by J.A. Campos-Ortega

Received June 10, 1986