Interaction of the mottler of white with transposable element alleles at the white locus in Drosophila melanogaster James A. Birchler , 1 John C. Hiebert , and Leonard R a b i n o w

Harvard University, Biological Laboratories, Cambridge, Massachusetts 02138 USA

The mottler of white (mw) locus has been determined to interact with alleles of the white (w) eye color locus which are a subset of the transposable element insertion mutants. The transposable e lements belong to six different types, including copia, and are located at several sites within the w gene. Three X-ray-induced revertants of white-apricot (w ~) no longer respond to row, indicating that the transposable e lement must be present for m w to act. The mottling property of the original allele was analyzed by combining the m w mutant with extra copies of w% either in a tandem duplication or in a transposable segment on chromosome two. Because neither duplication alters the mottling pattern, the event that results in the mottled pattern must occur at mw and not at w. The pattern of a deficiency for the locus heterozygous with the original allele differs from that of row~row females, confirming that this unique mottling property occurs at row. A new allele of m w was induced in hybrid dysgenic crosses. It is not mottled, slightly enhances w ~ as a heterozygote, and further enhances as a homozygote or hemizygote. An analysis of RNA from w ~ with m w shows a reduction of the full- length normal RNA and a concomitant increase in certain RNAs that terminate within the copia element. These results suggest that several retrotransposon-induced alleles share an RNA processing function encoded by m w .

[Key Words: Drosophila; retrotransposons; white locus; RNA processing]

Received August 15, 1988; revised version accepted November 14, 1988.

The mott ler of white (row) locus was first described by Muller in 1946. This mutant , near the center of the X chromosome, produces mosaic expression of the apricot allele of the white (w) eye color locus, at the tip of the X. The phenotype consists of nearly white sectors inter- spersed among those characteristic of apricot. In terms of sector size and distribution, the phenotype superfi- cially resembles those that result from position effect variegation; yet, there is no response of the wild-type alleles of w, and cytological examinat ion revealed no de- tectable chromosomal rearrangement. In many regards, m w acts as a point muta t ion that mimics position effect variegation.

In an effort to characterize genes that exert a trans- acting regulatory effect upon the w locus, a study of m w was initiated. We find that this unusual mot t l ing prop- erty is the result of a combinat ion of phenomena. The loss of function at the m w locus enhances a spectrum of transposon-induced mutan ts at w, caused primari ly by retrotransposons, and the random inactivation of the original allele of m w during development is responsible for the mosaic pattern of activity.

~Corresponding author.

The enhancer function of m w affects transposons lo- cated at various sites wi th in the structural portion of w. In the case of the white-zeste mo t t l ed { ~ 1 allele, the interaction with zeste (z) must occur for the effect of m w to be observed. The enhancement of white-apricot (w °) is additive with other modifying genes, such as sup- pressor of white-apricot [su(w~)], and suppressor of forked [su(f)], which are effective on this allele. An anal- ysis of the RNA profile from w a wi th and without m w indicates that the enhancement involves a reduction in the level of normal-sized w RNA with a corresponding increase in RNAs that terminate wi th in copia. There is no effect on the level of total copia RNA. The m w mu- tants identify a new RNA processing function util ized by transposon-induced alleles.

The mosaic ism of the w~/mw interaction is due to a unique mott l ing property of the original m w allele, rather than excision of copia or inactivation of the w locus, as evidenced by the observation that a duplication of w ~ does not alter the mot t l ing pattern and Southern blot analysis shows the retention of copia in w. The mott l ing allele heterozygous wi th a deficiency for the locus has a different pattern than homozygotes. A new allele induced on a wild-type chromosome is uniform for enhancement over the surface of the eye, in contrast to the original. These observations support the conclusion

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that the variegation is distinct from other types in- volving transposable elements (Fincham and Sastry 1974; Bryan et al. 1987).

R e s u l t s and d i s c u s s i o n

The first step in the characterization of m w was to com- bine it wi th a series of mutants at w that are hypomor- phic to determine the array of responsive alleles. This collection includes representatives of structural gene le- sions due to transposable e lement insertions, as well as those with undetectable lesions by the criterion of Southern gel analysis (Zachar and Bingham 1982). Also included are mutan ts in the 5'-noncoding regulatory se- quences. The rationale was that this series would be in- formative as to the interaction of the m w and alleles at w itself. To conduct this screen, an X chromosome was constructed that was carrying the following markers: y w c t m w f . This chromosome has the original w mutan t and therefore can be used to recover recombinants wi th various hypomorphic alleles between the w and cut (ct) loci. The ct and forked (f) loci flank the row; therefore, recombinants that exhibit the eye color of the allele under test and the two markers represent the successful combinat ion of the respective allele and the m w mu- tant. The recombinant individual males were mated to attached X females, C(1)DX, y w f/Y, to establish a stock of each allele wi th the row. The crossing scheme is il- lustrated in Figure 1. A further description of the mu- tants used can be found in Lindsley and Grell (1968).

The alleles affected by m w are the following: apricot, apricot-4, buff, honey, spotted-55, and zeste-mottled. These six alleles are all insertion mutants at various lo- cations wi th in w. They represent six different families of transposons; however, five of these are retrotran- sposons. Table 1 lists the location of insertion and tran- sposable e lement type for each of these alleles. Clearly, insertions at different sites can be affected, and mutants caused by several transposons will respond.

Although the affected alleles are all transposons, it is clear that not all insertion mutants at w are affected. For example, crimson, blood, eosm, and six alleles resulting from IR hybrid dysgenesis are all unaffected. These rep- resent insertions of a foldback (FB) e lement (w~), blood

element, and I e lements that belong to different c las se s of transposable e lements than those in the affected al- leles. On the other hand, white-spotted-1 is a B104 in- sertion in the 5 '-noncoding region of w and is not af- fected, whereas buff is a B104 insertion in the fourth in- tervening sequence and does interact.

The m w has no effect wi th three revertants of w ~. One of these, w ~Rsgkl, has been determined molecular ly to contain only a single long terminal repeat (LTR) of copia (Carbonate and Gehring 1985). The fact that this rever- tant does not respond supports the conclusion that m w requires the presence of the complete transposable ele- ment to be effective on w alleles.

Genetic analysis of mott l ing properties

The mot t l ing property was characterized further. First it was of interest to determine whether this effect was due to a random inactivat ion of the w locus in the early stages of development or whether there was a somatic excision or modification of the transposable e lement that generates a mosaic phenotype. In maize, snap- dragon, and Drosophila simulans, transposable e lement systems can produce mosaic phenotypes due to the transposition of the e lement away from the locus (Fin- cham and Sastry 1974; Bryan et al. 1987). In general, these involve normal sectors on a mutan t background that is fundamenta l ly different from the phenotype gen- erated by the combinat ion of w ~ and mw, which has nearly nul l sectors on an intermediate background. Re- gardless of these considerations, a prediction is made that the phenotype of a duplication of w ~ would have a dist inguishable phenotype in combinat ion with m w than the s implex alone. This is the case because both mechan i sms (inactivation/modification) involve random events at a set point in development. If the nul l phenotype is caused by an event at the w locus, a dupli- cation of w would have more tissue that exhibits the apricot level of pigment than the simplex form.

To test this, a tandem duplication of the w region car- rying two copies of the apricot allele (Green 1959a) was recombined onto the chromosome with the ct m w f markers, as described above, for tests of allele speci- ficity. The recombinants wi th a Dp(I : l )w ~ ct m w f con-

Figure 1. Genetic crosses to determine allele specificity at the w locus. Males carrying the markers y wct m w f were mated to virgin females from the collection of w allele stocks. The progeny were allowed to mate inter se, and the F2 males were scored for single recombinants between the w and ct loci. Re- combinant males were mated to attached females for confirma- tion and establishment of stocks.

y ~ c t ~ f T Y ® w x (white elleles)

l l I

l ~ z c t m~ f l y recombinants

® Cfl)D,,K, y ~ ,'<lY

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Table 1. Al le les o{ white tes ted w i th m w

No. of Allele recombinants Interaction Lesion Reference

Mottler o[ white

w ~ (apricot) I + copia insertion in second intron

w ass112 (apricot revertant) 3 - unknown w ~sn~z {apricot revertant) 2 - unknown "W "a59kl (apricot revertant) 3 - X-ray-induced revertant

solo copia LTR w ~ {apricot revertant) 5 + transposable e lement

insertion in copia 5' LTR w ~R84h (apricot revertant) 3 + I e lement insertion in

copia 3' LTR w ~ {ivory) 11 - duplication of intron-1/

exon2 sequences

w ~ (crimson) 3 - FB transposable e lement revertant of w ~

w,V {spotted) 16 - B104 insertion in S'

regulatory region

w ~va (spotted-4} 11

w ~p2 {spotted-2) 4

w~V s~ds (spotted-81 d5) 4

w ~pSs {spotted-55) 10 +

w °t2 (buff-2) 7 w ~ (tinged) 4 w ~° {coral) 6 w ~a {ecru-3) 5 w ~° {mottled-orange) 2 w ~t (coffee) 2 w ~3 {apricot-3) 11 w ~2 (apricot-2} 11 w *at (satsuma) 6 w ~°1 (colored] 6 w a n {Brownex) 8 w ~2 (blood) 4

vCv {buff) S

w ~4 {apricot-4) 6

w" {eosin) 4

w a (eosin-2) 2

w ~ (cherry} 8

w ~ (honeyl 7

w avl {apricot-like) 2

w ~m 4

deficiency in 5' cis regulatory region

deficiency in 5' cis

regulatory region deficiency in 5' cis

regulatory region retrotransposon insertion

unknown unknown unknown unknown unknown point point point point point point retrotransposon insertion

in intron 2

B104 transposable e lement insertion in intron 4

BEL insertion into intron 2

transposable e lement reversion of w I (Doc element)

derivative of w I {Doc

element) reversion of w ~ (Doc

element) deletion reversion of w ~

(Doc element) P-M hybrid dysgenic

revertant of w ~ (Doc

dement} I e lement insertion

( C o n t i n u e d on nex t page.)

Gehring and Paro (1980} Bingham and Judd (1981)

Carbonare and Gehring (1985)

Mount et al. {1988)

Mount et al. (1988)

Karess and Rubin (1982) Collins and Rubin {1982) &Hare et al. {1984} Collins and Rubin (1982) O'Hare et al. {1984) O'Hare et al. 11983} Zachar and Bingham {1982) O'Hare et al. {1984) O'Hare et al. {1983) Zachar and Bingham {1982) O'Hare et al. {1984) Zachar and Bingham {1982)

Davison et al. {19851

Zachar and Bingham 11982) O'Hare et al. (1984)

Zachar and Bingham (1982) Zachar and Bingham {1982) Zachar and Bingham (I 982) Zachar and Bingham (1982} Zachar and Bingham {1982) Zachar and Bingham {1982) Zachar and Bingham (1982) Bingham and Chapman

{1986) Zachar and Bingham {19821 O'Hare et al. { 1984) O'Hare et al. (1983) Zachar and Bingham {1982) Goldberg et al. {1983) Zachar and Bingham (1982) O'Hare et al. (1984) Hazelrigg (1987) O'Hare et al. {19841

Zachar and Bingham (1982) O'Hare et al. (1984) Zachar and Bingham (1982) O'Hare et al. (1984) C. McElwain {pers. comm.)

Sang et al. (1984)

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Tab le 1. C o n t i n u e d

No. of Allele recombinants Interaction Lesion Reference

w Ia2 2 - I element insertion Sang et al. {1984) revertant of w I (Doc element)

w IRa 5 - I element insertion Sang et al. (1984) w IR4 4 - I element insertion Sang et al. [1984) w ~Rs 4 - I element insertion Sang et al. {1984) w Ia6 4 - I element insertion Sang et al. (1984) z w is (isoxanthopterinless) 12 - unknown w ~ (zeste-mottled) 11 - 3S18 transposable element Zachar and Bingham (1982)

insertion in intron 1 O'Hare et al. (1984) z w ~1 (zeste-light) 4 - derivative of w ~r~ Judd (1963) z w ~ (zeste-mottled) 6 + 3S18 transposable element Zachar and Bingham (1982)

insertion in intron 1 O'Hare et al. (1984) z w ~ 3 + copia insertion in intron 2 Gehring and Paro (1980)

Bingham and Judd (1981) z a w ~ 1 + copia insertion in intron 2 Gehring and Paro {1980)

Bingham and Judd { 1981) z Dp(1;l) w +61e19 5 - duplication of w h i t e locus

The allele designations are given for each mutant recombined onto the y w ct m w f chromosome, as well as the number of recombi- nants replacing w. The third column notes whether the respective w allele was affected by row. For each genotype, the status of the knowledge of its molecular lesion is given along with the appropriate reference.

s t i tu t ion were mated to compound X females to produce stocks. When these males are compared to w ~ c t m w f, the pa t tern of p igment is the same; however, the inten- s i ty of p igmented regions is greater than wi th only a single copy of a p r i c o t present.

The second exper iment was to cross the m w stock wi th one carrying a t ransposed copy of w ~ in a Transpos- able Element , (Ising 1964) located on the second chro- mosome. For this, males of the cons t i t u t ion I n ( l ) w -

r s t - , y w r s t / y + Y ; I n ( 2 L ) C y I n ( 2 R ) C y , C y d p T E I ( w ~}

c n 2 / + were crossed to y2 w ~ c t m w f females. A m o n g the F~ males are those tha t are C y and carry an extra copy of w ~ w i th in the second c h r o m o s o m e balancer and those tha t have n o r m a l wings and only the w ~ present on the X. Both of these types of males were ident ical in the pat- tern of mosa ic i sm. Again, the dupl icated males differed only in tha t the in t ens i ty of color in the p igmented areas was greater. All copies of w ~ present in these cases be- come enhanced in the same deve lopmen ta l lineage. These resul ts suggest tha t the r andom mo t t l i ng property is a character is t ic of the original allele of m w and not of the w locus. The propert ies of a newly induced allele of m w , described below, are cons i s t en t w i th this v iew be- cause it does not exhibi t a mo t t l ed phenotype .

A sex difference exists in the mo t t l i ng pat tern. In gen- eral, females have por t ions of the p igmented areas tha t are l ighter than norma l w ~ compared wi th males. How- ever, w h e n females are produced tha t have a def ic iency opposite the m w allele (see below), the p a t t e m re- sembles tha t of the male. This resul t is in terpre ted such tha t the two copies of m w in a no rma l female become inac t ive in different cell l ineages. Because m w has a de- tectable d o m i n a n t effect, the r andom inac t iva t ion of the two copies versus one would produce this difference.

To test w h e t h e r enhancers of pos i t ion effect variega-

t ion could modify the pa t te rn of mot t l ing , males car- rying five different d o m i n a n t enhancers of variegat ion (Gsell 1971; Spofford 1976), heterozygous wi th I n ( 2 L ) C y

I n ( 2 R ) C y on c h r o m o s o m e two or C x D on ch romosome three, were crossed to females homozygous for w ~Rs4h c t

m w f. The male progeny were scored for any difference be tween the enhancer c h r o m o s o m e and the balancer. None of the tested enhancers , E(var)5 , E(var)7 , E(var)8 ,

or E ( v a r ) 1 2 on the second c h r o m o s o m e or E(var )13 on the th i rd ch romosome, produced any discernible effect on the mo t t l i ng pat tern. Thus, by this cri terion, as well as previous ones (Oster 1957, and those no ted in the in- troduction}, the mo t t l i ng is a d is t inct p h e n o m e n o n from pos i t ion effect variegat ion, despite the gross s imi lar i ty .

L o c a l i z a t i o n o f m w

To localize m w cytogenet ical ly , two types of aberra- t ions were used. First inser t iona l t rans loca t ions of por- t ions of the X c h r o m o s o m e in to au tosomal sites were crossed to a y2 w ~ ct m w f s tock to test for the comple- m e n t a t i o n of m w by the t rans located segment . The aberra t ions used were D p ( 1 ; 2 ) v + zsa, D p ( 1 ; 2 ) v +(ai, D p ( 1 ; 2 ) v +6sb, and Dp(1 ;3 ) sn laaL Males heterozygous for the dupl ica t ions were crossed to females of the afore- m e n t i o n e d m w stock. The F~ males were screened for n o r m a l w ~ phenotype , wh ich should be present in ap- p rox ima te ly half of the ma le progeny if the t ranspos i t ion carries the no rma l allele of r o w . In each case, several hundred progeny were scored. Only D p ( 1 ; 2 ) v +zsd com- p l emen ted the mo t t l ed pheno type in the male progeny.

Second, X c h r o m o s o m e deficiencies, v 11s, N l l 0 , N105, and C52 were crossed to males f rom a w ~ stock. The heterozygous females were backcrossed to males of y2 w ~ c t m w f cons t i tu t ion . If w ~ is r ecombined to the

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defic iency c h r o m o s o m e and i t is miss ing the no rma l al- lele of m w , t hen a f ract ion of the female progeny would exhibi t mot t l ing . Def ic iency C52 uncovered m w . A s tock was es tabl ished of the w ~ def ic iency c h r o m o s o m e by balancing it w i t h FM6. Collect ively, the dupl ica t ion and deficiency data place m w be tween 9A2 and 9B1 on the cytological map. These data are summar i zed in Table 2.

N e w al lele i n d u c t i o n

A new allele of m w was sought to unders tand the ac t ion of the original one. One poss ibi l i ty is tha t the m w migh t be a modif ied t ransposable e l emen t tha t encodes a t rans-ae t ing factor tha t is effective upon the e l emen t s present in the responsive w alleles. A sys tem of this type occurs w i th h u m a n T-cell l eukemia virus type 1 (HTLV- 1), in wh ich a regulator is encoded in viral sequences (Fujisawa et al. 1985). If such were the case, one would expect tha t new alleles could no t be generated f rom a no rma l c h r o m o s o m e at the same genet ic locat ion. If, on the o ther hand, the locus is one no rma l ly present in the Drosoph i la genome, which is in some m a n n e r involved in the expression of t ransposon- induced m u t a n t s at w, a new allele could be produced.

For the m u t a n t screen, Harwich P s train males were crossed to w ~ M strain females {see Fig. 2). This cross produces progeny tha t are act ive in the P-M sys tem of hybr id dysgenesis, thus act ing as a mutagen by mobi- l izing the P e l emen t s in the germ l ine of the FI (Rubin et al. 1982). The hybr id males are w ~ and dysgenic. They were backcrossed to ya w ~ ct m w f females. If a m u t a t i o n were induced in the dysgenic males, an except ional + w ~ + m w + female would be found among those tha t are o therwise exclus ively w ~. The screen was performed in this way in the event tha t newly generated alleles migh t be recessive lethals. In such case, they could be recognized by thei r pheno type in the he terozygote and yet recovered.

From a progeny of ~5000 flies, one except ional female was found tha t had w ~ mot t l ed eyes and was wi ld type for the markers y, ct, and f. This female was crossed w i th males f rom the w ~ stock. A m o n g the progeny were

Table 2. Cytological localization of mw

Aberrations Cytology m w + a

Dp{I;2) v +6ai 9F6-7-- 1 0 A 6 - 7 -

Dp(1;2) v +6sb 1 0 A 1 - - 1 1 A 7 -

Dp(1 ;3) sn13~1 6C 11 - 7C9 - Dp(1;2) v + zsa 9A2-- 10C2 +

Dr(l) v Lls 9B1-- 10A1 + Dill ) NI 10 9B3-4--9D1-2 + Dr(l) N105 10F7-8-- 11C4-D1 + Dr(I) C52 8EF--9CD -

Cytological determinations are by G. Lefevre, as provided by the Caltech Drosophila stock center. a Designations indicate the presence ( + ) or absence ( - ) of mw+ in the respective aberrant chromosome.

M o t t / e r of white

Harwich P o" ~ w a M strain 9

j dysgenic

1 Screen female progeny for ÷ ws÷ /~w ÷ exceptions.

Figure 2. Induction of a new allele of m w by P-M hybrid dys- genesis. Harwich P strain males were mated to w ~ virgin M strain females. The F~ w ~ males were crossed to females car- rying the multiply marked X chromosome with the original m w mutant, y2 w ~ ct m w f . The subsequent female progeny was screened for + w ~ + m w + exceptions among the + w ~ + + + normal females.

~,~ m w females and two major classes of males. These were y2 w ~ ct m w f and an enhanced w ~ class. The la t ter represents the new allele. In contras t to the original, it is not mot t led . Also, a l though there was no se lec t ion against l e tha l i t y in the screen, the m u t a n t is viable. The new allele has been designated raw 2.

When the new allele is backcrossed to w ~ females, the resul t ing progeny have females wi th l ighter eyes than males. Thus the m u t a n t exerts a par t ia l ly d o m i n a n t ef- fect.

The new allele was backcrossed to an a t tached X C(1)DX, y w f / Y from an M strain. Because there are P e l emen t s still present in the m u t a n t stock, this cross was dysgenic. If the m u t a n t is due to the inse r t ion of a P e lement , the m u t a n t wil l revert to normal . The progeny were a l lowed to ma te in ter se and the F2 males scored for reversions. From a tota l of 361 males screened, 6 were recovered tha t re turned to the apricot phenotype . These were ind iv idua l ly ma ted to C(1)DX, y w f / Y females to es tabl ish stocks.

To test the allele specif ici ty of the new allele, it was recombined w i th X markers to produce a w a~" ct m w a f chromosome. Because w a ~ has a un ique pheno type and is dominan t , it was chosen to test the specif ici ty of m w a. In an analogous scheme used for row, each allele re- placed w ~ on an o therwise ct m w a f chromosome, and these r ecombinan t s were crossed to C(1)DX, y w f / Y virgin females to es tabl ish stocks. The resul ts are shown in Table 3. The specif ici ty of the new allele is ident ica l to the original; however , in no case is the effect mot t led .

The new allele was p resumed to be a P-e lement inser- t ion because it arose during hybrid dysgenesis and is readi ly reverted by the same. It was local ized by in s i tu hybr id iza t ion of a biot in- labeled P-e lement probe. Sali- vary gland squashes of the m u t a n t males and of rever- t an t 3 were prepared and probed w i th the P e l emen t

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T a b l e 3. A l l e l e s of whi t e t e s t e d w i t h m w 2

No. of r ecombinan t s In terac t ion

w ~ {apricotl - - + w ~5sn2 (apricot revertant) 5 - w ~snn {apricot revertant) 2 - w ~sgkz {apricot revertant) 5 - w ~RM {apricot rever tant / 9 + w ~RSah {apricot revertant) 3 + w ~ (ivory) 3 - w ~ {crimson) 5 - w,V {spotted) 2 - w ~v# (spotted-4) 1 - w,t,z (spotted-2) 3 - w ~palas (spotted-8 ld5) 5 - w ~vss (spotted-55) 2 +

w #f~ (buff-2) 3 - w' (tinged) 2 - w ~° (coral) 2 - w ~ (ecru-3) 3 - w ~° (mott led-orange) 1 - w ~f (coffee) 1 - w '~a (apricot-3) 7 - w ~2 (apricot-2) 5 - w "~' (satsuma) 2 - w ~°l (colored) 3 - w #t (buff) 2 - w s~" (Brownex) 3 - w ~'l (blood} 3 - w "a (apricot-4) 4 + we {eosin) 1 - w "2 (eosin-2) 1 - w h (honey) 1 + w~W (apricot-like) 6 - w tRl 3 - w t a 2 3 -

w aRa 3 -

w aR# 3 - w 1Rs 3 -

w IR6 3 -

z" ~ (zeste-mottled) 2 + z ~ -~ (zeste-mottled) 10 +

(zeste-mottled) 9 - z" w ~ (zeste-light) 2 - z w ~ (zeste-light) 3 - w ~ {zeste-light) 3 - z a w ~ (isoxanthopterinless) 5 - z w ~s ( isoxanthopterinless) 3 - w ~' {isoxanthopterinless) 2 -

Table 1 describes the molecu la r lesion of each allele.

XP-19 (a g i f t f r o m D. Rio), a c o m p l e t e e l e m e n t w i t h o u t

f l a n k i n g s e q u e n c e s . T h e r e s u l t s of t h i s a n a l y s i s p l a c e t h e

g e n e in 9 A (see Fig. 3J. T h e m w e a l l e l e w a s u s e d to t e s t f u r t h e r t h a t t h e d i f fe r -

e n c e i n p a t t e r n b e t w e e n m a l e s a n d f e m a l e s d o e s n o t in-

v o l v e t h e d o s a g e of t h e w l o c u s . H e t e r o z y g o t e s a t w for

t w o r e s p o n d i n g a l l e l e s t h a t a r e p h e n o t y p i c a l l y d i s t i n -

g u i s h a b l e i n a b a c k g r o u n d of m w w e r e c o n s t r u c t e d . T h e

r a t i o n a l e is t h a t if t h e t w o w a l l e l e s a r e e n h a n c e d s i m u l -

t a n e o u s l y , t h e m o t t l i n g p a t t e r n w o u l d s h o w o n l y t h e

78 GENES & DEVELOPMENT

v

, ! :

Figure 3. In situ hybr id iza t ion of P e l e m e n t sequences to a hy- brid dysgenical ly induced m u t a n t of m w and a revertant . {Top)

Port ion of the X c h r o m o s o m e showing hybr id iza t ion in sect ion 9A f rom the m w e mu tan t ; {bo t tom} port ion of the X chromo- some showing loss of hybr id iza t ion in the rever tant strain.

h e t e r o z y g o u s c o l o r i n t e r s p e r s e d w i t h l i g h t e r s e c t o r s . If,

o n t h e o t h e r h a n d , t h e t w o a l l e l e s a r e i n d e p e n d e n t , t h e

p a t t e m w o u l d be a m i x t u r e of t h e t w o t y p e s . For t h i s

c ross , m a l e s of g e n o t y p e y2 s c w ~ c t m w [ / Y w e r e

m a t e d to y e w ~ c t m w e f v i r g i n f e m a l e s . T h e FI f e m a l e s

a r e h e t e r o z y g o u s a t w fo r t h e a p r i c o t a l l e l e a n d t h e m u c h

d a r k e r r e v e r t a n t of a p r i c o t , w ~gM. A t m w , t h e y a re h e t -

e r o z y g o u s fo r t h e o r i g i n a l m w a l l e l e a n d t h e n e w l y in-

d u c e d n o n m o t t l i n g o n e , m w e. If t h e t w o w a l l e l e s w e r e

a f f e c t e d r a n d o m l y , t h e r e s h o u l d be a p a t t e r n of r e v e r -

t a n t , a p r i c o t , a n d e n h a n c e d s e c t o r s . If n o t , t h e p a t t e r n

w o u l d r e s e m b l e t h e r e v e r t a n t a n d m w c o m b i n a t i o n

a l o n e . T h e l a t t e r r e s u l t w a s f o u n d , s u g g e s t i n g t h a t b o t h

a l l e l e s a t w a re e n h a n c e d in t h e s a m e ce l l l i n e a g e s . T h e

p a t t e r n r e s e m b l e s t h a t f o u n d in D f I l J C 5 2 / m w f e m a l e s

a n d i n m a l e s , b u t a l l o f t h e p i g m e n t e d a r e a s w e r e of a w ~ / w ~RM type , r a t h e r t h a n a n i n t e r s p e r s i o n of t h e t w o .

T h e n e w a l l e l e w a s u s e d to t e s t w h e t h e r e x c i s i o n of

t h e c o p i a e l e m e n t is r e s p o n s i b l e for t h e a l t e r e d p h e n o -

t y p e of w ~ m w d e s p i t e t h e f a c t s t h a t t h e p h e n o t y p e is

f u n d a m e n t a l l y d i f f e r e n t f r o m o t h e r s y s t e m s in w h i c h

m o s a i c p a t t e r n s r e s u l t f r o m t r a n s p o s a b l e e l e m e n t i n s e r -

t i o n a n d t h a t t h e m o d e of r e t r o t r a n s p o s o n t r a n s p o s i t i o n

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and excision also differs. In such systems, mu tan t tissue is interspersed wi th normal (or nearly so) tissue. In this case, a leaky phenotype is altered to a nearly null type. It is conceivable, however, that excision would routinely be such that the w locus would not be restored to a func- t ional state. For this to be the case, it could occur only in the soma, as no germ line change of w ~ to w has been observed wi th m w or m w ~. To test this, DNA from w ~, w ~ mw 2, and Canton S wild type was prepared and di- gested wi th the restriction endonucleases SalI and XbaI. Sites for these enzymes flank the w locus region in which the copia transposon is inserted and X b a I cuts once wi th in the element. The digested DNAs were sub- jected to electrophoresis, transferred to nylon mem- brane, and probed with a labeled subclone of w ex- tending from the SalI to X b a I sites ment ioned above. Be- cause m w ~ produces an enhancement of w ~ in all adult tissues in which w is expressed, any alterat ion in the size of the restriction fragments due to excision or rear- rangement of copia should be detectable. The w ~ + class serves as a control for the size of the two fragments with copia present and Canton S for the size of the fragment in the absence of copia. The results of this analysis indi- cate that w ~ + and w ~ m w 2 are indist inguishable (Fig. 4). Even after longer exposures, the w ~ mw ~ lane shows no additional fragments. It should be noted that these ex- periments could not detect the deletion of all sequences related to the probe. However, this possibility is un- likely, considering that the w ~ enhanced phenotype is not completely bleach white, which is characteristic of w deficiencies. Thus, there is no evidence on the DNA level for al terat ion of copia sequences in accordance with the genetic evidence.

eJ ¢Y) i

+ o ¢-

i

I

Figure 4. Southern analysis of w ° +, w ~ mw e, and Canton S. DNA was prepared from the three genotypes, digested with XbaI and SalI, separated in 1% agarose, transferred to nylon membrane, and probed with an oligolabeled DNA XbaI-SalI fragment. No difference was detected between w ~ + and w ~ mw e even after long exposures.

Mottler of white

In t e rac t i on w i t h z

m w is effective on the w ~ allele only when z is present. The w ~ allele is a 3S18 insert ion wi th in the first in t ron of w [O'Hare et al. 1984). It responds to z in a mosaic fashion in males (Becket 1960), in contrast to most other affected w alleles that require two copies to respond. The pattern of y2 z w ~ ct m w f males is such that wi th in the sectors that have reduced pigment due to z there is addit ional mot t l ing that l ightens the eye color even further. When a y2 z w ~ ct m w 2 f chromosome is made, the pattern is the same as wi th z w ~ , but the in tens i ty of the pigment in the lighter regions of the eye is reduced. In contrast, chromosomes wi thout z, namely, w " m c t m w f and w ~ ct m w 2 f, show no enhancement .

Interestingly, the zes te - l igh t allele of w h i t e (w ~) [Becker 1960) is a derivative of z e s t e - m o t t l e d (z w ~ ) but is not affected by m w . z w ~ has more area of the male eye responding to z than w ~m. Also, it is genetically un- stable (Judd 1963). The change from w ~m to w ~1 has elim- inated the ability to respond to m w .

The interact ion wi th z is specific to the w ~m allele since flies of the genotype z w ~ ct m w f / Y do not differ in the pattern of mot t l ing from w ~ ct m w f / Y males, z lightens w ~ (Becket 1960) but does not alter the pattern. z does not permit wild-type w to respond to m w because a genotype of z D p ( 1 ; 1 ) w +61e19 ct m w f shows a non- mott led z phenotype typical of duplications of the locus. Thus, ~ and z are both required for the phenotypic interact ion wi th m w .

In t e rac t ion of mw w i t h o ther m o d i f i e r s of w ~

The m w was combined with other mutants that have been implicated in interact ing with w ~. The three loci are su(w~), su(f), and E n h a n c e r of w ~ [E(w~)].

The s u ( w ~) locus specifically suppresses the apricot al- lele at w and alters the spectrum of RNAs that are gen- erated (Levis et al. 1984; Zachar et al. 1985). When a y2 s u ( w ~) ct m w 2 f chromosome is produced, the eye color is darker than the same combinat ion wi thout the sup- pressor muta t ion but lighter than a s u ( w ~) w ~ combina- t ion wi thout mw ¢. This indicates that the two mutan ts produce opposing effects but mw 2 is the stronger of the t w o .

The su(f) muta t ion acts to enhance the phenotype of w ~ and likewise has been shown to alter the pattern of RNAs produced from the apricot allele (Levis et al. 1984). The combinat ion w ~ su(f) has a much lighter shade of pigment than the normal w ~ const i tu t ion but is darker than w ~ m w 2. When a y2 w ~ ct m w 2 f su(f) chro- mosome is produced, the level of pigment is reduced to below the level wi th either su(f) or m w 2. Thus, the two enhancers are additive.

The third locus tested in combinat ion wi th mw 2, E(w ~) is also specific to transposable element insert ion alleles at w h i t e (J.A. Birchler and J.C. Hiebert, in prep.). When a y2 w ~ ct m w ¢ f male is crossed to females of y wa; E(w~)/CyO const i tut ion, the progeny can be divided into four classes: females heterozygous for mw ~ on the X

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Bitchier et al.

chromosome and for E(w ~) on the second chromosome, females heterozygous for rnw e and for the CyO balancer chromosome, males wi th w ~ on the X and heterozygous for E(w ~) on the second chromosome, and males wi th w ~ on the X and heterozygous for CyO on the second chro- mosome. From darkest to lightest, they are Cy male, Cy female, non-Cy male, and non-Cy female. This indicates that the rnw a allele has a dominant effect and is cumula- tive in combinat ion with the E(w~).

A reciprocal cross was performed that involved males of y w~/Y; E(w~)/CyO genotype and females of w ~ mw2/ w ~ m w 2. The hemizygous m w 2 sons have E(w~)/+ or CyO/+ second chromosome constitutions. The w ~ mw2/Y; E(w~)/+ progeny have lighter eye color than their w ~ mw2/Y; CyO/+ siblings.

RNA analysis

The w ~ muta t ion results from the insertion of a copia transposable e lement into the second intervening se- quence of the w locus (Gehring and Paro 1980; Bingham and Judd 1981). The mutan t phenotype is believed to re- sult from the terminat ion of transcription from the w promoter at sites wi thin the copia element, primarily in the 3' LTR. The su(f) and su(w ~) mutat ions alter the ratio of terminat ion products and the normal 2.6-kb mRNAs (Levis et al. 1984; Zachar et al. 1985).

To test the effect of m w on w ~, flies of w ~ +, w ~ m w 2 (produced from a segregating population), w ins (a defi- ciency for w), and Canton S were grown and separated according to sex. Previous phenotypic examinat ion of w ~ m w 2 flies showed that the mutan t effect of m w 2 occurs in all tissues where w is expressed (eyes, Malpighian tu- bules, and testis sheath); therefore, whole fly RNA anal- ysis is informative. RNA was isolated from each strain and analyzed on Northern gels. The blot was probed with an antisense RNA transcribed from a T7 vector containing a fragment extending from the XbaI site im- mediately preceding the second exon to the SalI site near the end of the third exon. Thus, it spanned the second intervening sequence where copia was inserted and de- tected RNAs initiated at the transcription start site of the white locus, as well as any initiated with copia and terminat ing at the 3' end of the w transcription unit. In addition, a second blot containing the same samples was probed with labeled RNA transcribed from a vector con- taining a SalI fragment including a small portion of exon 3 and extending into exon 6, which will only detect RNAs that contain sequences 3' to the site of copia in- sertion. Finally, a third blot was probed with RNA tran- scribed from a vector, containing sequences extending from the BamHI to HindIII sites, that includes only the first exon, which will detect transcripts initiating at w and terminat ing wi th in copia.

A complex array of RNAs is generated by the w ~ allele (Levis et al. 1984; Pirrotta and Brockl 1984; Zachar et al. 1985). The comparison of the w ~ + and w ~ rnw ¢ RNAs indicates that there is a drastic reduction in the level of 2.6-kb normal w message, but the levels of other species of RNA are unaffected, wi th the exception of two

species wi th homology to the 5' probe but not the 3' one and of a molecular weight slightly greater than the normal w message. The homology of these two species of RNA to the first two exons of w and their molecular weight suggest that they initiate at the w promoter and terminate wi th in copia, but do not contain all of the copia sequences. The results are shown in Figure 5.

The total RNA preparations from the population se- gregating for w ~ + and w ~ m w 2 was also probed wi th a single-stranded RNA antisense to a copia segment ex- tending from the HindIII to the ApaI restriction sites (Mount and Rubin 1985). There was no detectable quan- titative or qualitative difference among the genotypes (Fig. 6).

As an additional test, equal quantit ies of total RNA were dotted onto nitrocellulose and probed with the copia sequence. After hybridization, washing, and auto- radiography to check for gross background, the filters were counted by scintil lation spectrometry. The mean {--+S.D.) (n=10) of cpm in wa + /Y compared with w ~ mw2/Y males was 3012 ( _+ 449) and 3009 ( __ 504), respec- tively. In females, w ~ + / w ~ m w 2 versus w ~ mwalw ~ m w e gave values of 2600 (_+407) and 2334 {_+350). Nei- ther of these comparisons is significantly different in statistical tests at the 95% confidence level. These re- sults confirmed the Northern analysis, indicating no quanti tat ive modulat ion of total copia RNA levels by the m w 2 mutat ion.

An RNA species of low abundance is detected by the 3' w probe but not by the 5' probe, which is of the appro- priate molecular weight {-7.2 kb) and w homology to be a product initiated in the 5' LTR of copia and terminated at the 3' terminus of w. In Nor them blots of w ~, but not Canton S or other w ÷ strains, probed with copia se- quences, an RNA is detected of similar molecular weight. The quant i ty of this RNA is not modulated by m w 2.

Conclusions

An analysis of the m w locus indicates that it is a specific modifier of w alleles that are transposon-insertion mu- tants. Unlike other modifiers of insertion mutan ts at w, m w is effective on alleles induced by a variety of tran- sposons. The sites wi th in the w locus vary, but all are present wi th in the structural portion of the gene.

Five of the six affected alleles result from a retrotran- sposon insertion: apricot, apricot-4, buff, spotted-55, and zeste-mottled. The sixth affected allele is honey, which is a deletion derivative of the Doc element inser- tion in w 1. Several other derivatives of w ~, namely w ~, w ~2, w ~h, w ~pl, and w aR2 were also tested for interaction with row, but none of these responded. The allele speci- ficity of m w suggests that it operates primarily, but not exclusively, on retrotransposon insertion mutants .

The mott l ing property of the original allele is unique. It is unaffected by genetic modifications that influence position effect variegation despite the gross phenotypic similarities. Analysis of the pattern of a tandem duplica-

80 GENES & DEVELOPMENT

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U) 2

~ E E

,'~ 04 04 E ~

E 2 E E

5.8 kb

2.6 kb

m

-~ E CrJ

~ 04 04

E 2 E E co

+

+

5.8 kb

~ E m E ,,,,11...

~ 04 04 "g E ~ E 2 E E

. ,

Mottler of white

2.6 kb

oo +

T - -

+

" 5.8 kb

2.6 kb

5' exon probe 5' to 3' spanning probe 3' exon probe

Figure 5. Northern analysis of w ~ RNA products in the presence of the mwe mutant . RNA was isolated from flies (0-12 hr old) from a cross of w ~ m w ~ males to w a + / w a m w 2 females. Each blot was run separately and contains RNA from w ~ + males, w ~ + / w ~ m w 2 females, w ~ m w 2 males, and w ~ m w 2 / w ~ mw* females. Included in the center and right panels are lanes with RNA from wild type and from w l~s, a mutan t deficient for the 5' portion of w. (Left) The blot was probed with sequences homologous to the 5' exon; (center) the blot was probed with sequences that span the site of copia insertion in w; (right) the blot was probed with sequences homologous to regions of w 3' to copia. The position of the 2.6-kb normal w message is marked in each panel, as well as the major 5.8-kb RNA that initiates at w and terminates at the 3' of copia. In addition to these two RNAs, there is a pair with slightly greater molecular weight than the normal w message, which have homology to the 5' and spanning probes. An additional RNA species of lower molecular weight (1.2 kbl has homology to these two probes. Finally, a species of greater molecular weight than the 5.8-kb 3' LTR terminat ion product is detected with the spanning probe and the 3' exon. An additional RNA that has homology to all probes and is expressed at much greater levels in males than females is present in all cases, including wild type and the deletion strain w111s; thus, it is unl ikely to be a product of the w locus. The effect of the m w 2 mutat ion is to eliminate the normal 2.6-kb normal w message {most easily seen in the 3' exon panel) and to increase the two RNAs of slightly higher molecular weight, which have homology to the 5' and spanning exon probes.

tion of w ~, a deficiency for m w , and the induction of a new allele that is not mott led all suggest that the mot- tling is due to events at the m w locus and not w ~. It is postulated that each copy of m w in a female indepen- dently inactivates at different points in development and that this inactivation is mainta ined in a cell lineage manner. The inact ivat ion enhances transposon insertion mutan ts at w, thus producing the mosaic phenotype.

Possible mechan i sms of the action of m w on transpos- able e lement insertion alleles at w are as follows. The absence of the raw gene product would increase the de- gree of terminat ion of w-initiated RNAs wi th in the transposable element. The normal product of raw might therefore be involved in permit t ing readthrough of the terminat ion signals present in the respective tran- sposons to give the low level of functional RNA and pig- ment characteristic of each allele. Second, m w ÷ may be involved in RNA processing wi th in the transposable ele-

ment, and its e l iminat ion removes the processing pathway that leads to functional w message. Finally, the insertion of copia has been shown to alter the develop- menta l expression of w itself (Zachar et al. 1985). The m w product might be involved in regulating w under the influence of the unique set of regulatory information.

The fact that the m w 2 muta t ion does not alter either total w or total copia RNA levels, but reduces the level of the normal 2.6-kb message suggests a role other than modulat ing the transcription rates of copia or w. The apparent shift of the normal w message into one of the higher molecular weight forms present in w ~ suggests a role in alternative RNA processing.

While the available evidence suggests that m w is in- volved in a processing funct ion wi th the products of the w ~ allele, there is no evidence to suggest that this can be generalized to copia elements in other locations in the genome. In the absence of further data, it is important to

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Birchler et al.

O3

'~ E E & &

¢/3 (D 09 w

-~ E E

t 'Xl

2 kb

: : ~ - : Z z " =========================== . . . . . . . z : v

. . . . : : ? , , , : : . . . . . . . . . . . . : . . : . . . . . . . .

Figure 6. Northem analysis of copia RNA in the presence of the rnw a mutant. The RNA samples described in Fig. 4 were separated on formaldehyde-agarose gels, transferred to nylon membrane, and probed with sequences homologous to copia. The samples (left to right) are w ~ + males, w ~ +/w ~ mw e fe- males, w ~ mw a males, and w a m w a / w ~ m w 2 females. The posi- tion of the major 5.2-kb RNA is marked.

no te tha t sequences from w, as wel l as the respect ive t ransposable e lements , m a y be required to produce the observed shif t in R N A quant i t ies .

In the case of w ~ ' , the z m u t a n t m u s t be effective upon w for m w to produce a recognizable response in males . Because the t ransposon in th is case only has a m u t a n t effect in the presence of z, i t is possible tha t the t ransposon is on ly expressed then. It is po ten t ia l ly the case tha t the m w requires the express ion of the tran- sposon to be effective. Al terna t ive ly , the pheno typ ic re- duc t ion caused by z m a y a l low the cons tan t effect of m w

to be observed. Parkhurs t and Corces (1987) no ted tha t different

classes of re t ro t ransposons have different deve lopmenta l profiles of total RNA, as well as those of different molec- u lar we igh t f rom a single class of e lement , suggest ing cer ta in evo lu t ionary re la t ionships and diversi t ies among classes in t e rms of expression. Because m w acts on w m u t a n t s caused by e l emen t s w i t h diverse expression {namely, copia , B104, and 3S18), i ts i n v o l v e m e n t w i t h re t ro t ransposons appears to be an teceden t to the diver- s i ty observed. Modif iers of t ransposon- induced m u t a n t s have general ly been in terpre ted as represent ing no rma l cel lular func t ions tha t are paras i t ized by the retrotran- sposons. It is also possible tha t given the wide phyloge-

82 GENES & DEVELOPMENT

ne t ic r epresen ta t ion of re t roe lements , cer ta in func t ions have been selected to have specif ic i ty for e l e m e n t s and loss of i n v o l v e m e n t in o ther cel lular processes. Such a s i tua t ion could be selected for by i m p a i r m e n t of cel lular func t ions by compe t i t i on f rom re t roe lements . The ex- t en t of specif ic i ty m u s t awai t fur ther i n fo rma t ion about the role of var ious modif ier genes.

M e t h o d s

Drosophila culture

Flies were grown on Instant Drosophila Medium ICarolina Bio- logical Supply) at 25°C.

R N A e x t r a c t i o n

RNA was extracted from frozen flies by the method of Cox (1968). Homogenization was in 8 M guanidine-HC1 (Ultra- Pure, Schwarz/Mann), 0.01 M EDTA at a volume of 1 ml/gm of tissue, followed by ethanol precipitation with one-half volume. Four additional 4 M guanidine, 0.01 M EDTA/ethanol precipita- tions followed. The final pellet was extracted with diethylpyro- carbonate (DEPC)-treated water at a ratio of 1 ml/gram original tissue; a second water extraction was performed at 56°C; the third at room temperature. After ethanol precipitation from the water extractions, the RNA was stored frozen in chelexed, DEPC-treated water at -80°C.

Northern analysis

RNA was separated on formaldehyde- 1.5% agarose gels by the method of Lehrach et al. (1977). Formaldehyde was present in the tank buffer at the same concentration as that in the gel. RNA was transferred to Nytran nylon membrane, UV cross- linked (Church and Gilbert 1984; Khandjian 1986), and baked at 80°C for 2 hr.

Hybrid iza t ion

Filters of Northern blots were wetted in 5x SSC (1 x SSC = 0.15 M NAG1; 0.015 M Na citrate), 0.1% SDS and prehy- bridized (150 ~l/cm 2) in a solution of 50% formamide, deion- ized with AG 501-X8 (Bio-Rad) resin, 5 x SSC, 10 m/vi polyvin- ylpyrrolidine, 1% bovine serum albumin, 0.5% SDS, and 0.2 mg/ml calf thymus DNA (Sigma) for 4 hr at 60°C. Hybridiza- tion was started by the addition of 32P-labeled probe at 2 x 106 cpm/ml of solution and was conducted for 16 hr followed by four washes consisting of 0.1 x SSC, 5 mM NaH2HPO4, 0.015% pyrophosphate, 0.2% SDS (pH 7.0), each for 30 rain at 75°C. The filters were then washed at room temperature twice for 30 rain in 3 mM Tris-HC1 (pH 9.0). Filters were dried and subjected to autoradiography with Kodak XRP-1 film overnight at 70°C.

Dot blots

Pieces of nitrocellulose (8 x 20 cm) were marked in 2-cm squares with a lead pencil. The sheets were wetted in water and then soaked in 20× SSC (1 x = 0.15 M NaC1, 0.015 M Na ci- trate) for 4 hr prior to RNA application (Thomas 19801. The filters were then blotted to dampness on Whatman No. 3MM paper. The RNA 14 o.g/dot) was applied to the center of the squares in 4-ml aliquots in 10 replicas each for w a + males, w ~ mw ~ males, w~+/w ~ my& females, and w ~ m w ~ l w ~ m w a fe- males. The filters were placed between two sheets of Whatman

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No. 3MM paper and baked under vacuum for 2 hr at 800C. After hybridization and washing, the filters were subjected to autora- diography to check for gross background, cut into individual squares, dried under a heat lamp, and counted in 5 ml of tol- uene plus 4% Scintiprep (Fisher) in a Beckman LS 1801 scintil- lation counter.

DNA isolation

DNA from w ~ +, w ~ mw 2, and Canton S was prepared by the method of Bingham et al. (1981).

01igolabeling

Drosophila w locus DNA was labeled according to manufac- turer's {Pharmacia) specifications and the procedure of Feinberg and Vogelstein (1983).

Probe isolation

A fragment of Drosophila DNA, extending from the XbaI to SalI restriction sites surrounding coordinate 0 of Levis et al. {1982), was digested from the plasmid. The fragment and vector were separated in a 1% agarose gel, and the fragment was elec- troeluted and purified on an Elutip column (Schleicher and Schuell), followed by ethanol precipitation (0.33 M Na acetate and two volumes of ethanol).

Hybridization

Filter hybridization of Southern blots was conducted at 65°C, according to Church and Gilbert (1984), with the following modification. The hybridization solution was 50% (wt/vol) polyethylene glycol.

Probe preparation

Single-stranded RNA probes were generated from a T7/SP6 vector (IBI 76, International Biotechnology, Inc.)(Green et al. 1983) into which had been subcloned fragments of the white locus, extending from the BamHI to the HindIII site sur- rounding the first exon (designated pIBI 11.5 HB), from the XbaI to SalI sites surrounding the second and third exons (pIBI 12.5 XS), and a SalI fragment including a small portion of exon 3 and extending into exon 6 (pIBI 12.3 SS). The reaction mix consisted of the following: 40 mM Tris-HC1 (pH 7.5), 6 mM MgCI2, 2 mM spermidine, 0.5 rnM ATP, 0.5 mM CTP, 0.5 rnM GTP, -150 ~Ci [32P]UTP (3000 Ci/mmole) (New England Nuclear), 40 units RNasin (Promega Biotec), 15 units T7 Polymerase (New En- gland Biolabs), and 0.5 ~g linearized plasmid in a total volume of 20 ~1. The reaction was incubated at 37°C for 1 hr, at which point the volume was adjusted to 50 ~.1 with 0.01 M Tris-Cl, 0.01 M EDTA (pH 8.0). One microliter was removed and diluted to 50 ~.l in water. Twenty microliters of the diluted sample was spotted onto Whatman No. 1 2.3-cm disks. One was dried di- rectly and the second washed twice in 250 ml of 5% trichloro- acetic acid plus 1% sodium pyrophosphate, once in 250 ml of absolute ethanol and finally in 250 ml of anhydrous ether. The washed filter was dried under a heat lamp, and both were counted in a Beckman LS 1801 scintillation counter to deter- mine the percent of incorporation of label into RNA.

The completed reaction mixture was applied to a spin column of Sephadex G-50. The RNA recovered from the column was precipitated by addition of 1 ~g of Escherichia coli transfer RNA per 50 ~1 recovered from the column, one-tenth volume 2 M sodium acetate, and two volumes of absolute eth-

Mottler of white

anol. After centrifugation and resuspension in 100 ~1 of sterile DEPC-treated H20 , the appropriate volumes were added to the hybridization bags.

In situ hybridization

In situ hybridization was conducted as described by Langer- Safer et al. (1982), using a P-element probe biotinylated with biotin-16-dUTP (ENZO) and detected by reaction with a strep- tavidin-biotinylated peroxidase complex (ENZO) and staining with diaminobenzidine (ENZO), 0.03% H202 at 37°C for 20 rain.

A c k n o w l e d g m e n t s

Research was supported by a grant from the National Science Foundation. Discussions with Danielle Thierry-Mieg, Bob Levis, Steve Mount, Richard Linsk, and Mary Alleman were helpful. Plasmids of white and copia DNA were kindly supplied by Bob Levis. We thank the following individuals for fly stocks: Caltech, Bowling Green, Indiana, Umea stock centers, Danielle Thierry-Mieg, Bob Levis, Steve Mount, Paul Bingham, and Mel Green. We are also grateful to Ellie Valminuto and Karen McCree-Diaz for their help in preparing the manuscript.

References

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