Copyright 0 1990 by the Genetics Society of America
P Element Regulatory Products Enhance xeste’ Repression of a nWhitedup1icated3 Transgene in Drosophila melanogaster Dario Coen Micanismes Moliculaires de la Spiciation, Universiti Pierre et Marie Curie, F-75005 Paris, France Manuscript received March 24, 1990 Accepted for publication August 16, 1990 ABSTRACT Drosophila P element mobilization is subject to a complex array of regulatory mechanisms. A fruitful approach tostudy them is the use of insertion mutations whose expression is influenced by P regulation. In the present report, it is shown that P element somatic products may influence the expression of an unrelated geneinserted in a P transposon. T h e P[wd’9.3]19DE transgene carries an in vitro modified white gene harboringa duplication of the 5‘ regulatory sequences. Expression of this transgene is repressed in a P background. N o maternal effect is detected and repression can be relieved as soon as P chromosomes are replaced by M ones. T h e amplitude of repression is correlated to theP transposase activity of the individuals examined. Repression appears tobe exerted by somatic products of complete autonomous P elements or of in vitro modified P elements lacking the capacity to express the fourth P exon. T h e P repression of P[wd’9.3]19DE is strongly dependent on the insertion site of this transgene. This P repression effect occurs only in the presence of the zeste’ allele and is suppressed by Su(r)2mutations. No qualitative differencesof transcription pattern areobserved between white+ and P[wd’9.3]19DE in any backgrounds. P repression acts to reduce the amount of the major white transcript. This suggests that P regulatory products may act through cis-interactions at a distance of over 3 kb.
P
elements are a family of transposable elements responsible for the phenomenon of P-M hybrid dysgenesis in Drosophila melanogaster (for a review see ENGELS1989). This syndrome of correlated germline abnormalities (high rates of mutation and chromosome rearrangements, male recombination and a thermosensitive agametic sterility) occurs in the progeny of crosses involving females of an M strain (lacking P elements) and males of a P strain (containing numerous P elements in its genome). These traits are almost absent when the reciprocal cross is performed (as well as in P X P crosses). This is due to a cytoplasmic condition repressive for P element transposition called P cytotype. Transmission of P cytotype was shown to depend on both genomic P elements and maternal inheritance (ENGELS1979a,b, 198 1; KIDWELL 198 1, 1985). The P element family is heterogeneous in size. Its largest member is 2907 bp long and has 31-bp terminal inverted repeats. Shorter elements appear to derive from the 2.9-kbcompleteelement by internal deletions (O’HARE andRUBIN 1983). This complete 2.9-kb element is autonomouslyfunctional, as evidenced by germline transformation (SPRADLINGand RUBIN 1982).The transposase has been shown to be the 87-kD protein encoded by a single cistron comprising four exons (KARESSand RUBIN1984; LASKI, RIO and RUBIN1986; RIO, LASKIand RUBIN1986). P element mobilization is subject to two types of (;clrctir\ 1 2 6 949-960 (December, 1990)
regulation: astrain-specific one (cytotype) and a tissuespecific one (germlinelimitation of transposition). Tissue-specific regulation is achieved by alternative splicing; the third intron (2-3 intron) is spliced exclusively in the germline of both sexes, restricting the synthesis of transposase to this tissue (LASKI,RIO and RUBIN 1986). In somatic cells, lackof splicing of the 2-3 intron leads to a mRNA coding for a truncated 66kD protein that was postulated to be the (or one of the) repressor(s) of P transposition involved in P cytotype (RIo, LASKIand RUBIN 1986). Evidence that this may be the case comes from the results of NITASAKA, MUKAIand YAMAZAKI(1987) who have genetically and then molecularly cloned a particularinsertion of a P elementdeletedfor sequences in the fourth exon that mayspecify the P cytotype in a natural population. ROBERTSON and ENGELS(1989) have uncovered more direct evidence of the regulatory potential of some incomplete elements by using in vitro modified P elements. They have demonstrated that elements carrying mutations leaving the first three exons intact but altering the fourth exon or the splice-junction (KARESS andRUBIN1984; LASKI,RIO and RUBIN 1986) mimic some but not all aspects of the P cytotype (e.g., suppression of germline mutability and in certain conditions gonadal dysgenic sterility). However these modified P elements failed to show the reciprocal cross effect characteristic of the maternal inheritance of P cytotype.
950
D. Coen
One of the P cytotype features mimicked by the repressor-producing mutant P elements is their action on the expression of cytotype dependent P insertion mutations. The sterile phenotype of some singed alleles is enhanced by P cytotype, while the bristle phenotype of others is suppressed (ROBERTSON and ENGELS1989). WILLIAMS,PAPPUand BELL ( 1 9 8 8 ) have also described a vestigzal allele suppressed by the P cytotype. The present report shows that the cytotype repression can affect the expression of an unrelated gene inserted within a P transposon,instead of a P insertion mutation. P[wd’9.3]19DEis an insertion of a composite transposon carrying an in vitro modified white allele. The white gene is necessary in the adult for the pigmentation of the eyes, ocelli, malpighian tubules and thetestes sheath of males. The expression of white is repressed by the reste’ allele of the reste gene (GANS 1953). This repression depends not only on the presence of two copies of the white+ gene but also on their close proximity (transvection phenomenon, reviewed by WU and GOLDBERC 1989). T h e white region necessary for reste-white interaction has been defined genetically and molecularly and lies 1088-1859 bp upstream of the transcription start site (PIROTTA, STELLERand BOZETTI1985; LEVIS,HAZELRICC and RUBIN1985a; DAVISON et al. 1985). In P [ w d ’ ] ,the 5‘ sequences of the white+ gene, including this region have been duplicated in vitro (C. S. ZUCKER,personal communication). The P repression of P[wd’9.3]19DEis independent of the dysgenic state, displays no maternal effect and is reversible in onegeneration.This repression is proportional to P transposase activity. It can be exerted by somatic products encoded by the first three exons of P element, with no requirement for expression of the fourth exon. It is demonstrated that the P repression effect occurs through the enhancementof zpte’ repression on a single copy of the P[wd’9.3]1911E transgene. It is also strongly dependent on the chromosomal position of the P[wd’] insertion. P repression reduces the rate of the major white transcript but there is no qualitative modification of the transcription pattern in any backgrounds. These results suggest that the enhancement of zeste’ repression by P regulatory products involves cis-interactions at a distance of over 3 kb. MATERIALSANDMETHODS
The f l w ” ’ ] transgene: This transgene has been in vitro engineered by C. ZUCKER(personal communication). It carries the EcoRI-KpnI fragment containing the whole white gene including 5’ and 3‘ nontranscribed sequences sufficient for its correct temporal andspatial expression (HAZELRIGC,LEVISand RUBIN1984; LEVIS,HAZELRIGG and RUBIN 1985a; PIROTTA,STELLERand BOZETTI1985). In addition the 5’ EcoRI-Hind111fragment containing the5‘ regulatory
sequences and the first exon (PIROTTAand B R ~ C K1984) L has been duplicated in direct tandem order (Figure1). Droso hila stocks: T h e following stocks were used. y z’ wRI8 cu-M stock. w’lla is a viable partial deletion of the white locus (HAZELRIGG, LEVISand RUBIN1984). z 1 w1118 cu P[ud’9.3]19DE-A z1w1118 cv Mstrain was transformed with the DNA of P[wd’] to generate this line (C. ZUCKER and D. THIERRY-MIEG,personalcommunication; RUBINet al. 1985). This line contains an insertion of the P[wd’] element lying proximally on the X chromosome, in 19DE. This particular insertion will be named P[wd’9.3] 19DE. T h e z 1 w ” ’ ~ cu P[wd19.3]19DE X chromosome, or the line harboring it, will be abbreviated “z w 9.3.” Gruta-A typical M strain, lacking any P element sequences (ANXOLABEHERE et al. 1987). HS2-20 and other RPstrains-This set of P or M’ strains was derived from the Gruta stock by germline transformation with P element DNA(ANXOLABEHERE et al. 1987). They are therefore essentially isogenic to Gruta and between them. Harwich-A standard P reference strain (KIDWELL,KIDWELL and SVED1977). r2-An inbred P strain (ENGELSand PRESTON1979). M’ strains-”-Birmingham is an M’ strain (BINGHAM, KIDWELLand RUBIN 1982)that was described as being devoid of repressor making P elements (ROBERTSON and et al. 1988). Alma-Ata, BerlinENGELS1989; ROBERTSON 83, Chimkent,Ica, Kurume, and Uman-83 are wild-type M’ strain derived from natural populations. Their characteristics are described in RONSSERAY, LEHMANN and ANXOLABEHERE (1989). C ( I ) D X ,yf/FM7(P)/y+Y; r2-A P cytotype stock with all its autosomes derived from r2 (ENGELS1979a). C ( I ) D X , y w f/w v 1(1)44/Y-An M stock used to collect virgin females. l ( I ) 4 4 is a thermosensitive X-linked lethal (BUSSON et al. 1983). Thisstock will be abbreviated“C(I)DX y WAM).” #129-A C(I)DX, yf/w v 1(1)44/Y; r2-stock generated by substituting all the autosomes by a2 autosomes (M. GANS personal communication). C ( I ) D X , y w AP)/w v 1(1)44(P)/Y; Harwich-The X chromosomes of this stock were contaminated with P elements as described by ENGELS(1985). All the autosomes derive from Harwich. #625, #696 and #784 are differentsublines isolated at various steps of the process of P contamination. T h e #784 stock will be designated in the text as “C( I)DX y WAP).” rySo6P[ry+AIuI](86E),ry506P[ry+XhoI](47F), ry506P[ry+RI] stocks were gener(89A) and rySo6P[~I+SalI](89D)-These ated by transformation of a rySu6M strain with P elements in vitro mutated in each of the four exons by KARESSand RUBIN(1984). They were used by ROBERTSON and ENGELS (1 989)in their screen of repressor-producing P elements. They will beabbreviated in thetext as “P[AluI](86E),” “P[XhoI](47F),” “P[RI](89A)” and “P[SalI](89D),” respectively. rySo6P[ry+A2-3](99B)-This strain contains an essentially immobile P element which produces a high level of transposase somatically and germinally (ROBERTSON et al. 1988). It is designated hereafter as “A2-3(99B).” Su(z)2’/CyO and S~(z)2~/CyO-Twoalleles of the Su(z)2 complex (PERSSON1976; KALISCH and RASMUSON 1974). All genetic symbols not described here are in LINDSLEY and GRELL (1968) in or LINDSLEY and ZIMM (1985). Pigment assays: Pigment level determinations in extracts of adult heads were performed as described by HAZELRIGG, LEVISand RUBIN(1984). T h e mean of observed values of optical density is given in the tables, accompanied with its
P Repression of a P[w] Transgene
white
95 1
+
wild type RNA E
Bg
BgB
I
11
II I
X X U
I Is
5
I
S
IK
Probes exon 1
exons 4-5
P(w d l )
"
s confidence interval (calculated with theStudent t value corresponding to d.f. = (number of measures - 1) and (Y =
0.05). In this assay, absorbance mean value of pigment extracts from white males from a y z' w'"' N strain was 0.007 f 0.008.
P activity assay: Mobilization ofthe P[wd'9.3]19DE tranP activity sposon itselfwas used to determine the transposase of the tested strains. Males to be tested were crossed to z w 9.3 females (A type cross) and the resulting FI males were crossed to C( 1)DXy w AHarwich females (see Figure 2) in order to detect the occurrence of mutations in their germline. Mutation rates were calculated by the percentage of non-brown males occurring in the individual F2s obtained (see RESULTS). Generation of autosomal insertion of flwd'9.3]19DE: z w 9.3 females were mated to males from various P stocks or to A2-3(99B) males. F, males were individually crossed to y z I W/lln N females. Jumps to autosomal locations result in exceptional non-white F2 males. Males resulting from independent transposition events were individually back-crossed several times to y z ' w'"' cu females to generate lines to be analyzed. RNA preparation andanalysis: Crosses wereperformed at 2.5" and adult flies of the appropriate genotype and of 0- 12 hr of age were collected. Heads, separated by shaking flies frozen in liquid nitrogen, or whole flies were stored at -80" until extraction. Extraction, electrophoresis, transfer and hybridization wereperformed as described by RARINOW and BIRCHLER(1 989). Probes where synthesized from clones carried in an SP6/ T 7 vector (pIBI76)for white probes or in a BlueScript vector for ribosomal protein 49. T 7 R N A polymerase (Promega Biotech Gemini Kit) was used in conditions as described by RARINOWand BIRCHLER( 1 989). RESULTS
Eye phenotype of the z w 9.3 strain: T h e z ' w'll' P[w"'9.3]19DE strain, abbreviated hereafter z w Y.3, results from an insertion of the P[w'"] transposon (see Figure 1 and MATERIALS AND METHODS) after
cu
s
S
K
FIGURE 1 .--Structure of the while gene and of the P[w"'] transposon.
P Element Regulatory Products Enhance xeste’ Repression of a nWhitedup1icated3 Transgene in Drosophila melanogaster Dario Coen Micanismes Moliculaires de la Spiciation, Universiti Pierre et Marie Curie, F-75005 Paris, France Manuscript received March 24, 1990 Accepted for publication August 16, 1990 ABSTRACT Drosophila P element mobilization is subject to a complex array of regulatory mechanisms. A fruitful approach tostudy them is the use of insertion mutations whose expression is influenced by P regulation. In the present report, it is shown that P element somatic products may influence the expression of an unrelated geneinserted in a P transposon. T h e P[wd’9.3]19DE transgene carries an in vitro modified white gene harboringa duplication of the 5‘ regulatory sequences. Expression of this transgene is repressed in a P background. N o maternal effect is detected and repression can be relieved as soon as P chromosomes are replaced by M ones. T h e amplitude of repression is correlated to theP transposase activity of the individuals examined. Repression appears tobe exerted by somatic products of complete autonomous P elements or of in vitro modified P elements lacking the capacity to express the fourth P exon. T h e P repression of P[wd’9.3]19DE is strongly dependent on the insertion site of this transgene. This P repression effect occurs only in the presence of the zeste’ allele and is suppressed by Su(r)2mutations. No qualitative differencesof transcription pattern areobserved between white+ and P[wd’9.3]19DE in any backgrounds. P repression acts to reduce the amount of the major white transcript. This suggests that P regulatory products may act through cis-interactions at a distance of over 3 kb.
P
elements are a family of transposable elements responsible for the phenomenon of P-M hybrid dysgenesis in Drosophila melanogaster (for a review see ENGELS1989). This syndrome of correlated germline abnormalities (high rates of mutation and chromosome rearrangements, male recombination and a thermosensitive agametic sterility) occurs in the progeny of crosses involving females of an M strain (lacking P elements) and males of a P strain (containing numerous P elements in its genome). These traits are almost absent when the reciprocal cross is performed (as well as in P X P crosses). This is due to a cytoplasmic condition repressive for P element transposition called P cytotype. Transmission of P cytotype was shown to depend on both genomic P elements and maternal inheritance (ENGELS1979a,b, 198 1; KIDWELL 198 1, 1985). The P element family is heterogeneous in size. Its largest member is 2907 bp long and has 31-bp terminal inverted repeats. Shorter elements appear to derive from the 2.9-kbcompleteelement by internal deletions (O’HARE andRUBIN 1983). This complete 2.9-kb element is autonomouslyfunctional, as evidenced by germline transformation (SPRADLINGand RUBIN 1982).The transposase has been shown to be the 87-kD protein encoded by a single cistron comprising four exons (KARESSand RUBIN1984; LASKI, RIO and RUBIN1986; RIO, LASKIand RUBIN1986). P element mobilization is subject to two types of (;clrctir\ 1 2 6 949-960 (December, 1990)
regulation: astrain-specific one (cytotype) and a tissuespecific one (germlinelimitation of transposition). Tissue-specific regulation is achieved by alternative splicing; the third intron (2-3 intron) is spliced exclusively in the germline of both sexes, restricting the synthesis of transposase to this tissue (LASKI,RIO and RUBIN 1986). In somatic cells, lackof splicing of the 2-3 intron leads to a mRNA coding for a truncated 66kD protein that was postulated to be the (or one of the) repressor(s) of P transposition involved in P cytotype (RIo, LASKIand RUBIN 1986). Evidence that this may be the case comes from the results of NITASAKA, MUKAIand YAMAZAKI(1987) who have genetically and then molecularly cloned a particularinsertion of a P elementdeletedfor sequences in the fourth exon that mayspecify the P cytotype in a natural population. ROBERTSON and ENGELS(1989) have uncovered more direct evidence of the regulatory potential of some incomplete elements by using in vitro modified P elements. They have demonstrated that elements carrying mutations leaving the first three exons intact but altering the fourth exon or the splice-junction (KARESS andRUBIN1984; LASKI,RIO and RUBIN 1986) mimic some but not all aspects of the P cytotype (e.g., suppression of germline mutability and in certain conditions gonadal dysgenic sterility). However these modified P elements failed to show the reciprocal cross effect characteristic of the maternal inheritance of P cytotype.
950
D. Coen
One of the P cytotype features mimicked by the repressor-producing mutant P elements is their action on the expression of cytotype dependent P insertion mutations. The sterile phenotype of some singed alleles is enhanced by P cytotype, while the bristle phenotype of others is suppressed (ROBERTSON and ENGELS1989). WILLIAMS,PAPPUand BELL ( 1 9 8 8 ) have also described a vestigzal allele suppressed by the P cytotype. The present report shows that the cytotype repression can affect the expression of an unrelated gene inserted within a P transposon,instead of a P insertion mutation. P[wd’9.3]19DEis an insertion of a composite transposon carrying an in vitro modified white allele. The white gene is necessary in the adult for the pigmentation of the eyes, ocelli, malpighian tubules and thetestes sheath of males. The expression of white is repressed by the reste’ allele of the reste gene (GANS 1953). This repression depends not only on the presence of two copies of the white+ gene but also on their close proximity (transvection phenomenon, reviewed by WU and GOLDBERC 1989). T h e white region necessary for reste-white interaction has been defined genetically and molecularly and lies 1088-1859 bp upstream of the transcription start site (PIROTTA, STELLERand BOZETTI1985; LEVIS,HAZELRICC and RUBIN1985a; DAVISON et al. 1985). In P [ w d ’ ] ,the 5‘ sequences of the white+ gene, including this region have been duplicated in vitro (C. S. ZUCKER,personal communication). The P repression of P[wd’9.3]19DEis independent of the dysgenic state, displays no maternal effect and is reversible in onegeneration.This repression is proportional to P transposase activity. It can be exerted by somatic products encoded by the first three exons of P element, with no requirement for expression of the fourth exon. It is demonstrated that the P repression effect occurs through the enhancementof zpte’ repression on a single copy of the P[wd’9.3]1911E transgene. It is also strongly dependent on the chromosomal position of the P[wd’] insertion. P repression reduces the rate of the major white transcript but there is no qualitative modification of the transcription pattern in any backgrounds. These results suggest that the enhancement of zeste’ repression by P regulatory products involves cis-interactions at a distance of over 3 kb. MATERIALSANDMETHODS
The f l w ” ’ ] transgene: This transgene has been in vitro engineered by C. ZUCKER(personal communication). It carries the EcoRI-KpnI fragment containing the whole white gene including 5’ and 3‘ nontranscribed sequences sufficient for its correct temporal andspatial expression (HAZELRIGC,LEVISand RUBIN1984; LEVIS,HAZELRIGG and RUBIN 1985a; PIROTTA,STELLERand BOZETTI1985). In addition the 5’ EcoRI-Hind111fragment containing the5‘ regulatory
sequences and the first exon (PIROTTAand B R ~ C K1984) L has been duplicated in direct tandem order (Figure1). Droso hila stocks: T h e following stocks were used. y z’ wRI8 cu-M stock. w’lla is a viable partial deletion of the white locus (HAZELRIGG, LEVISand RUBIN1984). z 1 w1118 cu P[ud’9.3]19DE-A z1w1118 cv Mstrain was transformed with the DNA of P[wd’] to generate this line (C. ZUCKER and D. THIERRY-MIEG,personalcommunication; RUBINet al. 1985). This line contains an insertion of the P[wd’] element lying proximally on the X chromosome, in 19DE. This particular insertion will be named P[wd’9.3] 19DE. T h e z 1 w ” ’ ~ cu P[wd19.3]19DE X chromosome, or the line harboring it, will be abbreviated “z w 9.3.” Gruta-A typical M strain, lacking any P element sequences (ANXOLABEHERE et al. 1987). HS2-20 and other RPstrains-This set of P or M’ strains was derived from the Gruta stock by germline transformation with P element DNA(ANXOLABEHERE et al. 1987). They are therefore essentially isogenic to Gruta and between them. Harwich-A standard P reference strain (KIDWELL,KIDWELL and SVED1977). r2-An inbred P strain (ENGELSand PRESTON1979). M’ strains-”-Birmingham is an M’ strain (BINGHAM, KIDWELLand RUBIN 1982)that was described as being devoid of repressor making P elements (ROBERTSON and et al. 1988). Alma-Ata, BerlinENGELS1989; ROBERTSON 83, Chimkent,Ica, Kurume, and Uman-83 are wild-type M’ strain derived from natural populations. Their characteristics are described in RONSSERAY, LEHMANN and ANXOLABEHERE (1989). C ( I ) D X ,yf/FM7(P)/y+Y; r2-A P cytotype stock with all its autosomes derived from r2 (ENGELS1979a). C ( I ) D X , y w f/w v 1(1)44/Y-An M stock used to collect virgin females. l ( I ) 4 4 is a thermosensitive X-linked lethal (BUSSON et al. 1983). Thisstock will be abbreviated“C(I)DX y WAM).” #129-A C(I)DX, yf/w v 1(1)44/Y; r2-stock generated by substituting all the autosomes by a2 autosomes (M. GANS personal communication). C ( I ) D X , y w AP)/w v 1(1)44(P)/Y; Harwich-The X chromosomes of this stock were contaminated with P elements as described by ENGELS(1985). All the autosomes derive from Harwich. #625, #696 and #784 are differentsublines isolated at various steps of the process of P contamination. T h e #784 stock will be designated in the text as “C( I)DX y WAP).” rySo6P[ry+AIuI](86E),ry506P[ry+XhoI](47F), ry506P[ry+RI] stocks were gener(89A) and rySo6P[~I+SalI](89D)-These ated by transformation of a rySu6M strain with P elements in vitro mutated in each of the four exons by KARESSand RUBIN(1984). They were used by ROBERTSON and ENGELS (1 989)in their screen of repressor-producing P elements. They will beabbreviated in thetext as “P[AluI](86E),” “P[XhoI](47F),” “P[RI](89A)” and “P[SalI](89D),” respectively. rySo6P[ry+A2-3](99B)-This strain contains an essentially immobile P element which produces a high level of transposase somatically and germinally (ROBERTSON et al. 1988). It is designated hereafter as “A2-3(99B).” Su(z)2’/CyO and S~(z)2~/CyO-Twoalleles of the Su(z)2 complex (PERSSON1976; KALISCH and RASMUSON 1974). All genetic symbols not described here are in LINDSLEY and GRELL (1968) in or LINDSLEY and ZIMM (1985). Pigment assays: Pigment level determinations in extracts of adult heads were performed as described by HAZELRIGG, LEVISand RUBIN(1984). T h e mean of observed values of optical density is given in the tables, accompanied with its
P Repression of a P[w] Transgene
white
95 1
+
wild type RNA E
Bg
BgB
I
11
II I
X X U
I Is
5
I
S
IK
Probes exon 1
exons 4-5
P(w d l )
"
s confidence interval (calculated with theStudent t value corresponding to d.f. = (number of measures - 1) and (Y =
0.05). In this assay, absorbance mean value of pigment extracts from white males from a y z' w'"' N strain was 0.007 f 0.008.
P activity assay: Mobilization ofthe P[wd'9.3]19DE tranP activity sposon itselfwas used to determine the transposase of the tested strains. Males to be tested were crossed to z w 9.3 females (A type cross) and the resulting FI males were crossed to C( 1)DXy w AHarwich females (see Figure 2) in order to detect the occurrence of mutations in their germline. Mutation rates were calculated by the percentage of non-brown males occurring in the individual F2s obtained (see RESULTS). Generation of autosomal insertion of flwd'9.3]19DE: z w 9.3 females were mated to males from various P stocks or to A2-3(99B) males. F, males were individually crossed to y z I W/lln N females. Jumps to autosomal locations result in exceptional non-white F2 males. Males resulting from independent transposition events were individually back-crossed several times to y z ' w'"' cu females to generate lines to be analyzed. RNA preparation andanalysis: Crosses wereperformed at 2.5" and adult flies of the appropriate genotype and of 0- 12 hr of age were collected. Heads, separated by shaking flies frozen in liquid nitrogen, or whole flies were stored at -80" until extraction. Extraction, electrophoresis, transfer and hybridization wereperformed as described by RARINOW and BIRCHLER(1 989). Probes where synthesized from clones carried in an SP6/ T 7 vector (pIBI76)for white probes or in a BlueScript vector for ribosomal protein 49. T 7 R N A polymerase (Promega Biotech Gemini Kit) was used in conditions as described by RARINOWand BIRCHLER( 1 989). RESULTS
Eye phenotype of the z w 9.3 strain: T h e z ' w'll' P[w"'9.3]19DE strain, abbreviated hereafter z w Y.3, results from an insertion of the P[w'"] transposon (see Figure 1 and MATERIALS AND METHODS) after
cu
s
S
K
FIGURE 1 .--Structure of the while gene and of the P[w"'] transposon.
