Recombination Between Duplicated Elements Using a Site-Specific

Copyright 0 1994 by the Genetics Society of America

Local Transposition of P Elements in Drosophila melanogaster and Recombination Between Duplicated Elements Using a Site-Specific Recombinase

Kent G. Golic'

Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637, and Department of Biology, University of Utah, Salt Lake City, Utah 84112

ABSTRACT The transposase source A2-3(99B) was used to mobilize a P element located at sites on chromosomes

X , 2 and 3. The transposition event most frequently recovered was a chromosome with two copies of the P element at or near the original site of insertion. These were easily recognized because the P element carried a hypomorphic white gene with a dosage dependent phenotype; flies with two copies of the gene have darker eyes than flies with one copy. The P element also carried direct repeats of the recombination target (FRT) for the FLP site-specific recombinase. The synthesis of FLP in these flies caused excision of the FRT-flanked white gene. Because the two white copies excised independently, patches of eye tissue with different levels of pigmentation were produced. Thus, the presence of two copies of the FRT-flanked white gene could be verified. When the P elements lay in the same orientation, FLP-mediated recombination between the FRTs on separated elements produced deficiencies and duplications of the flanked region. When P elements were inverted, the predominant consequence of FLPcatalyzed recombination between the inverted elements was the formation of dicentric chromosomes and acentric fragments as a result of unequal sister chromatid exchange.

V AN SCHAIK and BRINK (1959) demonstrated that the Ac mobile element of maize exhibits a tendency to

transpose to chromosomal sites near its original site of insertion, and further, that the transposition events frequently produce chromosomes carrylng two closely linked elements: the original insertion and a second newly inserted element nearby. The determination of these transposition characteristics was facilitated by the phenotype of the particular Ac insertion analyzed. P- W is a variegating allele of the gene that controls pericarp pigmentation. This allele results from the insertion of an Ac element (also called M p ) in the P gene. Transposi- tion of Ac away from P reverts the phenotype. If this occurs su5ciently early in development a revertant spot encompassing several kernels will be produced on an ear. The pattern of variegation produced by P - W de- pends on the number of Ac copies resident in the genome (MCCLINTOCK 1948; BRINK and NILAN 1952). BRINK and NILAN observed that transposition events that caused reversion were frequently accompanied by a twin spot with the altered pattern of variegation that signaled the presence of two Ac copies. Genetic mapping of the second element revealed that it was usually linked to the P locus (VAN SCHAIK and BRINK 1959). Similar results have been obtained with the Tam3 element of Antirrhinum majus (COEN et al. 1989). Recently TOWER et al. (1993), ZHANG and SPRADLINC (1993), and DANIELS and CHOVNICK (1993) have shown that P transposable elements in Dro- sophila melanogaster are similarly disposed to transpose to sites near the original insertion.

Utah 841 12. I Current address: Department of Biology, University of Utah, Salt Lake City,

Genetics 137: 551-563 (June, 1994)

In a fashion similar to the Ac insertion that is P - W , I have used a P mobile element in Drosophila which al- lows the easy recognition of chromosomes that carry more than one copy of this element. Transposition of this element frequently results in chromosomes that carry two copies of the element very close to each other at the original insertion site. This element also carries direct repeats of the sequence (FRT) that is recognized by the site-specific recombinase (FLP) from the 2 p plas mid of Saccharomyces cerevisiae. The FLP gene, having been placed under the control of a Drosophila hsp70 promoter, can be induced with a short heat shock. The FLP that is made then catalyzes recombination between FRTs carried within a P element (GOLIC and LINDQUIST 1989). I show here that FLP can also mediate recombination between FRTs on separate elements. The trans position properties of P elements, combined with the availability of FLP-mediated recombination between FRT-bearing elements, may provide a useful tool for genetic investigations in D . melanogaster.

MATERIALS AND METHODS

Fly stocks: The plasmid P[>wh'>] that carries an FRT- flanked white gene and flies transformed with this plasmid are described by GOLIC and LINDQUIST (1989). FLP was provided by the hsFLP gene described therein. P[$, hsFLP12B is an insertion of this element on chromosome 2. It is homozygous lethal and is maintained over S2 Cy 0. In one experiment I also used a second similar heat-inducible FLP gene designated 70FLP3F located on the X chromosome (R. PETEFSEN and K. GOLIC, unpublished results). The A2-3(99B) transposase- producing stock is described by ROBERTSON et al . (1988). Other mutations are described by LINDSLEY and ZIMM (1992). Flies were raised at 25" on standard cornmeal-agar medium. Heat

552 K. G. Golic

J

J G2 pigmented Sco or Cy Sb+ d‘ or X w1118

(screen for altered eye color) I Screen for linkage to Xor 2

FIGURE 1.-The P[>whS>] transposition scheme. See text for details.

shocks were performed as described by GOLIC and LINDQUIST (1989).

Transposition of P[>wh”>]: Insertions of P[>whs>] are designated with a parenthetical indication of cytological location followed by an arbitrary isolation number. The crossing scheme for obtaining transposed copies of the chromosome 3 insertion P[>wh”>] (67A)lC is indicated in Figure 1. Trans- posed copies of the X-linked insertion P[>whs>] (SF) 73 and the chromosome 2 insertion P[>whS>] (60D)83 were obtained with analogous crosses except that, in the case of the X-linked insertion P[>whS>] (8F)73, when transposition to an autosome occurred in GI males it could be detected by the production of G, sons with pigmented eyes. To prevent the recovery of identical replicates of the same transposition no more than one transposed P[>whs>>l was recovered from each G, parent (although many progeny from a single parent may have been tested) unless there was an obvious indication that more than one independent transposition had occurred. Evidence for independent transpositions from the same parent was either different eye colors or linkage to different chromosomes. When the GI parent was female, G, Sb+ white mosaics were occasionally observed. I attributed this to flies that had received A2-3(99B) but not Sb owing to recombination in the G, female. These flies were discarded. The transposed copies were mapped to a chromosome using dominantly marked bal- ancer chromosomes or by looking for sex-linkage.

Cytology of neuroblast chromosomes: Metaphase chromosome spreads from larval neuroblasts were prepared as described by GATTI and PIMPINELLI (1983). The chromosomes were stained with 0.01 pg/ml of 4,6-diamindino-2- phenylindole (DAPI) (Sigma) in 1X phosphate-buffered sa- line (PBS) for 10 min, destained in l X PBS for 10 min, rinsed briefly with distilled water and air dried. They were mounted in 1 X PBS and stored at 4” overnight. They were then examined by fluorescence with W excitation on a Zeiss Axioplan equipped with an Optovar using a 100 X Plan-NEOFLUAR objective. Images were captured with a Zeiss ZVS47EC video camera and Colorsnap 32+ (Computer Friends, Portland, Oregon) capture board with RGB inputs in an Apple Macintosh Quadra 700.

Polytene chromosomes: Salivary gland polytene chromosomes were prepared as described by LEFEVRE (1976). For in situ hybridization the chromosomes were prepared as described by PARDUE (1986). Hybridization and detection were performed using the GENIUS system (Boehringer Mann- heim). Chromosomes were examined with brightfield and phase contrast. Images were captured as described above or by

using a Sony DXC-151 RGB video camera instead of the Zeiss camera.

Photography of developmental defects: For Figure 3 flies were photographed with Ektachrome 160T using an Olympus SZH microscope. For Figure 6 flies were mounted as described by LAWRENCE et al. (1986) and examined and photographed using a compound microscope. Slides were digitized to a PhotoCD (Eastman Kodak).

Printing images: Images for figures were assembled and la- beled in Photoshop (Adobe Systems, Mountain View, Califor- nia) running on an Apple Macintosh. The only manipulations made were whole-frame adjustments to brightness and contrast or digital sharpening. They were printed using a Tek- tronix (Wilsonville, Oregon) Phaser IIsdx dye-sublimation printer.

RESULTS

Chromosomes carrying two adjacent copies of P[>whs>] are a frequent product of A2-3 mediated transposition: The P element I used (P[>whs>>I) carries a hypomorphic allele of the white gene that confers an easily recognized dosagedependent phenotype (KLEMENZ

et al. 1987). A single copy of this gene in a w- back- ground typically produces flies with orange eyes, and two copies gives red eyes. The level of pigmentation produced by wh’ is also subject to chromosomal position effects. Some instances of P[>wh5>] integration result in red-eyed flies when only a single copy is present while some produce lighter yellow or pink-eyed flies. The phe- notypic variation apparent when whs is inserted at different chromosomal sites can be utilized when screen- ing for mobilization of a Pelement that carries this gene: altered eye colors immediately signal the occurrence of a transposase-mediated event without the necessity of mapping the element to a new location.

I generated new P[>whS>] insertion lines by using the transposase source P[ 9’. A2-3](99B) to mobilize single copies of P[>whS>] inserted at three different chromosomal loci. Each conditioned orange eye color when present as the only source of white gene product. The transposition scheme frequently produced flies with red eyes in G, instead of the parental orange-colored eyes. I recovered some flies with P[>wh’>] transposed to other chromosomes, which were identified by following the segregation of the whs gene. However, the majority of these flies carried P[>wh5>] on the same chromosome as the original insertion (Table 1).

I first considered the possibility that these flies had a darker eye color because they possessed an insertion at a new locus where the whs gene was expressed at a higher level. Since the whs gene in the construct used here is flanked by direct repeats of the FRT, each whr gene can be independently excised when synthesis of the FLP site- specific recombinase is induced. The eye-color mosaicism that results from intra-element recombination can be used to diagnose the presence of two whs genes. Ex- amination of mosaics quickly distinguished red-eyed flies that carried only one copy of whs from flies that had

Recombination Between P elements 553

TABLE 1

Transposition of p[>wh5]

Transposition type

No. of Independent Intra- Inter- Intrachromosomal Dicentric Insertion“ GI mated transpositions chromosomal chromosomal with two copiesb phenotype

(8F) 73 97 18; (60D)83 54 30 (67A) 1C 104 91

14 4 23 2 79 e 12

12/13 8/12 23/23 10/23 51/62 14/51

a The P[>wh’>] insertions that were transposed are indicated. These data represent the minimum number that carry two copies of P[>whs>>l. Not all of the intrachromosomal jumps were tested for copy

These were recognized by altered eye color in G2 or by scoring the progeny of mated G, animals for altered linkage. In these flies altered eye color of G, individuals was the only screen used-we did not attempt to recover transpositions by altered linkage.

e Seventytwo were darker and seven were lighter than the 1C insertion. The latter seven are not included in the 62 that were tested for two

number and of those tested with FLP a small number could not be determined.

Only 25/30 were tested further.

copies.

red eyes because they carried two copies of whs. In the first case mosaics had only red and white patches; in the second case the mosaic flies had red and orange and white patches owing to the independent excision of each whs gene.2 I found that almost all of the redeyed flies with P[>whS>] on the same chromosome as the original insertion had red eyes because they carried two copies of P[>wh5>] (Table 1). For instance, 15 of 18 transpositions recovered from an X chromosome location were darker than the original insertion, and 14 of those 15 were on the X. (These 15 were recovered by selecting G, individuals with darker eye colors: the remaining three transpositions were recognized by autosomal inherit- ance.) Of the 14 transpositions on the X, at least 12 were two-copy insertions. One was single copy and one was not determined. In sum, of 15 flies selected for darker eyes, at least 12 carried two copies of P[>whs>>1 on the same chromosome as the original insertion. I also recovered two-copy insertions at high frequencies when P[>whS>] was transposed from a site on chromosome 2 and a site on chromosome 3 (Table 1).

The two copies are usually adjacent one another at or near the parental insertion site: In the original transposition experiment I did not at first realize that the darker eyed flies I collected carried two copies of P[>whS>]. I therefore made no attempt to prevent recombination on the chromosomes that now carried two elements. In the course of mapping and making stocks of the two-copy insertion lines, the insertion-bearing chromosomes were present in females heterozygous with a normal chromosome yet I very rarely observed

catalyzed recombination occasionally produces cells that have gained an addi- ‘This is a slight simplification of the mosaicism actually observed. FLP-

tional copy of W” in tandem with the first. This can result in three levels of pigmentation in a fly that originally camed only a single d” gene (Gout and LINDQUIST 1989). This potential confusion is in practice only a minor problem when diagnosing copy number. The patches with tandem copies of whs produced by amplification in situ are, first, darker in color than the eye of the mon-mosaic parent, and second, relatively infrequent compared to the two- copy patches that result when neither copy of 4’ in a twc-copy fly is excised

After looking at a few such mosaics it becomes fairly easy to distinguish flies under the conditions used here (37” or 38” 1-hr heat shock to induce hrFLf2B).

M n g one copy of 4 > u k 1 from flies carrying two copies of this element.

segregation of progeny with lighter eye colors. This im- plied that the two copies were near one another making recombination between them rare.

When I performed in situ hybridization to polytene chromosomes to localize P[>whS>] I found that the insertions in two-copy lines were indeed very close. Eight of the X chromosome two-copy lines, 11 of the chromosome 2 two-copy lines, and 14 of the chromosome 3 two-copy lines were examined. In only one instance were the P elements far apart (at 2A and at SF on the X). In the majority of cases hybridization occurred about the site of the parental insertion and there was only a single band of hybridization (5/8 X lines, 4/11 chromosome 2 lines, 11/14 chromosome 3 lines). In some cases there were two barely separable bands (2/8 Xlines, 5/11 chromosome 2 lines, 1/14 chromosome 3 lines). Infre- quently the two bands were easily separable cytologically but still genetically quite close (2/11 of the chromosome 2 lines, 2/14 of the chromosome 3 lines). Figure 2a shows the localization of elements in the two-copy line P[>whS>] (67A)85. This line exhibited the largest separation of all the two-copy lines that were examined (excepting the X chromosome line noted above).

Independently derived twocopy insertions are not identical: As the majority of two-copy insertions localize to but a single band, it is possible that most transposition occurs to a single hotspot adjacent to the original copy. Several lines of evidence suggest that this is not the case. First, a given set of the double-copy lines (derived from a single parental insertion) vary in their degree of pigmentation, producing eye colors ranging from only slightly darker than the original insertion up to an almost wild-type dark red. Second, some of the the two- copy lines are homozygous viable, some are lethal, and some are viable but sterile when homozygous (data not shown). Third, the two-copy lines fall into two classes with respect to the relative orientation of the two elements: those which are directly repeated and those in which the copies are inverted.

554 K. G. Colic

Deficiencies and duplications are produced by exchange between directly repeated elements: FLP- mediated exchange between the directly repeated FRTs within a single copy of P[>w"'>] results in the excision and loss of the 7uh,' gene. Unequal exchange between the FRTs on sister chromatids can result in the amplification (and deletion) of whr (GOLIC and LINDQUIST 1989). Simi- larly, I find that FLP can catalyze recombination between the FRTs of separated P[>w"'>] elements that lie in the same orientation and that this recombination can generate either a deficiency of the chromosomal material between the two elements, or a duplication of that same material.

I noticed that when flies carrying the homozygous viable two-copy insertion P[>w"">] (67A)85 and h.sFLP were heat-shocked and then mated, a small number of their progeny exhibited a Minute phenotype; that is, they had small fine bristles, were delayed in development, and the females were sterile. The chromosome 3 derivative of P[>w"'>] (67A)85 that these flies carried had lost 7 d r and was lethal when made homozygous. These are the expected phenotypes of a heterozygous Minute (M) deficiency and a M gene is present in the region of the #85 insertion (M(3)67C). I concluded that the two copies of P[>wh'>] carried by strain #85 flank this M locus, and that FLP-mediated recombination deleted the M gene between them to produce a deficiency designated Df(3Z.)85, M.

The frequency of deficiency formation by FLP- mediated recombination between the FRTs of separated elements is much less than the frequency of wkr excision from P[>w*">] under the same conditions. In one experiment wlll' 70FLP3F; P[>wh'>] (67A)85/Sb males were heat-shocked for 1 hr at 38" during the first 3 days of development then mated to w"'8females. I recovered deficiencies of the region (recognized as Minute Sb+ progeny) with a frequency of 0.7%. Three of 20 males produced Minute offspring; 5/767 Sb+ progeny were Minute. Excision of wb' measured in the same flies was greater than 99% (763/767 were 7uhr). In a second experiment (but using hsFZJ2B instead of 70FLP3F) I recovered deficiencies of the region with a frequency of 1.8%. Twelve of 22 males produced Minute progeny which occurred with a total frequency of 19/2070. How- ever in this experiment the normal 3 was not marked so only half the progeny would be expected to inherit the chromosome that carried P[>w"">] (67A)85 or its deriva- tives: the corrected frequency of Minute progeny is then 19/1035. One factor contributing to the decreased frequency of excision of the entire region compared to simple excision of 7uhr may be the much greater distance between the FRTs that flank the region; this separation probably makes it less likely that the FRTs will recombine (GRONASTAISKI and SADOWSKI 1985). In addition, the frequencies measured by these experiments undoubt- edly underestimate the true frequency of deficiency for-

b FIGURE 2.-The polytene cytology of P[>w"'>] (67A)85. ( a )

Arrows point out the black bands o f hybridization from the in situ localization of the two I'[>7crh'>] copies. (b) The same region of chromosome 3 from flies heterozygous for Dp(3L)85 and a normal chromosome. Landmark bands are indicated.

mation because the M flies are considerably less viable than M+ flies and take much longer to develop. I counted the progeny of M/Sb males and estimated that approximately 85% of these M flies do not eclose (there were 355 M progeny and 2427 Sh progeny).

Since the Minute phenotype is elicited by halving the dosage of a M gene, it is a simple matter to select for amplification of the region flanked by the two P[>w'">] insertions of strain #85: a duplication should suppress the Minute phenotype. I screened for Minute-suppressing duplications by crossing wlllx; hsFLP2B/+; P[>&>] (67A) 85/ + females that had been heat- shocked (37" for 30 min) to males that were 7u"18;

Df(3L)85, M/Sb. Ten M+ Sb+ offspring were recovered from approximately 50 female parents. Six retained at least one copy of whr. These were mated to w'"'; cu knr Sb/TM6, Ubx e. Whiteeyed Minute offspring were recovered from all six indicating that the M+ parent carried the Mdeficiency but did not show the phenotype. The &'-bearing offspring were M+. To show that the Minute-suppressing chromosome carries a duplication of the region flanked by P[>&5] elements (Dp(3L)85) I crossed the putative 7~""; Dp(3Z3)85/ TM6, Ubx e females back to 71J'"'; Df(3L)85, M/Sb males. In all cases the Ubx+ Sb+ progeny were also M+. Finally, I examined one of the Minute-suppressing chromosomes cytologically and found that it did carry a duplication of the region flanked by P[>r(/1s>] elements (Figure 2).

Dicentric chromosomes are formed by exchange between inverted repeats: In many of the two-copy insertion lines the induction of FLP synthesis in late larvae produced adults with a number of unexpected phenotypes. These included scalloped wings, rough and dis- ordered eyes, etched tergites and sternites, Minute


FIGURE 3.-The phenotypes produced by dicentric chromosome formation. Flies that carried hsFLP2B and a two-copy insertion of P[>wh3] on the third chromosome were heat-shocked for 1 hr at 38" as larvae and the adults were photographed. Panels (a, c and e) are from flies canying a two-copy insertion that does not make dicentrics and serve as controls. Panels (b, d, and fl are &om flies carrying an insertion that does make dicentrics. In panels (e and f ) the wings were removed to allow photography of the tergites.

bristles and missing bristles (Figure 3), sterility (not over a limited developmental period as indicated in Fig- shown), and lethality (Table 2). Most of these pheno- ure 4. The portion of two-copy lines which produced types were produced when FLP synthesis was induced these phenotypes is indicated in Table 1.

556 K. G. Golic

TABLE 2

Effect of X chromosome dicentric/acentric formation on viability

- HS + HS

Male Female Male Female Insertion Progeny viability viability Progeny viability PQ viability Pa

(8F)73' 317 0.61 0.53 271 0.56 0.36 0.52 0.82 (8F) 105 501 0.48 0.57 496 0.03 <0.0001 0.52 0.16 (8F) 110 228 0.64 0.56 188 0.23 <0.0001 0.51 0.43 (8F)lll 156 0.41 0.38 170 0.27 0.08 0.50 0.10 (8F) 115 482 0.55 0.53 565 0.05 <0.0001 0.54 0.67 (8F) 120A 327 0.55 0.54 338 0.15 <0.0001 0.53 0.77 (8F) 120B 191 0.56 0.55 271 0.10 <0.0001 0.52 0.60

Viability was scored among progeny,of the cross will8; hsFLRB/S2Cy0 , cn bw males X W " ' ~ P [ > ~ ~ ~ > ] females where the specific single or double insertion of P[>w '>I 1s indicated. After 7 days of egg-laying the parents were removed and the progeny were heat-shocked at 38" for 1 hr (+HS). Controls were treated identically except not heat-shocked (-HS). Flies that eclosed were then scored as Star' Curly or FLP (Star' Curly +). Male viability is expressed as (FLP males)/(FLP males + Curly males). Thus a value of 0.5 indicates equal viability. Female viability is similarly determined.

a Probability ( P ) values were determined with a 2 X 2 contingency test comparison of FLP males: Curly males under -HS and +HS conditions. ' This is the parental single-copy insertion from which the double-copy insertions were generated. AI1 other insertions are two-copy. - scalloped wings - rougheyes Minute and missing bristles

etched tergites white mosaicism

1 d

0 1 2 3 4 5 6 7 8 9 1 0

days of development FIGURE 4.-The dicentric phenotypes are produced over

limited developmental periods. The bars indicate the approxi- mate stages during which the induction of FLP synthesis produces that phenotype. Staging was done by collecting adults daily as they eclosed and then counting backwards to deter- mine the stage at which they were heat-shocked. This does not provide a very precise measure of the developmental stage, however, the progress of the morphogenetic furrow across the eye could be tracked during some of this period (see GOLIC and LINDQUIST 1989) increasing the accuracy of the determination. Staging is given in days of development after fertilization. Lar- vae pupate on day five and adults eclose on day 10.

In the previous section I showed that FLP can be used to generate deficiencies and duplications of the material flanked by copies of P[>wh5>>I. I inferred from these results that the two copies were in the same orientation. However, when the original P element transposed, there is no reason to suppose that the second copy is of necessity in one or the other orientation with respect to the first copy. When the two elements are inverted with respect to one another, it is expected that FLP will cause the inversion of intervening material. FALCO et ul. (1982) provided evidence that in yeast an alternative FLP-mediated recombination event occurs between inverted chromosomal FRTs: namely, an exchange between FRTs that lie in oppo- site orientation on sister chromatids. The predicted result of this unequal sister-chromatid exchange is the fusion of sister chromatids at the point of exchange, generating a dicentric chromosome carrying all the

material proximal to the site of exchange and an acentric fragment that carries the material distal to the site of exchange (Figure 5).

To test the hypothesis that FLP is catalyzing the formation of dicentric chromosomes and that this produces the phenotypes described, I generated females carrying the recessive yellow ( y ) and forked" (f ") mutations on a normal X chromosome heterozygous with one of the two-copy insertion chromosomes (at locus SF between the y andfloci) that produced developmental defects with FLP and is marked with y+ and f +. If FLP catalyzes sisterchromatid fusion as diagramed in Figure 5, then the acentric fragment carrying the y+ genes should not be stably transmissible and its loss should uncover the recessive y mutation. It is expected that the dicentric would also be unstable-a number of fates can be imagined: the dicentric may be stretched between the two poles and broken delivering a centric fragment to each daughter; it may remain at the metaphase plate and be lost; or it may be pulled to one pole or the other. Any of these possibilities predicts that occasionally at least one of the daughter cells will lose the t h e y allele as well, uncovering the recessive f mutation. If the cells in which such loss events occur are able to differentiate then one should be able to detect them by the presence of yellow bristles and forked bristles.

I heat-shocked flies of the genotype indicated in Fig- ure 5, and which carried hsFLP2B, during late third- instar. I inspected the females that eclosed for yellow or forked bristles. In these flies yellow bristles were frequent. The yellow bristles were also Minute: a phenotype expected for cells that have lost the acentric fragment (Figure 6, a and c) as there are several M loci distal to SF on the X chromosome. Less frequently, these flies also had bristles that were forked (Figure 6, b and c). Thus, the loss of markers on both the acentric and the dicentric is confirmed. It was not possible to score the yellow phenotype with confidence in bristles that were


FIGURE 5.-The mechanism of FLP-mediated dicentric formation. Solid ovals and circles indicate centromeres. Arrows indicate FRTs. See text for details.

forked. Even flies from the y f" stock had bristles that looked yellow'. I did, however, occasionally observe in these flies bristles that were yellow and forked (not shown).

In a similar experiment I tested whether P[>wh5>>I two- copy insertions on chromosome 3 can also cause loss of the chromosomal material distal to their location at 67A on 3L following hsFLP induction. The recessive mutation multiple wing hairs (mwh) on the tip of 3L can be used to mark wing cells. I inspected the wings of heat- shocked flies that were will8* , hsFLRB/+; mwh+P[>whs>] (67A) /mwh. The three P[>whs>>1 insertions I used were twocopy insertions at 67A that FLP acts on to produce the phenotypes of Figure 3. I observed abundant multiple wing hair spots on the wings of the flies of all three strains when they were heat-shocked in third-instar or the first day of the pupal stage, indicative of loss of the tip of 3R.

Cytological observation of mitotic chromosomes from larval ganglia provided further evidence of dicentric/ acentric formation. Chromosomes were obtained from larvae that carried one of the twocopy insertions on the Xchromosome and hsFLRB, 6-8 hr after a 60 min 38" heat shock. In almost 90% of the metaphase nuclei, one

FIGURE 6.-Bristle phenotypes confirm dicentric and acentric formation. Flies are of the genotype indicated in Figure 5. P[>whS>] (8F) 105 is the insertion used. In (a) the arrows indicate yellow Minute bristles that result from loss of the acentric fragment. In (b) the arrow indicates the location of a forked bristle on the humerus of the same fly shown in (a). Several yellow bristles are visible in the vicinity. In another fly (c) both yellow Minute bristles (small arrow) and forked bristles (large arrow) were found next to each other on the head. There are other yellow and forked bristles visible but not indicated.

of the Xchromosomes had separated into two parts (Fig- ure 7, Table 3), no doubt as the result of a FLP-mediated sister-chromatid fusion event. I also found figures that showed the expected dicentric anaphase bridge and a lagging acentric fragment (Figure 7, e and f ) . Metaphase figures from flies treated similarly, but carrying one of the chromosome 3 twocopy insertions that cause loss distal 3R, showed chromosome 3 dicentric/acentric figures. In control spreads (heat-shocked larvae carrying hsFLP and the original singlecopy insertion) I saw no abnormalities in metaphase or anaphase figures.

I believe that the adult phenotypes shown in Figure 3 are sufficiently indicative of an inverted orientation. It is conceivable that were two directly repeated Pelements to flank a region required in two copies for cell viability, its deletion might cause cell death leading to the above phenotypes. Since I find that in most cases the two P elements are very close to one another any deficiencies generated would be quite small. In Drosophila small deficiencies are almost always tolerated in the heterozygous condition (LINDSLEY et al. 1972). Thus, it is unlikely that deletions would cause all the phenotypes

TABLE 3

Frequency of dicentric chromosome formation

(SF) 105 204 26 (13)" 178 (87) (8F)11.5 195 24 (12) 171 (88) (SF) 120R 40 9 (23) 31 (77) " Nuclri \wre scored only if all four pairs of homologs codd

" Prrcentagcs are given in parent~~cses. he seen.

described. If the hvo copies flank a M gene (as with P[>ro"'>] (67A)85) then a deletion could of course generate Minute bristles but would not produce the entire array of phenotypes shown in Figure 3.

It should also be noted that there was substantial variation in the severity of the aforementioned phenotypes among the two-copy lines derived from a given parental insertion. I n some of the hvo-copv lines all the phenotypes shown in Figure 3 were strongly manifest; there were also hvocopv lines derived from the same parental insertion that produced only a few Minute bristles and moderately etched tergites, but little obvious effect on the eyes and wings; and at the other end of the spectrum there were those lines that showed none of these phenotypes. This variation might be accounted for by the distance between inverted FRTs. Those insertions that are farther apart probably recombine less frequently (GRONASTAISKI and SADOWSKI 1985) and thus elicit milder expression of the phenotypes shown in Figure 3. So, although these phenotypes may snffice to indicate that P [ > d ' > ] elements are inverted when those elements lie near each other, their absence cannot be taken to in-

FICIIRE 7.-Cytological ohsenration of dicentric chromosomes and acentric fragments Experimental details are given in the text. Control metaphase (a) and anaphase (h) from flies carrying hsl;l.P that were heat-shocked hut that have normal X chromosomes (indicated in (a)) . For (c-f) chromosome spreads were prepared from females carrying the P[>rcrh'>](8F)105 two- copy insertion and a normal X homolog. (c and d ) Metaphase figures from flies showing the acentric fragment (small arrow) and the dicentric (large arrow). In (d) the sister chromatids are well separated and the fusion of sisters can he clearly seen. ( e and f ) Anaphase figures showing the dicentric stretched between the two polesand the lagging acentric fragment (indicated by the small arrow). The acentric appeared as a single fragment in some instances ( f ) and a doublet in others (e).

dicate that the elements are directly repeated. There was also an obvious difference in the severity of these phenotypes produced by twocopy insertions at cytological site 60D Z I S . those at 8F and 67A: the former, when considered as a group, had a less severe effect.

Finally, I generated 13 chromosome 2 lines with two copies of P[>Io"'>] by recombining two independently isolated single insertionsof P[>ru"'>]. These elements lay anywhere from approximately 2 to 50 or more map units apart. When larvae carrying both hsFLP and the recombinant chromosomes were heat-shocked I did not oh serve the severe abnormalities described above. In some of the recombinant lines I did infrequently find flieswith developmental abnormalities. When I examined the macrochaete of the dorsal head and thorax perhaps one fly in 10 would have one or two defective bristles; a few flies exhibited etched tergites and, rarely, I saw small nicks in the wing margins. In contrast to the striking occurrence of these phenotypes in the twocopy lines that result from local transposition, the observation of the phenotypes in the recombinant lines required close inspection. If recombination between widely separated FRTs were to remove a large piece of chromosome then the same array of phenotypes that follow the formation of dicentric chromosomes might be observed (albeit less frequently). So, in the case of insertions that are far apart their relative orientation can probably not be deduced from these phenotypes.

DISCUSSION

The utility of local transposition: TOWER et nl. (1993) and ZHASG and SPRADLING (1993) have previously shown that the region about a P element insertion site can be


further mutagenized by taking advantage of the P element’s tendency to transpose locally. The recovery of local transpositions can be quite efficient if some phe- notypic screen is available. These groups looked for increased P-borne rosy+ gene expression, or for reversion of a lethal mutation caused by a P element insertion. I recovered lines with increased whs expression at a high rate following A2-Smediated mobilization of P[>whS>].

The results presented here can be compared with those obtained by the Spradling groups on two levels: first, the overall frequency of transposition; and second, the frequency of intrachromosomal transposition. The recovery of transposed P[>whs>>I varied; I obtained 91 independent jumps from 104 G, parents with P[>whs>>l (67A) 1C while I found 18 from 97 parents with P[>whs>>l (8F)73. Although most of the data presented by the SPRADLINC groups are not reported in a way that al- lows a determination of independent transpositions per parent, in one instance ZHANG and SPRADLINC report re- covering 75 transpositions from 11 1 parents. There were 13 independent intrachromosomal jumps among these 75. The overall frequency of transposition is similar to that I obtained with P[>whS>] (67A) lC, but the ratio of intrachromosomal to interchromosomal jumps is very different. Approximately 20% of the transpositions they recovered were intrachromosomal-more than 80% of those I recovered were intrachromosomal (1 16/134 for all three sites). Almost all of the intrachromosomal jumps I obtained were to sites very close to the parental insertion and most of these were on chromosomes that now carried two copies of P[>whS>]. It could be argued that those intrachromosomal events I recovered that did not carry two copies of P[>whs>>I were not genuine mobilization events, but resulted from defective events that alter the chromosomal context of P[>whS>] thereby al- tering the level of whs expression. This is certainly a possibility but it can only serve to explain a small fraction (a little over 10%) of the intrachromosomal jumps. The explanation for the higher proportion of intrachromc- soma1 jumps found with P[>whS>] is not known. Most of the intrachromosomal jumps obtained by the Spradling groups were, of necessity, local jumps, since they transposed a P element on a very small free duplication chromosome and perhaps P elements do not reinsert in that minichromosome as efficiently as they do in a normal chromosome.

In another set of experiments, TOWER et al. (1993) transposed an element located on chromosome 2 and recovered a high frequency of intrachromosomal jumps of which approximately one-fourth were local jumps. DANIELS and CHOVNICK (1993) performed a similar experiment using a P element inserted on the X chromosome. They found locally transposed elements in one quarter of the lines they analyzed. These are again lower frequencies than I observed but in the experiments of the two other groups the only lines analyzed were

those in which the P element’s transposition altered the expression of the locus at which the element was inserted (making the mutant phenotype more or less severe) and this may have biased the results. Another reason for the difference may be that the particular sequences carried by P elements influence their frequency of local insertion.

HAWLEY et al. (1988) and RAYMOND and SIMMONS (1981) provided earlier evidence that P elements inserted at singed tend to transpose to nearby sites: both within the same locus (HAWLEY et a l . ) or within a few map units (RAYMOND and SIMMONS). Because singed is a hotspot for Pelement insertion (ROIHA et al. 1988) these results might have been thought to be unique to this locus. However, the recent results of the SPRADLINC groups, DANIELS and CHOVNICK, and the data herein ex- tend those observations to encompass P elements inserted at a variety of loci. The mechanistic basis of high frequency local transposition is a matter for speculation. In that vein, SCHWARTZ (1989) has put forth a delightful model to explain short range transposition of A c in corn that may serve equally to explain the basis of local P transposition. He suggests that the ends of an excised transposon may be held together and could, in some cases, encircle the chromosome from which it excised. This association would then preferentially direct its re- insertion to the same chromosome. The rates of integration and diffusion along the chromosome could gov- ern the distance an element tends to move before reinserting. If excision and reintegration were to occur in G, of the cell cycle, then a chromosome carrying two copies of the element could be produced by restoration of the original insertion by gene conversion using the sister chromatid as a template (ENGELS et al. 1990) or by integration into the sister chromatid.

In any case, it is clear that the region about a P element insertion can be efficiently mutagenized by trans- posing that P element with the A2-3(99B) transposase source. The results presented here show that the region about a P[>whS>] insertion can be further manipulated by using FLP to make deficiencies and duplications of the region flanked by the double insertions. Deficiencies of a region flanked by P elements can also be produced with P transposase (COOLEY et aZ. 1990). It is difficult to compare the efficiency of FLP-mediated deficiency formation with that produced by transposase. COOLEY and co-workers used insertions that were approximately 800 kb apart. The insertions I used were separated by perhaps 400-500 kb. [Both distances were estimated by re- ferring the cytologically determined insertion sites to SORSA’S (1988) estimates of DNA content in different regions.] They recovered deficiencies of the region flanked by P elements with a frequency of 1.2% when using A2-3(99B). The highest frequency at which I recovered deficiencies was 1.8%. However, because the deficiency-bearing flies produced by the experiments

560 K G. Golic

described here are Minute and most do not eclose, the actual frequency of their formation with FLP may be much higher. The frequency of FLP and transposase- mediated events would have to be compared using the same insertions to resolve the matter. The FLP-mediated process probably has the advantage of a more predict- able outcome and FLP can also be used to duplicate precisely the same material as is deleted.

Why are dicentric chromosomes formed so efficiently? In earlier work we observed that unequal sister chromatid exchange between the directly repeated FRTs within a single element resulted in the amplification of the FRT-flanked whs gene. The amplified product was recovered only about 1 % of the time (GOLIC and LINDQUIST 1989). Here I show that the unequal sister- chromatid exchange that FLP catalyzes between two inverted copies of P[>whs>>l can be extremely frequent- approaching 90%. There are probably two reasons for the difference. In the case of direct repeats, if FLP first catalyzed intrachromatid exchange between the repeats the result would be reduction to a single FRT on that chromatid. Further sister-chromatid exchange would then be without consequence. If the unequal sister- chromatid exchange event were to occur first, further FLP-mediated intrachromatid exchanges would then still produce the original or reduced versions. Once the whs gene is excised from the chromosome (on a circle with an FRT) it may be free to diffuse away and this would greatly reduce its chance for reintegration. The fact that the frequency of whr excision approaches 100% with high levels of FLP argues that FLP drives the reaction in this direction. If however there were two inverted copies of P[>whS>] present then FLP-mediated excision of the FRT-flanked whs genes would leave two inverted FRTs on the chromosome. These are still the substrates for the unequal sister-chromatid exchange that gener- ates a dicentric. When a dicentric chromosome and acentric fragment are produced they may be free to diffuse away from each other preventing restoration of the chromosome by additional FLP-mediated recombination events. Thus, at high levels of FLP the reaction would tend to drive the chromosomes to this dicentric/ acentric configuration.

FLP-mediated recombination can be used to turn a gene on or off by placing inverted FRTs so that they flank a portion of the gene. The induction of FLP synthesis will catalyze the expected inversion leading to a revers- ible switching of the gene’s activity (K. AHMAD and K. G. GOLIC, in preparation). However the potential for generation of dicentric/acentric chromosomes should be considered when the use of inverted FRTs is contem- plated. There are alternative methods for using FLP to switch genes on that will not produce dicentric chromosomes (discussed in GOLIC 1993). Sequences that cause transcriptional termination have been placed between directly repeated FRTs and then inserted as a unit

into the coding sequence of a gene (O’GORMAN et a,!. 1991) or between the promoter and coding sequence of a gene (STRUHL and BASLER 1993). The gene is inactive until FLP excises the FRT-flanked portion from the chromosome. FLP has also been used to activate a gene by mediating mitotic recombination between FRTs in specially constructed P elements at allelic sites (HARRISON and PERRIMON 1993).

What is the fate of dicentric and acentric chromosomes? The acentric fragment generated by FLP- mediated unequal sister-chromatid exchange is probably lost from most cells. Results presented elsewhere show that an acentric extrachromosomal circle is not maintained through mitotic divisions (GOLIC and LINDQUIST 1989; K.. AHMAD and K G. GOLIC, in preparation). The experiments presented here directly demon- strate loss of the acentric fragment. Our results do not rule out the possibility that a cell may occasionally retain an acentric fragment simply because it happens to be in the right spot to be enclosed by the nuclear membrane as it reforms at telophase. We expect this result to be rare, especially if the cell goes through more than a single round of mitosis with the acentric. The aneuploidy that results from acentric loss can be substantial and its extent will depend on the site of sister-chromatid fusion.

A number of fates may await the dicentric formed by sister-chromatid fusion. The experiment diagramed in Figure 5 and the cytology of dicentrics give some insight into which of those possible fates actually ensue. One fate that may befall the dicentric chromosome is that it remains at the metaphase plate and is lost. This appears to be the fate of X chromosome dicentrics generated by meiotic exchange between a normal and an inverted X chromosome (STURTEVANT and BEADLE 1936). It seems unlikely that this is the fate of the X chromosome dicentrics produced here. The anaphase figures of dicentrics suggest that this is not the case-they showed that the centromeres of the dicentric progressed to both poles. The dicentric did not appear to lag at the metaphase plate. Furthermore, if this were the usual fate of dicentrics produced in mitotic cells then all bristles that were yellow should also have been forked and they were not: in fact there were many more yellow than forked bristles. However, the allele of forked used here ( f ”) is not a strong allele and so bristles produced by cells that were f -/ f x may not all have exhibited the forked phenotype.

Another possibility is that the dicentric is dragged to one pole. This also seems unlikely. NOVITSKI (1952) o b served this behavior with dicentrics that arose by meiotic exchange between an inverted and a normal sequence X that differed by the attachment of a heterochromatic arm derived from the Y chromosome. The heteromor- phic centromere regions acted as though they were of different strength and Novitski concluded that the dicentric was pulled to one pole. In the present case the


two centromeres are identical-they should be of the same strength. And again, anaphase figures did not show the configuration one might expect (an asymmetrically located bridge) if this were the usual fate of a dicentric.

It also might happen that the dicentric is stretched between between daughter nuclei, but not broken. I did in fact observe nuclei that appeared to be connected by a drawn out thread of chromatin when examining the DAPI-stained squashes (not shown). It is not obvious whether such cells would be able to divide again or differentiate. If this is the fate of some of the dicentric chromosomes it cannot be the only possible fate, because if such cells produced bristles they should be forked' bristles, and bristles that were forked were also produced.

Finally, the dicentric might be stretched between poles and break. NOVITSKI (1952) proposed this fate for dicentrics generated in meiosis that have two strong centromeres (by virtue of the appended heterochromatic arms). Although the Xused in these experiments is normal, and should therefore have a weak centromere, the mitotic spindle may exert a stronger pull than the meiotic spindle and cause breakage. If breakage were to occur at a site between one of the centromeres and the nearest f + allele then the daughter cell that received the shorter fragment would be f -/f x and the other daughter cell would be f ' / f '. This event can account for the production of yellow bristles that are forked or forked' and is consistent with the cytology of anaphase bridges. It seems probable that the daughter cells that do losef +

arise in this way because the other two routes off + loss (dicentric loss from one or both daughter cells without breakage) are unlikely for the reasons discussed above.

Why do animals with dicentrics survive? It seems im- probable that an animal could survive when dicentric chromosomes are formed in 90% of its cells, and yet, in at least some cases, they do. One trivial explanation is that the 90% figure may overestimate the overall frequency of dicentric formation. When scoring metaphase spreads for dicentrics I looked only at cells reaching mitosis 6- 8 hr after the heat shock. It is possible that if cells were in G, when FLP was produced they might not reach G, (when they would be susceptible to unequal sister- chromatid exchange) until most of that FLP was de- graded. They might then pass through G2 and mitosis unscathed. So I may have missed a population of cells that were fortunate to be in a part of the cell cycle where they were not susceptible to dicentric formation.

A second reason animals might survive is that they have finished their developmental program of mitoses. Although dicentrics may be formed in cells that are no longer dividing (assuming that they have replicated their chromosomes after the last division) the dicentric should be harmless unless a round of division ensues. In Figure 3, c and d, the boundary between the anterior mosaic zone and the posterior pigmented zone corre-

sponds to the division between dividing cells in the anterior and mitotically quiescent cells in the posterior (READY et al. 1976; GOLIC and LINDQUIST 1989). The pos terior of the eye in Figure 3d is unaffected by dicentric formation (even though FLP does mediate recombination in the non-dividing cells in the posterior of the eye, K. AHMAD and K. G. GOLIC in preparation) while the anterior portion shows obvious abnormalities in the pattern of facets. But this cannot serve to explain the survival of females with a dicentric X as shown in Table 2 because in this experiment the animals had not reached the stage at which most mitoses cease.

Most significantly, the results of Table 2 suggest a third reason that animals in which dicentrics are formed can survive. The animals survive because the cells with dicentrics are still able to participate in development. This can be deduced by comparing the survival of males whose only X becomes dicentric with the survival of females, one of whose two Xs becomes dicentric. The survival of females is unaffected by dicentric formation but the survival of such males is greatly reduced. The loss of the distal acentric portion of the X in males will produce cells completely deficient for a large portion of the X chromosome while in females such cells will still have one copy of this portion of the chromosome. One can conclude that the complete deficiency for a large chromosomal segment that is induced in male cells affects them so severely that they cause death of the animal, but in females the hypoploid cells function sufficiently to avoid this developmental catastrophe. The form of their participation in development is not known but it is clearly not the case that they continue to divide and differentiate normally.

Several points of evidence show that the cells in which dicentrics are formed have limited mitotic capacity. First, animals in which dicentrics are induced early in development show little evidence of developmental defects (Figure 4). Neither of the two indicators I used for loss of the acentric piece (yellow bristles and multiple wing hair spots) was seen in animals heat-shocked in the first two or three days of development. Thus, the con- tribution these cells and their descendants would normally make has given way to cells in which dicentrics were not formed. These healthy cells must have under- gone extra mitoses to compensate. Second, close exami- nation of eyes such as that shown in Figure 3d reveals that the cells which were undergoing their final mitotic division when FLP was made (in the middle of the eye) show the most severe defect, while the ommatidial pattern in the very anterior of the eye is close to normal. This indicates that the anterior of the eye is somehow able to recover from dicentric formation, most likely by allowing the cells that do not have dicentrics to undergo extra rounds of division. GATTI (1979) also concluded that cells with chromosome breaks divide poorly based

562 K. G. Golic

on his analysis of the chromosome aberrations that occur in somatic cells of mutagen-sensitive and meiotic recombination mutants of D. melanogaster. This limitation may be a consequence of the aneuploidy that these cells experience. RIPOLL (1980) examined the viability of clones of cells that were terminally hypoploid to varying extents. The hypoploid clones were much smaller and less frequent than the hyperploid twins or the euploid parental cell type. It follows that the limited mitotic potential of the daughter cells produced by a mitosis with a dicentric chromosome and an acentric fragment can be explained as resulting from their aneuploidy. This correlates well with my observation that the flies with two copies of P[>whS>] at 8F and 67A were, in general, more severely affected by the induction of dicentrics than were flies with two copies at 60D. In the former instances the daughter cells will necessarily be hypoploid for a much larger segment of chromosome than will the latter flies. An alternative explanation for this difference may be that the insertions at 600 tend to be farther apart and as a consequence they recombine and make dicentrics less frequently. Cells that receive a broken chromosome may also be subject to mitotic ar- rest or delay as a result of a cell-cycle checkpoint. In yeast the rad9 mutation identifies a cellular mechanism that detects chromosome breaks and halts the progress of the mitotic cycle until those breaks are repaired (HARTWELL and WEINERT 1989). A similar mechanism may operate in Drosophila. The phenotypes produced by the generation of acentric fragments and dicentric chromosomes are similar to those that can be produced by other agents: among them X-rays (WADDINGTON 1942; GARCIA-BELLIDO and MEW 1971), temperature-sensitive cell-lethal mutations (POODRY et al. 1973), and expression of an activated oncogene (BISHOP and CORCES 1988). The autonomous nature of each agent may be quite different yet the developmental consequences are reminiscent of each other. The response of cells of D. melanogaster to dicentric chromosomes, the fate of that dicentric during subsequent mitoses, and the relationship this bears to other agents with similar developmental effects should be fruitful ground for further inquiry.

I thank SUSAN LINDQUIST for generously providing lab space and facilities for portions of this work. I also wish to acknowledge DAN L~NDSLEY for suggesting the duplication screen. I thank KAMI hm for constructive comments on the manuscript. This work was partially supported by the Howard Hughes Medical Institute. The author is partially supported by American Cancer Society grantJFRA-370. Work in the laboratory is supported by American Cancer Society grant IRG 178B and grant HD28694 from the National Institutes of Health.

LITERATURE CITED

BISHOP, J. G., and V. G. COKCES, 1988 Expression of an activated ras gene causes developmental abnormalities in transgenic Drosoph- ila melanogaster. Genes Dev. 2: 567-577.

BRINK, R. A,, and R. A. NILAN, 1952 The relation between light variegated and medium variegated pericarp in maize. Genetics 37: 519-544.

COEN, E. S., T. P. ROBBINS, J. ALMEIDA, A. HUDSON and R. CARPENTER, 1989 Consequences and mechanisms of transposition in Antir- rhinum majus, pp. 413-436 in Mobile DNA, edited by D. E. BERG and M. M. HOW. American Society for Microbiology, Washington D.C.

COOLEY, L., D. THOMPSON and A. C. SPRADLING, 1990 Constructing deletions with defind endpoints in Drosophila. Proc. Natl. Acad. Sci. USA 87: 3170-3173.

DANIELS, S. B., and A. CHOVNICK, 1993 P element transposition in Drosophila melanogaster: an analysis of sister-chromatid pairs and the formation of intragenic secondary insertions during meiosis. Genetics 133: 623-636.

ENCELS, W. R., D. M. JOHNSON-SCHLITZ, W. B. ECCLESTON and J. SVEI), 1990 High frequency P element loss in Drosophila is homolog dependent. Cell 62: 515-525.

FALCO, S. C., Y. LI, J. R. BROACH and D. BOTSTEIN, 1982 Genetic properties of chromosomally integrated 2 p plasmid DNA in yeast. Cell 29: 573-584.

GARCIA-BELLIDO, A,, and J. R. MERRIAM, 1971 Parameters of the wing imaginal disc development ofDrosophila melanogaster. Dev. Biol. 24: 61-87.

GATTI, M., 1979 Genetic control of chromosome breakage and re- joining in Drosphila melanogaster: spontaneous chromosome a h errations in X-linked mutants defective in DNA metabolism. Proc. Natl. Acad. Sci. USA 76: 1377-1381.

GATTI, M., and s. PIMPINELLI, 1983 Cytological and genetic analysis of the Y chromosome of Drosophila melanogaster. Chromosoma 88: 349-373.

G o l . ~ c , IC G., 1993 Generating mosaics by site-specific recombination, pp. 1-31 in Gllular Interactions in Development: A Practical Approach, edited by D. H A K ~ Y . Oxford University Press, New York.

GOLIC:, K. G., and S. LINDQUIST, 1989 The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59: 499-509.

GKONASTAJSKI, R. M., and P. D. SADOWSKI, 1985 The FLP protein of the 2-micron plasmid of yeast: inter- and Intramolecular reactions. J. Biol. Chem. 260 12328-12335.

HAKKISON, D. A,, and N. PERRIMON, 1993 Simple and efficient generation of marked clones in Drosophila. Curr. Biol. 3: 424-433.

HAKTWEIL, L. H., and T. A. WEINERT, 1989 Checkpoints: controls that ensure the order of cell cycle events. Science 246: 629-634.

HAWLEY, R. S., R. A. STEUBER, C. H. MARCIJS, R. SOHN, D. M. BARONAS et al., 1988 Molecular analysis of an unstable P element insertion at the singed locus of Drosophila melanoga.rter: evidence for intracistronic transposition of a P element. Genetics 119: 85-94.

KLEMENZ, R., U. WEBER and W. J. GEHRING, 1987 The white gene as a marker in a new P-element vector for gene transfer in Drosophila. Nucleic Acids Res. 15: 3947-3959.

LAWKENCE, P. A,, P. JOHNSTON and G. MORATA, 1986 Methods of mark- ing cells, pp. 229-242, in Drosophila: A Practical Approach, edited by D. B. ROBERTS. IRL Press, Washington, D.C.

LEFEYKE, G., 1976 A photographic representation and interpretation of the polytene chromosomes of Drosophila melanogastersalivary glands, pp. 31-66 in The Genelics and Biology ofDrosophila, Vol. la, edited by M. ASHBUKNER and E. NOVITSKI. Academic Press, New York.

LINDSLEY, D. L., and G. G. ZIMM, 1992 The Genome of Drosophila melanogaster. Academic Press, San Diego, Calif.

LINDSIXY, D. L., L. SANDLER, B. S. BAKER, A. T. C. CARPENTER, R. E. DENELL et al., 1972 Segmental aneuploidy and the genetic gross struc- ture of the Drosophila genome. Genetics 71: 157-184.

MCCIJNTOCK, B., 1948 Mutable loci in maize. Carnegie Inst. Wash. Year Book 47: 155-169.

NOVITSKI, E., 1952 The genetic consequences of anaphase bridge formation in Drosophila. Genetics 37: 270-287.

O'GOKMAN, S., D. T. Fox and G. M. WAHL, 1991 Recombinase- mediated gene activation and site-specific integration in mam- malian cells. Science 251: 1351-1355.

PARDUE, M. L., 1986 In situ hybridization to DNA of chromosomes and nuclei, pp. 111-137 in Drosophila: A Practical Approach, edited by D. B. ROBERTS. IRL Press, Washington, D.C.

POOUKY, C. A., L. HALL and D. T. SUZUKI, 1973 Developmental properties of Shibire"': a pleiotropic mutation affecting larval and adult locomotion and development. Dev. Biol. 3 2 373-386.


RAYMOND, J. D., and M. J. SIMMONS, 1981 An increase in the X-linked lethal mutation rate associated with an unstable locus in Drosoph- ila melanogaster. Genetics 9 8 291-302.

READY, D. F., T. E. HANSON and S. A. BENZER, 1976 Development of the Drosophila retina, a neurocrystalline lattice. Dev. Biol. 4 3 217-240.

RIPOLL, P., 1980 Effect of terminal aneuploidy on epidermal cell viability in Drosophila melanogaster. Genetics 9 4 135-152.

W. K. BENZ et al., 1988 A stable genomic source of P element transposase in Drosophila melanogaster. Genetics 118: 461-470.

RoIm, H., G. M. RUBIN and K. O’HARE, 1988 Pelement insertions and rearrangements at the singed locus of Drosophila melanogaster. Genetics 119: 75-83.

~CHWARTZ, D., 1989 Pattern of Ac transposition in maize. Genetics 121: 125-128.

SORSA, V., 1988 Chromosome Maps of Drosophila, Vol. 11. CRC Press, Boca Raton, Fla.

ROBERTSON, H. M., C. R. PRESTON, R. w. PHILLIS, D. M.JOHNSON-SCHLITZ,

STRUHL, G., and K. BASLER, 1993 Organizing activity of wingless protein in Drosophila. Cell 72: 527-40

STURTEVANT, A. H., and G. W. BEADLE, 1936 The relations of inver- sions in the X chromosome of Drosophila melanogaster to crossing over and\ disjunction. Genetics 21: 554-604

TOWER, J., G. H, KARPEN, N. CRAIG and A. C. SPRADLING, 1993 Prefer- ential transposition of Drosophila P elements to nearby chromosomal sites. Genetics 133 347-359

VAN SCHAIK, N. W., and R. A. BRINK, 1959 Transpositions of Modu-

Genetics 44: 725-738 lator, a component of the variegated pericarp allele in maize.

WADDINGTON, C. H., 1942 Some developmental effects of X-rays in Drosophila. J. Exp. Biol. 19: 101-117

ZHANG, P., and A. C. SPRADLING, 1993 Efficient and dispersed local P element transposition from Drosophila females. Genetics 133: 361-373

Communicating editor: M. J. SIMMONS

Documents

Recombination Between Duplicated Elements Using a Site-Specific