Copyright 0 1991 by the Genetics Society of America

Cloning andCharacterization of the scalloped Region of Drosophila melanogaster -

Shelagh D. Campbell,*9’Atanu Duttaroy,* Alisa L. Katzent and Arthur Chovnick**‘ *Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-2131, and +The George Williams Hooper Foundation, Universityof Calfornia, Sun Francisco, Calfornia 941 43-0552 Manuscript received June 12, 1990 Accepted for publication October 12, 1990 ABSTRACT Viable mutants of the scalloped gene ( s d ) of Drosophila melanogaster exhibit defects that can include gapping of the wing margin and ectopic bristle formation on the wing. Lethal sd alleles characterized in the present study now implicate this gene in a genetic function essential for normal development. In order to further characterize the developmental role of this gene, we have undertaken to clone and characterize the region where sd maps. A P[ry+] transposon insertion at 13F associated with sdl’Y+22‘6] served as the starting point for a 42-kb chromosomal walk. Molecular lesions associated with viable and lethal sd alleles werecharacterized by genomic hybridization analysis asa means of defining the extentof the gene. DNA rearrangements associated with 11 viable sd alleles map to a 2-kb interval which appears to be a “hotspot” for P element activity. Four of five recessivelethal sd mutations were mapped by denaturing gradient gel electrophoresis to a region 12- 14 kb away from the region of viable lesions. In a sd+ genotype, at least two structurally related and developmentally regulated transcripts hybridize to the genomic region where several sd lethal alleles have been localized. A viable mutation, sd5*, used for comparison in the transcript analysis, makesat least two slightly smaller transcripts that also hybridize to this region. Preliminary analysis of cDNA clones hasidentified three structurally related transcripts that hybridize to this genomic region. The 5’ end of these transcripts extends into the 2-kb genomic region wherein DNA rearrangements were seen in the P element rearrangements. We favor the view thatthe transcripts represented by these cDNAclones are products of the sd gene. If this is true, the sd gene would include genomic sequences extending over at least 14 kb of the described chromosomal walk, and would appear to be subject to alternative splicing.

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HE scalloped locus (sd: 1-5 1.5/13F) (GRUNEBERG and GRELL1968; DANIELS et 1929; LINDSLEY al. 1985) is associated with a viable, mutant phenotype involving the loss of structures thatnormally comprise the adult wing margin, creating a gapped or scalloped appearance. Some sd alleles are phenotypically weak, exhibiting only occasional slight nicking of the distal wing margin, whereas phenotypically extreme alleles can have their wing marginal structures completely eroded and thesurface area of the wing blade greatly reduced (see DANIELS et al. 1985 for illustration). In addition to wing margin scalloping, some sd alleles exhibit diminution of the halteres, an uplifting of the post-scutellar bristles and thepresence of ectopic bristles on the wing blade (the last observation is made in this report). In this report we describe several recessive lethal alleles of sd that implicate this gene in some vital process(es) in development. Bristles, which are structures commonly affected in sd mutants, are the cuticularexternalstructures of innervated sensory or-

’ Present address: Department of Biochemistry, University of California, S;m Francisco, California 94 143-0448.

To whom correspondence should be addressed. Genetics

127: 367-380 (February, 1991)

gans. The sensory organs of the peripheral nervous system in the Drosophila wing have been analyzed by histological, electrophysiological and genetic methods and have served as a model system for studying neurological development (LEESand WADDINGTON 1942; and MERRIAM 1971; MURRAY, LEES 1942; BELLIDO SCHUBIGER and PALKA1984; DICKINSON and PALKA 1987; RIPOLLet al. 1988; GARCIA ALONSO and GARand CARTERET 1989; HARCIA-BELLIDO 1988; SIMSON TENSTEIN and POSAKONY 1989;PALKA, SCHUBIGER and SCHWANINGER1990). Two genes with demonstrated roles in neurological development are Notch and cut (reviewed in CAMPOS-ORTEGA and KNUST 1990;XU etal. 1990; BLOCHLINGER et al. 1988). Certain mutant alleles of Notch and cut are associated with wing phenotypes that resemble those of viable sd mutants. It is reasonable to consider therefore, whether the wing phenotype associated with sd also indicates a role in neurological development. A study of t w o temperature-sensitive sd alleles indicated a temperature-sensitive period for the wing phenotype thatextendsthroughout larval developLAWRENCE and MASCHAT 1981). The ment (SIMSON,

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and GRELL(1968). They were obtained from the Bowling Green Drosophila Stock Centre. sd‘is the original X-rayinduced viableallele (GRUNEBERG 1929). sd2 is an X-rayinduced viable allele reported to be associatedwith In( I)sd2 (PANSHIN1935). sdssd is a a-ray-induced viableallele reported to be associated withIn( I)sdssd(IVES 196 1). sd6“ is a viable allele obtained from the Bowling Green Stock Centre (we have been unable to find information regarding the origin of the stock). D f ( 1 ) ~ d ~(CRAYMER ~”~ and ROY 1980) is an X-ray-induced deletion removing 13F1-14Bl obtained from C. POODRY at the University of California, Santa Cruz, and is referred to as D f ( 1 ) ~ d ~ ~ ~ . D isf (covered l ) ~ d ~ by ~* Dp(I;Y) shi+Y#3 but not by Dp((;Y)shi+Y#l(distaI breakpoint of both du lications is 13F1-4 (POODRY 1980). a P-M dysgenesis-induced insertion at 13F of sd[ry+2216Pis a P[ry+] transposon (RUBINand SPRADLING 1982), and sd[~y+22/6-547] . 1s a P-M dysgenesis-induced mutation located in the sd[V+22’61insert (DANIELS et al. 1985). These, as well as sd2A3, sdllD, Sd299ASd2YYD , sd’”, and sdY3were generated by P-M dysgeniccrosses in theCHOVNICK laborator (M. MCCARRON,personal communication). wl’” (LEVIS,HAZELRIGG and RUBIN1985) is a chromosome carrying a P[ry+ w+] transposon insertion at 13F obtained from R. LEVIS(Fred Hutchison Cancer Research Institute, Seattle). ~ d ~is a~P-M ~ dysgenesis-induced ” ’ ~ alleleobtained from T. ORR-WEAVER (Whitehead Institute, Cambridge). sdJMis a P[ry+ lacZ] insertion at 13F obtained from J. MOORE (University of Texas, Austin). A screen for lethal alleles in the region uncovered by Df(I)sd7” (described below) generated the following sd al, l( I)sd47M,E( I)sd68Mand l( I)sd3IH. leles: l( I ) s ~ ’ ~ E(I)sd’”-, The lethals are covered Dp(I:Y)shi+Y#I by and Dp( I:Y)shi+Y#3. The following rearrangements are reported (LINDSLEY and ZIMM 1987) to carry breakpoints near 13F as indicated: T(I;Y) P12 (13F1-2; YS), In(I)AB (9F;13F1-10) and T(I;Y) 240 (14A; YL). Results of segmental aneuploid crosses performed withT(1;Y) 240 indicate that the breakpoint is in factdistal to sd, rather than 14Aaspreviously reported CORCES1986), rudimentary (TSUBOTA,ASHBURNER (NICOLETTIand LINDSLEY 1960). All balancer chromosomes usedinthis study are described in LINDSLEY and ZIMM a n d SCHEDL1985) and Sgs-4 (McGinnis, Shermoen 1987. 1983). a n d BECKENDORF Lethal screen for mutations uncovered by Of ( Z ) S ~ ’ ~ * : Inordertoaddressthe role(s)played by sd in Adult males from a w sd+ stock, referred to subsequently as Drosophila development, and to generate material for w ( K ) , were mutagenized with EMS. Mutagenized males were examining theissues raised in theP element work, we mated to an attached-X stock carrying Dp( 1;Y) shi+Y#Z and first generation male progeny collected. Of the progeny have undertaken to clone the sd region by transposon carrying X-linked lethals, only those complemented by tagging (BINGHAM, LEVISand RUBIN1981) and chro(BENDER,SPIERERa n d HOGNESS Dp(1;Y) shi+Y#I survive. These males were mated singly to mosomalwalking females of genetic constitution Df(l)~d’~’/FM7c and the 1983). Fortunately, the sd region appears tobe a “hot resulting progeny were scored for the presence of white+ spot” for P elementrearrangements,and thishas females. Those cultures producing only white- femalespreprovided opportunity for characterization of an array sumably carry a lethal mutation on the treatedchromosome Females from such cultures (preuncovered by Df(l)~d~’~. of sd alleles derived from P-M hybrid dysgenesis a n d sumed genotype: I(I)/FM7c) were allowed to mate with transformation experiments. Additionally, an array of sibling males (FM7c/Dp(I;Y) shi+Y#l) creating a balanced radiation and ethyl methanesulfonate (EMS)-induced stock. A complete description of the lethal screen will appear reviable and recessive lethal sd alleles and elsewhere. One lethal complementation group was identiarrangements are included in the study. The present fied that is comprised of five sd mutations, and these are molecular analysis characterizes a cloned genomic rediscussed in the present report. Genomic DNA analysis: Adult genomic DNA was exgion towhich a number ofsd region lesions have been tracted essentially as described in BENDER, SPIERERand mapped and describes transcriptional activity of this HOGNESS (1983) with the omission of diethylpyrocarbonate region. and the addition of a phenol/chloroform extraction step. DNA aliquots of approximately 4 pg were digested withthe MATERIALS AND METHODS appropriate restriction enzyme and fractionated on 0.8% agarose gels.Hind111 digested wild-typelambda phage Stocks used in the analysis: Unless otherwise indicated, references to the following stocksmay be found in LINDSLEY served as molecular weight standards. Genomic blots were same study established by somatic mosaic analysis that t h e wing margin phenotypeis a cell-autonomous property of the gene. Histologicalanalysis of wing disc development in sd larvae implicatedcell death during imaginal wing disc larval development as a determi(sd””: JAMESa n d BRYANT nantofthephenotype 1981). The present molecular characterization of the sd gene representsa further effort toward understanding the functionof this gene in development. In prior work ofthis laboratory, a sd allele, sd[ry+22f61, was isolated from a P-M dysgenesis-mediated transformation experiment, andwas characterized as a P[ry+] transposon insertion at polytene segment 13F (DANIELS et al. 1985; 1986). Subsequent hybrid dysgenic allele produced secondperturbation of the sd[ry+22’61 ary derivative alleleswith sd and/or ry phenotypic alterations. These, in turn, wereassociated with DNA rearrangements within thetransposon which remained in place at 13F. Significantly, the scalloped-phenotype could be reverted virtuallyt o wild type by deletions that appeared to be entirely internal tothe transposon. An important implication of these observationsis that the mutagenic effect of the P[ry+] transposon insertion is not due to the perturbation of sd coding sequences, but rather to the disruption of regulatory sequences, or of inproperly spliced. tronicsequenceswhichmustbe Mechanisms of transposable element mutagenesis involvinginsertionsintononcodingregionsofgenes have been documentedfor a number of loci including white (MOUNT, GREENa n d RUBIN1988), the Bithorax complex (PFEIFER a n d BENDER 1986), RPII215 (SEARLESet al. 1986), yellow (GEYER,SPANA a n d

The scalloped Region made by capillary transfer of fractionated DNA (SOUTHERN 1975) to Nytran (Schleicher and Schuell) or to ImmobilonN (Millipore) membranes according to the manufacturer’s instructions. Probes for genomic blots were made by labeling DNA restriction fragments isolated in low melting point VOagarose gels either by random priming (FEINBERG and GELSTEIN 1983) or by nick translation (RIGBY et al. 1977). Denaturing gradient gels (FISCHER and LERMAN 1983) were prepared and run according to GRAYet al. 199 1. Cytological analysis: Salivary gland polytene chromosome spreads were made from mature third instar larvae grown at 18” in uncrowded cultures. We used nick-translated, biotinylated DNA probes and a streptavidin-alkaline phosphatase complex for detection. The hybridization and detection protocol followed the recommendations ofENGELS et Ul. (1986) Construction and screening of sdq2216-5471 and wild-t e genomic DNA libraries: Genomic DNA from sd”’’ 2 x 7 1 adults was partially digested with SauSA, and fragments of 15-25 kb were selected by sucrose gradient fractionation. A genomic library was constructed by ligation of fractionated DNA to BamHI-digested EMBL4 lambda vector DNA (FRISCHAUF et al. 1983). The ligation mixtures were packaged in vitro (HOHN1979), and transfected into host strain KH802 for screening ata densityof -1 X lo4 plaqueforming units (pfu) per 82-mm plate according to procedures outlined by BENTONand DAVIS(1977). Screening of a wild-type (Canton S strain) genomic library (MANIATISet al. 1978) obtained from W. BENDER,Harvard Medical School, was performed following identical procedures. Hybridizing clones werereselected twice to ensure theisolation of single clones, and phage DNA was isolated by polyethylene glycol precipitation followed by phenol/chloroform extraction (MANIATIS, FRITSCH and SAMBROOK 1982). cDNA library screening: A set of cDNA libraries representing sequential stages of Drosophila development (POOLE et al. 1985) was obtained from A. HILLIKER (University of Guelph) who had received them from T. KORNBERG(Universityof California, San Francisco). The cDNA libraries were screened following procedures identical to those described above for genomic libraries, except that thebacterial strain C600Hfl was used. Two additional cDNA libraries, obtained from N. BROWN (Harvard University), represent late embryonic and third larval instar imaginal disc transcripts (BROWN and KAFATOS 1988). These libraries, constructed in the plasmid vector pNB, were screened by transforming competent DH5a strain bacteria (HANAHAN 1983). Colonies were initially screened at high density (2-3 X l o 4 per 82-mm plate). Standard protocols were used to perform colony lifts and hybridizations (MANIATIS,FRITSCHand SAMBROOK 1982). Hybridizing clones were rescreened twice before plasmid DNA from the clones was isolated by an alkaline lysis miniprep procedure (BIRNBOIM and DOLY1979). RNA analysis: Flies were grown at25” on standard cornmeal-sugar-agar media. Embryo collections were made on grape juice-agar plates for the indicated interval of egg deposition. For third instar larvalcollections,eggswere collected over a 12-hr period, and allowed to develop at 25” until they reached the “wandering larvae” stage (4-5 days). Adults were collected from 1 to 4 days posteclosion. Samples for R N A analysis were prepared by phenol/chloroform extraction in a buffer consisting of 0.2 M NaCI-0.2 M Tris, pH 7.5-2% sarcosine, followed by batch adsorption to oligo-dT cellulose (BRL). The bound poly(A+)RNA was washed four times with binding buffer (0.5 M NaCI-IO mM Tris, pH 7.5), eluted from the oligo-dT cellulose in 10 nlM Tris, pH 7.5, and precipitated withsodium acetate and

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ethanol. The precipitate was resuspended in water and a portion quantitated byOD260 absorbance. Poly(A+)R N A was fractionated on I % agarose-0.66 M formaldehyde gels, then transferred by capillary action to Nytran (Schleicher and Schuell) according to the manufacturer’s instructions. A single-strand, antisense R N A probe was synthesized from a cDNA clone isolated from the BROWNlibrary (BROWNand KAFATOS 1988). The antisense transcript of the sdcE2l clone (Figures 6 and 7) was synthesized from the T 7 promoter of the pNB vector using Pharmacia Transprobe reagents. RESULTS

Chromosomalwalking in the scalloped region: T h e transposon insertion associated with ~ d [ 9 ~ ~ was used as a “tag” to enter the region (BINGHAM, LEVISa n d RUBIN 1 9 8 1). A genomic library was constrain that carriesa structed from the sd[922’6-5471;ry506 P[ry+]transposon insertion at 13F associated with a sd phenotype (DANIELSet al. 1985). T h e library was screened with a probe of ry+ DNA derived from the second ry+ exon, a region deleted in the ry506 allele (COTEet al. 1986). The ry+ probe was a 1.2-kb SstIBamHI fragment that includes +1501 to +2780 of the ry gene (LEE et al. 1987). A positive control for this probe having a single site of hybridization in the sd[~2216-547];ry506 genome was a hybridizationexperof ~ d [ ~ Y,ry506 ~ ~ with ~ ~ - ~ ~ ~ iment that compared DNA DNA of ~ d + ; r y ~The ” ~ .probe hybridized to one fragment in the sd[rY2216-5471;ry5u6 samples and showed no hybridization signal in the sd+;ry506 samples (data not from the shown).Thus, all positiveclonesisolated sd[ry2216-547],.ry506 librarywereexpectedtohybridize uniquely to thesite of the P[ry+]insertion at 13F. One positive clone, designated sdX 19a, was recovered in a screen of approximately 10‘ phage clones from the sdlry22/6-547];ry51)6 genomiclibrary(Figure 1). A 3.2kb EcoRI fragment subcloned from the sdX19a clone (Figure 1) hybridizesin situ t o polytene region 13F as sdX19a clone expected. Sequencingof a portion of the (D. CURTIS, personalcommunication)revealedthe presence of a 454-bp deletion within the first intron is consistent with of the ry gene. This observation previous genomic hybridization experiments that mapped a deletion in this region (DANIELSet al. 1985; DUTTONand CHOVNICK1988) and further confirms the origin of the sdX19a clone from the transposon insertion at 13F. For the purpose of chromosomalwalking, a genomic DNA library, constructed from the wild type Canton S strain (MANIATISet al. 1978), was screened with the 3.2-kb EcoRl fragment subcloned from the sdX19a clone (Figure 1). A screen of 2 X lo5 phage clones yielded seven positive clones. These were initially mapped with EcoRI to determine the terminal fragmentsofeach,andtwoclones(sdhl-40aand sdX1-40b) were chosen for more extensive restriction analysis. T h e twodistal EcoRI fragmentsfromthe

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FIGURE1.-Restriction map of the sd locus and representative phage clones isolated in the chromosomal walk. The coordinates are given in kilobases (kb). The zero coordinate corresponds to the KcoR1 site nearest the site of the transposon insertion in ~ d l which was used to clone the entry point for the walk (the sdX19a clone).The black box shown in the sdX19a clone represents P element sequences from the pry1 vector used to construct the P[ry+] transposon (DANIELS et al. 1985; RUBINandSPRADLINC1982). Restriction sites illustrated on the map are: R = EcoRI, H = HindIII, and S = SalI. The bars at the bottom of the figure represent the relative extent of the designated phage clones.

genomic region representedin these clones were used as probes for asecond step in each direction. Samples of 1.5 X 1O5 phage were screened with each probe to yield three and four positive clones for the leftward and rightward steps, respectively. A clone designated sdX3-14a extends farthestin one direction and a clone designated sdX2-4 extendsfarthest in the opposite direction on the chromosomal walk. Figure 1 depicts the restriction map of the 42-kb chromosomal walk. It illustrates three phage clones isolated fromthe Canton S library which are discussed in the text, as well as the sdX19a clone isolated from the ~ d [ ~ ;rySo6 genomic library. The zerocoordinate of the map representsthe EcoRI restriction site closest to the transposon insertion associated with the ~ d [ ’ Y ~ allele. This restriction site has been conserved in all stocks examined in this study. Two genes previously cloned in the 13F cytological region are the Drosophila c-myb oncogene homolog (KATZEN, KORNBERC and BISHOP 1985)and a Gprotein@subunit (YARFITZ, PROVOSTandHURLEY 1988). Cloned genomic DNA from eachof these genes was provided by the respective authors for the purpose of determiningwhetherthe sd chromosomal walk included sequences from either of these genes. Probes derived from these clones do not hybridize to restriction digests of cloned genomic sd region DNA nor do the genomic restriction patterns of these genes correspond tothose seen in the walk herein described. Genetic and phenotypic characterization of viable sd alleles: Stocks of sd mutants were obtained from the sources listed in MATERIALS AND METHODS for the purpose of mapping DNA lesions within the cloned sd region. Before inclusion in the molecular analysis described below, each stock was tested for comple-

mentation with at leasttwo “tester” sd stocks (one being the original sd’ allele, others included sd2, sdi8, Sd621, and Sd[rYz216-5471 ). In all cases, allelism withsd was inferred by failure of complementation, scoring the sd wing phenotype. “Strong” sd/“weak” sd transheterozygotes have an intermediate wing phenotype, implying that “weak” sd alleles are hypomorphic relative to “strong”alleles. Of(1 )sd7”/sd heterozygotes involving “strong” sd alleles have extreme vestigialwings (the phenotype is more severe than the respective sd/ sd genotype). In addition, the capitellum of the haltere is eliminated. These results indicate that even “strong” sd alleles are hypomorphic. Of(~1 )sd7”/sd+ heterozygotes, as well as synthetic ~ ~ ’ ~ ~ ~ ~ ~ ~ deletions of 13F constructedby segmental aneuploidy, have an incompletely penetrant wing margin phenotype similar to sd. Inaddition, females of the Of(1 )sd”’/sd+ genotype often have twisted third thoracic legs. These observations suggest haploinsufficiency for genetic functions uncovered by Of(l)sd7”. Haploinsufficiency for sd+ function may be responsible for the dominant wing phenotype. The possibility remains however, that an adjacent,previously uncharacterized genetic function is involved. It remains unclear what relationship might exist between genetic functionsunderlying the wing and leg phenotypes uncovered by Of ( l)sd7”. The expressivity and penetrance of the dominant wing and leg defects seen in Of(1 )sd7”/sd+ heterozygotes appears to be sensitive to genetic background but we have not attempted to characterize this phenomenon. in phenotypic y ~An ~effect ~ not ~ -previously ~ ~ ~recognized ~ descriptions of sd mutants, is the occurrence of ectopic bristles on the dorsal and ventral surfaces of the wing wing ~ ~blade. ~ - ~Ectopic ~ ~ 1 bristles, oftenclusteredalong veins, were seen in sd’, sdIg9, ~d~~~~and Of( 1)sd7”/ sd+, as well as in sd[rY+22161and its P-M dysgenesisinduced internal deletion derivatives. Ectopic bristles have never beenseen in the following genotypes however: sd’, sd6“, sdi8. The occurrence of ectopic bristles does not appear to be concordant with the relative severity of the wing margin phenotype among the sd alleles examined. A comparison of three radiationinduced alleles, sdi8, sd2 and sd6”, makes this point. sd58 has an extreme wing margin phenotype (vestigiallike) but ectopic bristles have never been observed, while sd2 has a weak (nibbled) wing margin phenotype and ectopic bristles are frequently seen on the wing blade. sd6“, which has a weak margin phenotype comparable to sd2, has never shown ectopic bristles on the wing, however. Of(l ) s d / s d + heterozygotes also exhibit the ectopic bristle phenotype, suggesting that sd might be haploinsufficient for both the wing margin and ectopic bristle traits.

37 1

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Molecular characterizationof scalloped viable alleles: Each of the sd alleles listed in MATERIALS AND METHODS was examined by genomic DNA blot analysis with at least three restriction enzyme digests (EcoRI, PstI and HindIII), andin most cases seven restriction digests (also including BamHI, PuuII, Sal1 and BglII). In addition, stocks carrying chromosomal rearrangements with published cytological breakpoints in the vicinity of 13F were examined. Genomic blots of restriction-digested DNA from each of the mutant stocks were prepared as described in MATERIALS AND METHODS. These blots were serially hybridized with labeled restriction fragments excised from the genomic clones illustrated in Figure l . T h e specific genomic restriction fragments used as probes in this study, most of which have since been subcloned into plasmid vectors, are illustrated in Figure 3. Two examples of the results we observed are presented in Figure 2. Stocks chosen for this experiment represent five independently derived sd mutations (MATERIALS AND METHODS), each associated with aconfirmed DNA rearrangement within the sd region. Confirmed DNA rearrangements were inferred from the observation of restriction fragment polymorphisms (relative to wild type) in at least threedifferentrestriction digests. In this experiment, the E probe (Figure 3) including genomic region -0.1 to +3.5 kb, hybridizes to DNA restriction fragments of altered mobility in both EcoRI and HindIIIdigests in each mutant sample compared to the sd+ sample (Canton S, lane 6). DNA rearrangements were detected with the E

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93

FIGURE 3.-Sumnl;1ry map of nine viable sd mutations associated withDNA rearrangements. The units at the top refer to walk coordinates. Restriction sites illustrated on the map are: R = EcoRI, H = Hindlll. and S = Sall. In addition, a Puu 11 site ( 8 ) at genomic coordinate +2.0 kb is indicated by “V”(Puull sites are not illustrated for the rest of the map). The map summarizes data obtained by serially hybridizing genomic digests ofDNA fromeach of the indicated genotypes with probes A. B, C and D. Horizontal lines designate regions where restriction fragments seen in sd mutants are identical to those seen inwild type (ie., no detectable rearrangement is present). The region of observed DNA rearrangements is depicted in each case by a hatched box. Deletions defined in this analysisare illustrated by gaps in the horizontal lines. A question mark denotes uncertainty as to the relative position of the deletion endpoint in the case of sd”.

probe in 11 of 20 sd alleles tested. Two alleles, ~d~~~~~~ and sdM, were examined only with this probe and a DNA rearrangement was seen in both cases (Figure 2). The entire interval between -10 kb and +14 kb was examined for the remaining mutations described in MATERIALS AND METHODS. The genomic hybridization dataonnine extensively analyzed sd alleles whichshow DNA rearrangements within the chromosomal walk are summarized in Figure 3. All of these alleles show a DNA rearrangement within a 2kb interval defined by the EcoRI site atthe zero coordinate and a PuuII site at coordinate +2.0 kb, as indicated in Figure 3. No other rearrangements were sd[”*9”]3,~ d ~sdZA3 * ~ , noted in five mutations (sd[’Y+2216], and sd””). In four cases, (sd””, sd””, sd93 and ~ d * ’ ~ ) , the combined sizes of fusion fragments detected by the E probe suggest for each case that either a large insertion (greater than 20 kb) or the endpoint of a more complex rearrangement is responsible for the observed restriction fragment polymorphism. Chromosomal deletions were inferred forthree mutations ( ~ d ’ ~ ~ ” , and sd93) from the absence of hybridization signals when blots of DNA from these mutants were probed with cloned DNA of the regions indicated (Figure 3). These same blots were rehybridized with cloned DNA from the 0 to + I 5-kb region as a positive control for DNA in the samples. Signifi-

cantly, as much as 10 kb of chron~osom;lID N A rnav bedeleted without compromising maleviability or fertility (e.g.,sd"). sd9' homozygous females also survive, but their fertility may be impaired. An important implication of the sdV3 deletion is thatthedeleted region cannot be associated with a vital function of any kind, including a vital sd function. Regulatory sequences necessary for the normal expression of sd could, nevertheless, liewithin this region.Indeed, such an argument is favored by the fact that extreme sd wing phenotypes are associated with chromosomes carrying such deletions. Two P-M dysgenesis-derived alleles, sdPv"'" and Sd2'"/1) , exhibit related sd region rearrangements. The ,d.?'/'):\ allele exhibits D N A rearrangements at two separate places within the cloned region in the locations indicated (Figure3). The sd2vy'J allele, which was derivedfrom the stock carrying the sd2""." allele, has a D N A deletion within the interval bounded by the two rearrangement sites. A distinctly moreextreme sd wing phenotype is observed in sd2""" mutants relative to Sd2'J'J.,\ During the genomic hybridization analysis it was apparent that the D probe (Figure 3)includes repetitive sequences. Major bands corresponding to fragments identical in length to wild type controls were seen, as well as multiple fainter bands. In order to confirm this deduction, and to further map the purative repetitive element(s) within this region, a "reverse Southern" experiment was carried out ( H A L L , MASON and SPIERER 1983). Total Canton S genomic D N A was labelled and hybridized to a blot comprised of a set of restriction digests of cloned genomic D N A enconlpassing the entire walk. A dilution series of ry plasmid D N A was included on the blot as an internal controltorepresentthe signal from a single-copy gene. Prominent signals, corresponding to two separate restriction fragments, localize the repetitive D N A sequences within the genomic interval (datanot shown). One of these is within a 1.5-kb EcoRI fragment (map coordinates+9.7 to +11.2 kb) included in the D probe. No restriction fragment heterogeneity was noted in this genomic region among any of the stocks examined, suggesting that these repetitive sequences do not result from arecent transposition event. The reverse Southern analysis alsorevealed the presence of a second .repetitiveelement mapping within a 6-kb interval (mapcoordinates +22.2 to +28.2) contained in the sdX2-4 clone (Figure 1) that had not been included as a probein the genomic DNA analysis. This region has not been further characterized. DNA rearrangements were not detected within the interval described in Figure 3 forany of the remaining sd mutations listed in MATERIALS AND METHODS [sd', sd2, sd'"' , I( I )sd"., I( I )sd"'., I( I )sd"j,", I( I )~d"~",

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I.'I(;I.RI. l . - ( ~ \ ~ o l o x i ( ~ l l ; l r l ; t l \ \ i b o l 111c. .\d rc.gio11 \ I I O \ \ I I I X the inversion l o o p ;lssoc-i;llc.tl \vi111 .w/'~'. ( x ) ' 1 ' 1 1 ~ n l o l y t 1 o l o ~ \ o f rile

invrrsion in ;I sd'~\'/sd+heterozygote. I'lw prosimal t)rcakpoint of t h e i n \ w s i o l l is a t 131; ; u ~ dt h e distal 1wc;lkpoint is a t I 1 F / I ' L A . (13) Tllc F 1~roI)c(I;igurc 3) hylwitlircs t o t h e proxit1l;ll hordcr of the inversion l o o p i t 1 a sd'*/sd+ Ilctcrongote. ((:) I Iyl,ritlir;ltion of t h e (;prohe (I;igurc :%);It I I F/I 'LA. the distal Iwahpoint of t h e sd'.' inversion. IOIlolno7\gotcs.

I ( I)sd3"' or Df(l)sd7'*]. Presurnably these radiationinduced or EMS-induced mutations are single base substitutions or rearrangements that are too small to be detected by conventional genomic restriction analysis (COTEet al. 1986). sd+ D N A rearrangements with breakpoints mapping near 1SF [T(I;Y)P12, T( I;Y)240, In( I ) A B , Df(I )sd7'* and Dp( I;Y)shi'Y#3] were similarily not found within the region examined. Cytological analysis: Two questions raised by the molecular analysis of the sd region were addressed by in situ hybridization to polytene chromosomes. The first of these concerns the y-ray-induced, viable allele s d S NThis . allele was characterized as a probable inversionwhen it was first described, due toa reduced crossover frequency observed between the flanking markers ras andf(1vEs 1961). Cytological analysis of sdSN/sd+heterozygotes shows an inversion loop with breakpoints at 13F and 11F/12A (Figure 412). I f the breakpoint of the sd5" inversion and the molecular rearrangement in the sd region that we have characterized are identical, then sd region DNA that is proximal to the lesion should hybridize to the proximal side of the inversion loop. The F probe (Figure

The scalloped Region

3), which maps to the “right”of the molecular breakpoint in the sd region of sd58, hybridizes to the proximal side of the inversion loop (Figure4B).This result indicates that our chromosomal walk is oriented such that “right” is toward the centromere (see Figure 3). This interpretation was further confirmed by hybridization of the G probe (Figure 3)to sd58 homozygotes. In this case, a single site of hybridization at 1 1 F/12A was observed (Figure 4C). It had been proposed that the sd2 allele was also associated with an inversion due to observations of crossover suppression in the region flanking sd (PANSHIN 1935).The cytological examination of sd2/sd+ failed to reveal a visible rearrangement(datanot shown). P element seWe further questionedwhether quences were presentat 13Fin sd alleles derived from P-M hybrid dysgenic crosses carried out in the CHOVNICK laboratory (MATERIALS AND METHODS). Previous cytological studies of sd[v+22161 (DANIELSet aZ. 1985), ~dI”*‘Y”13 (LEVIS, HAZELRIGG and RUBIN 1985) andsdJM (J. MOORE, personal communication)indicatedthat these alleles do have P sequences at 13F. We have examined sdS3,sdI8’, sdZA3, ~d~~~~ and ~d~~~~ homozygotes by hybridization to the P element clone pa25.7 wc (KARESSand RUBIN 1984). All of these alleles show hybridization of the P element probe at 13F (as well as other sites). In favourable preparations,two sites of P element hybridization can be detected at 13F in sdzYYA homozygotes (data not shown). These may correspond to the two rearrangements seen in our genomic hybridization analysis of this allele (Figure 3). T h e deletion associated with sd93 (Figure 3) is not cytologically detectable however. The in situ hybridization results are consistent with the molecular rearrangements described in Figure 3, and confirm the association of sd mutations with P element mobilization. We also examined sd58 and sd2 with the pa25.7 wc probe and saw no hybridization to 13F or any other location, except at 17C. Hybridization at 17C is due to genomic sequences from the original cytological location of the P element cloned in ~ ~ 2 5 wc, . 7 and thus serves as a positive control for the hybridization reactions. This result is consistent with genomic hybridization data indicating that these stocks did not contain P sequences. Genetic and phenotypic characterization of lethal scalloped alleles: The original characterizationof five members of a recessive lethal complementation group as being alleles of sd was based upon failure of two alleles (sd3L and sd3IH) to complement the sd wing phenotype associated with ~ d ~ [ ( ~ and 9 9 )sd’. ~ 1 Extend~ ing the complementation analysis, all five members of the group [Z( 2)sdjL, 1( 1)sd’IL, I( Z)sd68L,I( 1 ) ~ d ~and ~‘” Z ( l ) ~ d - ) were ’ ~ ] examined as heterozygotes with each

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of four different sd viable alleles (sd’, sd2, sd6”, and Sd[ry22Z6-547] ). Four of the lethal alleles [ l ( l ) ~ d ’ ~ ,

I( 1)sd68L,Z( I)sd47Mand I( 1)sd3lH]failed to complement the wing phenotype of each of the viable sd alleles. In these cases, the wing phenotype associated with sdZ(1)sd heterozygotes wasalways more severe than that seen in the respective sd/sd genotype. One member of the group, Z(1)sd1lL, did fully complement all of the viable sd alleles tested, however. The time of lethality was determinedfor males carrying each of the lethal alleles in hemizygous condition. In order to facilitate this analysis, a type 1 allele of y (NASHand YARKIN1974) was crossed onto each of the lethal-bearing X chromosomes to serve as a larval marker. Balanced stocks marked in this way generate Z(1)sd male larvae that are phenotypically distinguishable from sd+ siblings. Two classes of lethal sd alleles were apparent from this analysis. The first ” ~ 1 ( 1 ) ~ dcan ~ ~ hatch ~, class, comprised of l ( l ) ~ d and and survive as larvae for several days. Such larvae are noticeably feeble and slow growing relative to their sd+ siblings. T h e remaining lethal alleles [ Z ( l ) ~ d ” ~ , Z ( I ) S ~ @and ~ Z ( l ) ~ d ” ~are ] late pupal lethals (larval and early pupal lethality is seen also). Gross morphological distortions of the pupal head are commonly observed among late survivors of the pupal lethal class. T h e two members of this class that fail to complement ~ ’ ~l ( l ) ~ dex~ ~ ~ ] the visible sd phenotype [ l ( l ) ~ d and hibit vestigiai-like pupal wings at this late lethal stage whereas the pupal wings seen in I( 1)sd1lLlate survivors appearnormal, consistent with interallelic complementation data discussed earlier. We also examined each of the lethal alleles in females as transheterozygotes with Of( I)sd7”. The I( 1 ) ~ d ~ ~ / D f ( l genotype ) ~ d ’ ~ ’ appears to be embryonic 1 ) ~ d genotype ~~’ usulethal, whereas the Z( 1)47M/Df( ally hatches and is otherwise similar to the 1(1)47M male hemizygotes. All three members of the pupal and I( I ) s ~ ” ~can ] surlethal class [I(I)sd3lH, Z(l)sd68L vive as femalehemizygotes until the pupal stage (none were observed to develop as completely as the male hemizygotes however). These observations are consistent with those based on sd male hemizygotes in indicatingthat two distinct (albeit loosely defined) lethal phases are represented by the two classesof lethal sd alleles. The exceptional allele Z( I)sd’lL which complements viable sd alleles for thewing phenotype, is considered to be an authentic allele of sd on the basis of two criteria. First, this allele fails to complement the recessive lethal phenotype of all other Z( 1)sd alleles and Of( 1 ) ~ d ~Secondly, ~’. late pupal sdllL male hemizyotes exhibit head defectsidentical to those seen in sdslH or sd68Lmale hemizygotes that survive to a similar developmental stage. Together theseobservations imply

Cfo 1 Alu 1 that a common vital function is defective in these 6 5 4 3 2 1 1 2 3 4 5 6 mutants. The interallelic complementation data raises the possibility that w e are dealing with two closely linked genes encodingtwo separate functions: one associated with the wing, and another serving a vital function. T h e possibility that four EMS-induced lethal alleles might have two lesions, one in a previously unknown vital gene and thesecond lesion in the nearby sd locus, seemed remote in view of the fact thatthe same mutagenesis screen produced 59 additional mutations of 14 other lethal complementation groups, all complementing the viable sd mutations. Nevertheless, a recombination experiment was carried o u t to test this possibility. I( l)sd"l"/Binsn females were crossed to F I G U R E 5.-lknaturing gradicnl awlysi\ 01' letl~;~l sd alleles. A males carrying a marked, non-balancer chromosome: s;lnlple of 5 pg of genomic DSI\ was digested w i t h the indicated rcstriction C I I Y ~ I I I Cfor r;lch S ~ I I I I and ~ ~ , I'lactionatetl o n ;I 20% t o y u ma-1. Virgin, non-Bar female progeny of this cross, 80% ; ~ c r y l ~ ~ ~ ~tlcnaturitlg ~ i ~ l e -gradient. ~ ~ r ~ ~ Thc ;Illtor;ltliogr;lnl I( l)sdJIFf/yv ma-1, were then crossed to Binsn males. s h o w n rcl~rcsents;I I ) l o t hyhridi~etlto ;I probe derived from ;I cl)NA Male progeny (20,000) of this cross were scored for clone (sdcl 1 I O ) t h a t I I I ; I ~ St o gcnomic coordinates+ 12.2 to + I I . I . sd but none were found. If the Z( 1)sd""' chromosome "Shifted"I)antls c1l;lr;Icteristic of fr;Igmcnt melting point differenct*s are intlic;lted by arrows. The I;lne designationsrepresent the followwere carrying a separate, recessive sd mutation, then ing genotypes: ( I ) r u ( ~ ) s d +( 2 ) /( /)sd"'., (:%)/( I ) S ~ " ~ ' (4) . . /( I)sd:"". the maximum genetic distance separatingthe two ( 5 ) /( I)sd:".; 1 n d (6) /( I)Sd'".". putative genes on the basis of a 95% Poisson Confidence Interval is 0.0015 cM.Given that there is a we have not attempted to characterize this phenomeminimum 10 kb distance between the molecular lenon further. Denaturing gradient analysis of lethal sd alleles: sions associated withviable and lethal alleles of sd The denaturing gradient gel method has proven to mutations (see below), this result yields a cM/kb estibe an eflective means of mapping point mutations in In contrast,large scale rosy locus mate of 1.5 X the rosy gene (GRAYet al. 1991; CURTISet al. 1989). intragenic recombination experiments, involving muThe method is suitable for the localization of DNA tations at known sites in the molecular map have lesions when the parental wild-type DNA is available yielded cM/kb values an order of magnitude greater for comparison. Fortunately, this was the case for the thanthat estimated above (A. J. HILLIKER,S. H. I( l ) s d alleles (MATERIALS AND METHODS). Six different CLARK and A. CHOVNICK, unpublished results). This four-cutter restriction enzymes were used in this comparison is particularly significant since rosy region analysis: CfoI, AluI, HueIII, MspI, RsaI and TuqI. recombination is somewhat reduced, due to its cenStocks carrying all l( 1 )sd alleles (as balanced heterotromere proximal location. On the basis of these obzygotes)plus the parental chromosome were tested servations, we believe that lethal sd mutations reprefor each enzyme. Our initial expectation was that the sent a class of single lesions of the sd gene. l ( l ) s d alleles would map within or near the genomic The pupal lethal allele sd31if represents a special interval wherein rearrangements were mapped in the case in that it appearsdominant(although incomviable mutations. Accordingly, restriction fragment pletely penetrant) with respect to wing margin nicking probes encompassing genomic coordinates -0.1 to and ectopic wing bristle phenotypic traits. A similar +7.9 kb (probes E and F, Figure 3) were hybridized dominant phenotype is also observed in D f ( l ) ~ d ' ~ ~ / to the set of denaturing gradient genomic blots. No sd+ hemizygote females. In that case, haploinsufficonvincing examples of "shifts" indicating sequence ciency for the genetic function(s) underlying the wing alterations in this interval were seen (data not shown). phenotype was suggested. Haploinsufficiency is unsatWhen these blots were re-probed with a cDNA clone isfactory as an explanation for the I( 1)sd""' dominant designated sdcH 10, convincing band shifts were obwing phenotype, however. This mutationdoesnot served in at least one digest for each of four of the behave as a null allele. I t is a member of the pupal five lethal sd alleles examined. The sdcH 10 clone was lethal class, implying that an earlier requirement for recovered in a cDNA library screen for the -0.5 to sd function (defined by the early lethal classof sd +15 kb genomic region and is discussed below. An mutations) has been satisfied. The expressivity and example of the denaturing gradient hybridization repenetrance of the dominant wing defect associated sults with the sdcH 10 probeis shown in Figure 5. The with sd31"/sd+ heterozygotes appears to be sensitive altered fragments in this autoradiogram are indicated to genetic background, like that of Df(l)sd'2h/sd+,but by arrows andinclude: the apparentloss of a fragment

T h e scalloped Region

'

"

? I I

n

1 2 3 4 5 6 7 8 9 1011 i n two instances (CjioI digest, and AluI digest; sd"",/ 1 1 1 1 1 1 1 1 1 1 l FM7c sample), a "shifted" fragment (AluI digest; ~ d " ' ~ / FM7c sample) and the appearanceof a novel fragment - 5.9 A (AfuI digest; sd"'"'/FM7c sample). In total, sequence - 4.8 alterations were detected by the sdcH10 probe for -- 3.7 3.5 four of the five lethal sd alleles. sd3', showed no shifts 3.0 i n any of the restriction digests. sd"'. showed alterations with Rsal. sd:'"' showed alterations withAluI - 2.3 and TaqI. ~d'"~'' showed alterations with Alul and - 2.0 . . RsaI. sd"", showed alterations with C'ol, AluI, RsaI - 1.6 and HaeIII. These results locate the DNAlesions 1.4 associated with four 1( I)sd alleles to the DNA encoded by the sdcH 10 clone. - 1.0 cDNA analysis of the scalloped region: As discussed earlier, the observation thatdeletion of genomic DNA sequences from 0 to -10 kb was not B c lethal implies that coding sequences associated with 4 . 0 - .vital sd function cannot occupy this region of the chromosomal walk. Thus, we focused our attention 2.7- A: to the "right" of the zero coordinate for screening - 5.9 cDNA libraries, as a means of identifying potential sd' gene products. cDNA libraries representing em-- 3.6 4.0 bryonic to pupal stages of Drosophila development (POOLEet al. 1985) were screened with labeled BcoRI genomic fragments from the sdX1-40a clone encompassing coordinates -0.1 to 14.3 kb (Figure 1). Five cDNA libraries representing the following stages were screened: first and second instar larvae (G library), early third instar larvae (H library), late third instar 1 2 3 4 5 6I 72 83 4 larvae (1 library), early pupal ( P library) and late pupal FIGURE6.-\lqqing of three c D N A clones to restriction di(Q library) (POOLE et a f . 1985). Approximately 2 X gested sd region genomic DNA. The molecu1;lr nlass (kh)of rcstriction fmgnlents \vas determined using Nindlll-digested A DNA m t l IO5 phage clones were screened from each library. A isshown a t the side of e;lch panel. (A) is an autor;ltliogr;lIn o f single clone, referred to as sdcH10, was recovered. genomic D N A hybridized with a 1.5-kh EcoRl fragment from the T h e sdcH 10 clone, retrieved from the H library (early sdcF.21 clone. I.ancs 1-3 are samples of sdX1-37 D N A (see Figurc third instar larvae), was mapped to genomic coordi7 for n ~ a pof this clone). Restriction digests i n lanes 1-3 \verc: nates 12.2 to +14.3 kb by hybridization to a blot of EroKl/flamHI, I:'coKI/Hindlll, and EroRl, respectively. I.;~ncs4-6 are s;miples ofsdX2.hV4 DNA (digests were: EcoKI. F~coKl/\Iindlll, restriction-digested cloned genomic DNA (Figure and /:'roKI/flamHI. respectively). Lanes 7-1 1 are sanlplcs of stlAIGB). Initially, it was not apparent that the transcript 40a D N A (digests were: EcoKI/fsfl, EroKl/l'mll, IhKl/flg111, represented by this clone bore any relevance to the sd EroRI/HindIII and EroKI, respectively). (R) is a11 autoradiogram of gene. However, once it became clear that the denagenomic D N A hybridized w i t h a 1.6-kh EcoKl fragment fro111thc sdcHlO clone. Lanes 1-5 are samples of stlA1-40;1 D N A (digests turing gradientanalysis had localized four of the lethal BcoRI/Putrll a n d KroKI/ were: BroKI, EroRI/Hindlll.BcoRl/~~1II, sd alleles to sdcH 10, its potential importance for our Psfl, respectively). Lanes 6-8 are samplcs of sdA2-4 DNA (digests characterization of the sd region was recognized. were: EcoRl, BcoRI/Hinldlll, F~coRI/RarnlHl, rcspcctivclv). Faint Subsequently, two further cDNA libraries (BROWN b ; d s seen i n this autor;ltliogr;lm are tluc to contanlinant l a n ~ h d : ~ and KAFATOS 1988) representing late embryonic and D N A sequences in the probe. ( C ) is XI autot-;tdiogramof genomic DNA hybridized with ;I 2.5-lib EcoKl fragnwnt from the stlcF.7 imaginal disc transcripts were screened using the clone. Imws 1-2 are samples of stlA2-4 D N A (digests \\'ere: I:'coKI. sdcH 10 insert as aprobe. Approximately 5 X IO5 and Hindlll, respectively). I.;tnes 3-4 arc s ; ~ n ~ p l ofsdA1-4Oa es DS'4 clones were screened for each library and a total of (digests were: EroRI, ;md Hindlll, respectively). 32 positive cDNA clones were isolated in this screen (1 7 from the embryonic library and 15 from the disc digested genomic clones in order to map the cDNA library). Our preliminary analysis indicates that sevsequences to the cloned genomic region. The reader eral cDNA clones recovered in this screen include will find it helpful to consider the data presented in exons that span the genomic interval between 0 and Figures 6 and 7A together in order to follow the discussion of these results. Figure 7B illustrates our +14.3 kb. Figure 6 illustrates these data. The sdcH 10 interpretation of these data. clone and two other cDNA clones isolated by hybridsdcE2l is a cDNA clone isolated from the late ization with sdcH10, were hybridized to restriction-

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B-10

- 5

-

0

5'

5'

+5

=

+15

+10

=

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sdXcH10 sdcEZl

I sdcE7

FIGURE7."Mapping of cDNA clones to restriction digests of cross-hybridizing genomic DNA clones. (A) Genomic map of the - 0 . 1 to +I6.2 region. illustrating the hybridizing restriction fragments seen i n the autoradiogr;ms depicted in Figure 6. Numbers ahove the horizontal hars at the top of the genomic map indicate the size (kb) ofeachof the fragments shown. Restriction sites illustrated on the map are: R = EcoRI, H = Hindlll, B = BamHI, G = BgllI, V = Puull, T = Psll. S = Sall. Below the genomic restriction map, horirontal lines represent the extent of genomic clones used in the experiment. Vector-encoded EcoRI sites at the ends of the genomic clones are illustrated as (R)to indicate that they are not genomic BcoRl sites (FRISCHAUFel al. 1983). (B) Summary ofsdcH IO. sdcF.21 and sdcE7cDNA clone genomic 1oc;llization data. The large open box encompassing -0. I to 16.2 lih indicates the region depicted in (A). Hatched boxes represent the genomic localization of cross-hybridizing sequences from each ofthe indicated cDNA clones, determined from the autoradiograms slwwn i n Figure 6. Below the genomic restriction map, horizontal lines represent the extent ofgenomic clones used in the experiment. Restriction sites illustrated on the mapare: R = BcoRI, H = Hindlll, I3 = BamHI. G = BglII, V = PuulI. 7'= P s f 1 , S = Sall. Restriction sites for E a m H I , Rglll. Puull, and Pstl are shown only for the 0 to 14 kb region of the map.

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embryonic BROWNand KAFATOS library (1988). Restriction analysis of this clone indicated an internal EcoRI restriction site and the insert was subcloned as two EcoRI fragments of 1.5and1.7kb(thepNB vector has EcoRIsites flanking the cDNA inserts). The relative 5'-3' orientation of clones isolated from this library can be determined by restriction analysis. By this means it was established that the 1.5-kb EcoRI fragment from sdcE21 represents the 5' end of the mRNA (data notshown). Figure 6A showsthe hybridization of this 1.5-kb EcoRI restriction fragment to

restriction-digested genomic DNA from sdX1-37 (see Figure 7B for a description of this clone), sdX1-40a and sdX2-4.EcoRI fragments of 5.9, 2.3 and 1.0 kb from the sdX1-40a clone are labeled, as well as a 1.Okb EcoRI fragment in sdX2-4 and a 4.8-kb fragment in sdX1-37. Double digests further localize the crosshybridizing sequences to a 3.0-kb EcoRIIPstI fragment,a 1.4-kb BgZIIIEcoRI fragmentanda 1.0-kb EcoRI fragment from the sdX1-40a clone (Figures 6A and 7, A and B). A further hybridization experiment, utilizing the entire sdcE2 1 clone as a probe, detected a 2.1-kb EcoRI fragment from the sdX1-40a clone in addition to the fragments detected above (data not shown). These results localize sdcE2 1 cross-hybridizing sequences to the genomic map as shown in Figure 7B. Figure 6 B shows the hybridization of a labeled 1.6kb EcoRI fragment including the entiresdcH 10 cDNA clone, to restriction-digested genomic DNA from the sdX1-40a and sdX2-4 clones (sdX1-37 was also tested but no hybridization was seen, data not shown). The sdcH 10 probe hybridizes to a 2.1-kb EcoRI fragment from thesdX1-40a clone and to a1.9-kb EcoRI/BamHI fragment from the sdX2-4 clone. These results localize sdcH 10 cross-hybridizing sequences to the genomic map as shown in Figure 7, A and B. Figure 6Cshows the hybridization of a 2.5-kb EcoRI fragment representing the entire sdcE7 cDNA clone to restriction-digested genomic DNA from the sdX140a and sdX2-4 clones. The sdcE7 probe hybridizes to a 4.0-kb EcoRI fragment insdX2-4 and to EcoRI fragments of 5.9, 2.3 and 2.1 kb in sdX140a. I t also hybridizes to a Hind111 fragment of 3.6 kb from the sdX140a clone (the larger Hind111 fragments represent fusions of vector and insert DNA). These results localize sdcE7 cross-hybridizing sequences to the genomic map as shown in Figure 7, A and B. The relative 5'-3' orientation of the sdcE7 and sdcE2l clones is depicted in Figure 7B. These data indicate that the sdcE7 andsdcE2l cDNA clones represent transcripts derived from a primary transcription unit that extends from somewhere near the 0 coordinate to atleast 14 kb. Neither of the restriction fragments previously shown by "reverse Southern" analysis to contain repetitive sequences hybridize to any of the cDNA clones we have examined. There is no evidence therefore that eitherof these repetitive elements are included in the processed forms of transcripts represented by these cDNA clones. Transcript analysis of the scalloped region: A transcriptional analysis of the sd genomic region encompassed by the viable and lethal sd mutations was undertaken with the intent of identifying putative sd transcripts. Figure 8A shows transcripts observed when a Northern blot of poly(A+)RNA (isolated from embryonic,third instar larvae andadult develop-

+

'The scalloped Region

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- 2.0

C

- 1.7

I;IC;L'RE X.-~l'r;ltlscriptiotl~ll analysis of the sd region. Panels A , I3 ;IIKI C r ~ ~ p r c s ;turol-;ltliogr;Ims e~~t of the same blot. Approximately I O p g of poIy(A+) R N A isolated from the indicated developmental st;~ges;IIKI genotypes were fractiotwted in each lane. Lane desigtlations refer t o tl~efollowing samples: ( 1 ) sd', 0-8 hr AEL (after egg laying); (2) sd'"", 0-8 hr AEL.; ( 3 )sd', 8-24 hr AEI.; (4) sd'Xd, 8-24 11r A I . 1 . ; (5) sd+, late third instar; (6) sd"", late third instar; (7) sd*, a d u l t ; ; ~ n d(X) ~ d ' " ~adult. , ( A ) Autol-;ldiogr;m of a blot I~ybridixdt o :I 2. I - k b EroRl restriction fragment (the H probe, l;igure 3 ) encon~passingthe genomic interval +12.2 to +14.3. (B) w a s hvhritlized to the 5C actin probe (FYRRERCel al. 1983). ((:) 'l'he b l o t w a s hybridized with the E probe(genomic interval - 0 . 1 t o +J.5, Figure 3 ) . Numbersontherightindicate

.l'l~cb l o t

111olecu1;1r weight (X 1000) estimates for the respective transcripts

tletrrminetl by comparison with RNA molecular weight standards (BKI.).

;IS

mental stages respectively) was probed with a genomic fragment encompassing the + I 2.2 to +14.3 kb interval wherein the sd lethals map (probe H, Figure 3). Two prominentadulttranscripts were detected by this probe in the sd' samples, estimated as 4.5 and 3.3 kb (Figure 8A, lane 7). Samples of sdfiXdpoly(A+)RNA (equivalent amountstothe sd+ samples, estimated 260 nm) were analyzed at the by absorbanceat same developmental stages for comparison. At least two slightly smaller transcripts (relative to the major transcripts seen in the sd+ samples) were seen in the Sd5Hd samples (Figure 8A, lanes 2, 4, 6 and 8). These transcripts are undetectable in adults, in contrast to two clearly defined transcripts presentin the sd+ sample. The transcripts which hybridize to the H probe may be even more heterogenous than our discussion of these results implies. Faint bands which may represent further transcripts can be seen in the sd+ embryonic samples on this autoradiogram (Figure 8A). Figure 8 B shows an autoradiogram of the same blot rehybridized to an actin probe (actin 5C; FYRBERG et

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al. 1983) as an internal control for sample clegr;ld;ttion. The overexposure of the autoradiogram shown in Figure 8 B precludes quantitative statements about the relative abundance of the transcripts which hybridize with the H probe. I t shows that the apparent sample is not absence of adult transcripts in the sdScqd to dueto sample degradation, however. These results also suggest that the transcripts hybridizing with the H probe are not abundant (note that the exposure that time for the autoradiogram in Figure 8 B was for Figure SA). Two genomic probes, the E probe and the F probe (Figure 3) representing coordinates -0.1 to +7.9 kb gave inconclusive results for sd' samples, i n that no consistent signals were seen in our transcript analysis. The blot used in Figure 8, A and B, was hybridized with the E genomic probe (Figure 3) and the autoradiogram is shown in Figure 8C as an example of such data. Unexpectedly, a 1.7-kb transcript is seen in embryonic and adult poly(A+) R N A extracted from sdfixd mutants. This transcript may originate within the D N A rearrangement associated with the sd'"" mutation. One issue to consider is that this ectopic transcript may interfere with transcript initiation from sd this region,perhapsaccountingforthemutant phenotype observed. However, we have no data that directly addresses this issue. Given that our transcription data indicates that at leasttwo transcripts originate from the +12.2- to +14.3-kb genomic region, it is of interest to consider whether they might be structurally related. T o address this issue, we asked whether the two major transcripts that hybridize to the H probe (Figure 8A) in sd+ adults hybridize with an antisense probe synthesized from one of the cDNA clones isolated from this region (sdcE21, Figures 6 and 7). The sdcE2 1 cDNA clone was used as a template to make a single-strand antisense probe (MATERIALS A N D METHODS) that was hybridized to a blot containing samples of sd+ adult poly(A+)RNA. The resulting autoradiogram is shown in Figure 9. The autoradiogram showstwo major transcripts estimated to be 3.3 and4.5 kb. These transcripts probably overlap since they both hybridize to thesame cDNA probe andmust both be transcribed in the same 5'-3' orientation. Restriction analysis and genomic hybridization of the sdcE2l clone indicates the orientation of these transcripts to be as indicated in Figure 7. One apparent discrepancy in our interpretation of these results is the failure to detect transcripts with the E genomic probe,althoughthesdcE2l cDNA clone clearly hybridizes tothe E fragment(Figure 6A). This discrepancy is likely due to the presence of introns, making detection of these rare transcripts difficult. Efforts are now underway to further char-

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IICURE 9.-An autoradiogram is shown of sd+ adult poly(A+) R N A hybridized with a single-strand antisense RNA probe synthesized from the cDNA clone sdcE2 1. Approximately 8 and 2 pg of

poly(A+) RNA were loaded( i n the left andright lanes, respectively). .. I ranscript molecular weight (x 1000)estimates are indicatedon the right.

acterize the structural relationships of transcripts from this region and to determine their codingpotential. DISCUSSION

A major issue of the present report concerns the nature of the sd region. One interpretation suggests that that we are dealing with two separate genetic units, one of which is the sd gene whose mutations are homozygous viable. T h e second unit,according to this notion, is a vital gene located in close proximity to sd. Alternatively theentire sd region may bea single functional genetic unit. In view of the experimental evidence, we believe the former model to be unlikely, and prefer to believe that we are dealing with a single, vital sd gene. There remains the vexing issue of the complementation analysis of I(l)sd””. This lethal mutation fails to complement the vital function missing in all other lethal sd alleles and Of(I ) S ~ ” ~It. is, however, phenotypically wildtype in transheterozygotes with alltested viable sd alleles. These observations suggests that the sd+ gene might be associated with morethanone product(perhaps via differential processing) or it might reflect differential tissue and/or temporal regulation. The resolution of these questions awaits further molecular analysis now in progress. A 42-kb chromosomal walk has been described that was initiated at thesite of a P[ry+]transposon insertion at polytene section 13F (DANIELSet al. 1985). Genomic analysis of DNA from twenty different sd alleles was undertaken for the purpose of identifying genomic regions within the chromosomal walk that might delimit the sd gene. One genomic region (coordinates 0 to +2.0 kb) is the site of a number of independently derived DNA rearrangements involving P element sequences. We propose that this region P element mobilization events. defines a “hot spot” for

Since the sd mutations examined in this analysis were of diverse origin, we are not able to quantify the rate of new insertions into the region however. This genomic region also encompasses one breakpoint of an X-ray-induced chromosomal inversion associated with sdsXd (IVES 1961) that juxtaposes sequences from our walkwith sequences from cytological region 11F/ 12A, enablingus to orient thechromosomal walk with respect to the centromere. Three of the hybrid dysgenesis-induced alleles carry deletions within the genomic region described. The largest of these deletions, sd”, is at least 10 kb in size, yet males are viable and fertile. This deletion serves to define a chromosomal region across which coding sequences of the sd gene are not expected to map since our genetic analysis indicates that sd is an essential gene. Several lethal sd alleles were mapped by denaturing gradient electrophoresis to a cDNA clone, sdcH10. Hence, they map to a genomic region of our walk that is 10- 12 kb from the region perturbed in P-induced lesions. These lethal alleles are likely to represent changes, either in coding regions or at splicejunctions, by analogy with an extensive study of ry alleles using this technique (GRAYet al. 1991; CURTISet al. 1989). These observations require that some of the coding sequences associated with the sd genebe localized within the +12.2 to +14.3 genomic region. Screening of two cDNA libraries with the sdcH10 clone (which maps within this genomic interval) hasyielded 32 clones. Two of the cDNA clones are described in this report (sdcE2l and sdcE7). They represent transcripts that extend across 14 kb of the chromosomal walk herein described,and encompass two genomic regions where molecular lesions associated with sd mutations have been localized. Our preliminary sequence analysis of these clones indicates that they represent structurally related, yet distinct transcripts (data not shown). While all the cDNA clones recovered from this region appear structurally related, the possibility that other, unrelated transcripts might exist has not been eliminated. The simplest interpretation of these experimental results is that the structurally related transcripts represented by the cDNA clones we describe are alternatively spliced forms of a primary sd transcript that extends across a 14-kb interval, and perhaps beyond. The 5’ ends of these cDNA clones map within a region defined by P element-associated DNA rearrangements. It has been noted before that P element insertion events are often clustered in regulatory regions atthe 5‘end of gene transcription units (ENCELS 1989). Such a distribution may reflect a preference for a particular chromosome structure shared by such genomic regions. While many questions remain, the present report

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ENGELS,W. R., 1989 P elements in Drosophilamelanogaster, pp. 437-487 in Mobile DNA, edited by D. BERGand M. HOWE. American Society for Microbiology, Washington, D.C. ENGELS,W.R., C. R. PRESTON,P. THOMPSON and W.B. EGGLESTON, 1986 In situ hybridization to Drosophila salivary chromosomes with biotinylated DNA probes and alkaline phosphaT h e authors gratefully acknowledgethe help and adviceof tase. Focus 8: 6-8. WELCOME BENDER, DAN CURTIS,STEVEDANIELS and MARK PEIFER. FEINBERG,A. P.,and B. VOGELSTEIN,1983 A techniquefor We also thank those who provided stocks and libraries. Anonymous radiolabeling DNA restriction endonuclease fragments tohigh reviewers provided helpfulsuggestions. Someof thiswork was specific activity. Anal. Biochem. 132: 6-13 completed in partialfulfillment ofrequirementsforthePh.D. FISCHER, S. G . ,and L. S.LERMAN, 1983DNA fragments differing degreeatthe University ofConnecticut by S.D.C andatthe by single base-pair substitutions are separated in denaturing University of California, San Franciscofor A.L.K. T h e investigation gradient gels: Correspondence with melting theory. Proc. Natl. was supported by research grants GM09886 from the U.S. Public Acad. Sci. USA 8 0 1579- 1583. Health Service to A.C and CA44338 to J. M. BISHOP. FRISCHAUF,A. M., H.LERACH, A. POUSTKAand N. MURRAY, 1983Lambdareplacement vectors carrying polylinker seLITERATURE CITED quences. J. Mol. Biol. 170: 827-842. E. A,, J. W. MAHAFFEY,B. J. BONDand N. DAVIDSON, FYRBERG, BELLIDO,A., and J. R. MERRIAM, 1971 Parameters of the wing 1983 Transcripts of the six Drosophila actin genes accumulate imaginal disc development of Drosophilamelanogaster. Dev. in a stage- and tissue-specific manner. Cell 33: 1 15-1 23. Biol. 24: 61-87. BENDER,W., P. SPIERERand D. S. HOGNFSS, 1983 Chromosomal GARCIAALONSO,L. A,,and A. GARCIA-BELLIDO, 1988 Extramacrochaetae, a trans-acting gene of the achaete-scute complex walking and jumping to isolate DNA from the Ace and rosy of Drosophila involved in cell communication. Wilhelm Roux’s loci and the bithorax complex in Drosophilamelanogaster. J. Mol. Biol. 168: 17-33. Arch. Dev. Biol. 197: 328-338. BENTON,W. D., and R. W. DAVIS,1977 Screening Xgt recombiP. K . , C . SPANA and V. G. CORCES,1986 On the molecular GEYER, nant clones by hybridization to single plaques in situ. Science mechanism of gypsy-induced mutations at the yellow locus of 196: 180-182. Drosophila melanogaster. EMBO J. 5: 2657-2662. BINGHAM, P. M., R. LEVISand G. M. RUBIN,1981 T h e cloning GRAY,M., A. CHARPENTIER, K. WALSH,P. Wu and W. BENDER, ofthe DNA sequences fromthe white locus of Drosophila 1991 Mapping of point mutations in the Drosophila rosy locu3 melanogaster using a novel and general method. Cell 25: 693using denaturing gradient gel blots. Genetics 127: 139-149. 704. GRUNEBERG,H.,1929 Ein Beitrag zur Kenntnis derRontgenH . C., and J. DOLY,1979 A rapid alkaline extraction BIRNBOIM, mutationen des X-Chromosoms von Drosoph.ila melanogaster. procedure for screening recombinant plasmid DNA. Nucleic Biol. Zentralbl. 49: 680-694. Acids Res. 7: 1513-1523. HARTENSTEIN, V., andJ. W. POSAKONY, 1989 Development of J. JACK, L. Y. JAN and Y . N. JAN, BLOCHLINGER, K., R. BODMER, adult sensilla on thewing and notumof Drosophila melanogaster. 1988Primarystructureand expression of a productfrom Development 107: 389-405. cut, a locus involved in specifying sensory organ identity in HALL,L.M. C., P. J. MASONand P. SPIERER, 1983 Transcripts, Drosophila. Nature 333: 629-635. genes and bands in 3 15,000 Base-pairs of Drosophila DNA. J. BROWN,N. M., and F. C. KAFATOS,1988 Functional cDNA liMol. Biol. 1 6 9 83-96. braries from Drosophila embryos. J. Mol. Biol. 203: 425-437. HANAHAN, D., 1983 Studies on transformation of Escherischia coli CAMPOS-ORTEGA, J. A., and E. KNUST,1990 Molecular analysis with plasmids. J. Mol. Biol. 166: 557-580. of a cellular decision during embryonic development of DroHOHN,B., 1979 I n vitro packaging of lambda and cosmid DNA. sophila melanogaster: epidermogenesis or neurogenesis. Eur. J. Methods Enzymol. 6 8 299-309. Biochem. 1 9 0 1-10, IVES,P. T . , 1961 New mutants. Drosophila Inform. Serv. 35: 46. COTE, B., W. BENDER,D. CURTIS and A. CHOVNICK,1986 JAMES, A. A., and P. J. BRYANT, 1981 Mutations causing pattern Molecular mapping of therosy locus in Drosophila melanogaster. deficiencies and duplications in the imaginal wing disk of DroGenetics 112: 769-783. sophila melanogaster. Dev. Biol. 85: 39-54. I.., and E. ROY, 1980 New mutants. Drosophila InCRAYMER, KARESS,R. E., and G. M. RUBIN, 1984 Analysis of P transposable form. Serv. 55: 200-204. elements in Drosophila. Cell 38: 135-1415, andW. BENDER,1989