Copyright 0 1986 by the Genetics Society of Anierica

MOLECULAR MAPPING OF THE ROSY LOCUS IN DROSOPHILA MELMOGASTER

BABETTE COTE,**’ WELCOME BENDER,*.* DANIEL CURTIS* AND ARTHUR CHOVNICKt

*Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02 115, and ?Department of Molecular and Cell Biology, The University of Connecticut,

Storrs, Connecticut 06268

Manuscript received August 30, 1985 Revised copy accepted December 9, 1985

ABSTRACT T h e DNA from the chromosomal region of the Drosophila rosy locus has been

examined in 83 rosy mutant strains. Several spontaneous and radiation-induced alleles were associated with insertions and deletions, respectively. T h e lesions are clustered in a 4-kb region. Some of the alleles identified on the DNA map have been located on the genetic map by fine-structure recombination experiments. T h e genetic and molecular maps are collinear, and the alignment identifies the DNA location of the rosy control region. A rosy RNA of 4.5 kb has been iden- tified; its 5’ end lies in or near the control region.

HE cytogenetics of Drosophila has made it possible to isolate the DNA T for genes that have gene products that are rare or unidentified. The first task in the molecular analysis of a cloned locus is to define the DNA changes responsible for various mutations. Such mutation mapping serves to define the size of the locus, and it sometimes indicates the positions of such features as introns and protein coding regions. Some mutations in Drosophila have peculiar properties, such as variegation, instability or distortion of the recombination map. The DNA changes in such mutations must be defined to understand the molecular reasons for these properties.

The rosy locus in Drosophila codes for the enzyme xanthine dehydrogenase (XDH). Rosy mutants which totally inactivate XDH are unable to synthesize the red drosopterin eye pigments and, thus, have brownish eye color. There are presently hundreds of rosy mutations, including spontaneous, radiation- induced and chemically induced alleles. Many rosy alleles have been mapped relative to each other, using the purine selective scheme for wild-type recom- binants (CHOVNICK, BALLANTYNE and HOLM 197 1). Sites responsible for elec- trophoretic variation of the XDH enzyme have been positioned on the fine structure map (GELBART, MCCARRON and CHOVNICK 1976). These sites, and the sites of other mutations which make an altered XDH peptide, define, on the genetic map, the XDH coding region or “structural element.” Wild-type

’ Deceased. ’ To whom reprint requests should be addressed.

Genetics 112: 769-789 April, 1986.

770 B. COTk E T AL.

strains have been found that make different levels of XDH. Two sites respon- sible for overproduction or underproduction of the XDH enzyme have been mapped to the left of the structural element, in what has been called the “control element” (CHOVNICK et al. 1976; MCCARRON et aE. 1979; CLARK et al. 1984). These control sites are the best defined cis-acting regulatory sites in Drosophila; they are of particular interest in a molecular analysis.

The chromosomal region including the rosy locus has been isolated as part of a chromosomal walk in the 87DE region of chromosome 3R (BENDER, SPIERER and HOGNESS 1983; SPIERER et al. 1983). The mapping of deletion breakpoints flanking the rosy region served to locate the locus within about 30 kb. We report here the mapping of DNA lesions in a number of rosy mutants. This allows us to match the DNA restriction map with the genetic fine struc- ture map and to identify the rosy RNA product.

MATERIALS AND METHODS

Mutant strains: The rosy mutations used are listed in Table 1. All mutants were obtained from the Storrs collection, with the exception of r~’~?‘ , which was obtained from M. M. GREEN.

DNA probes: Recombinant phage containing DNA from the Canton-S wild type were collected in a chromosomal walk covering the rosy region (BENDER, SPIERER and HOC- NESS 1983). The 8.1-kb Sal1 fragment and the 4.6-kb EcoRI fragment from the rosy locus (see Figure 3) were subcloned into the plasmid vector pBR322. The probes for blots or plaque screening were made by nick translation (RICBY et al. 1977) of whole phage or plasmid DNA. Labeled DNA was added to a probe mix containing 50% formamide, as described by MANIATIS, FRITSCH and SAMBROOK ( 1 982), and hybridiza- tions were performed at 42”.

Strand-specific probes for RNA blots were made using rosy DNA fragments cloned into the M 13 phage vector MP11. The 17-base sequencing primer (New England Bio- labs) was annealed to single-stranded DNA from the M13 recombinants, and it was extended with the Klenow fragment of DNA polymerase I (New England Biolabs) in the presence of labeled deoxynucleotide triphosphates. The reaction mix was heated to melt the labeled extension product from the M13 template, and the mix was separated on a 1% agarose gel. The gel region containing the separated product was melted at 100” and was mixed directly with formamide and salt solutions to generate a probe solution as above.

Blots of genomic DNA: DNA was extracted from adult flies by a rapid homogeni- zation and ethanol precipitation procedure, as described in BENDER, SPIERER and Hoc- NESS (1 983), except that diethylpyrocarbonate was omitted from the homogenization solution. DNA aliquots of about 3 fig were digested with appropriate restriction en- zymes, and the digested fragments were separated on 0.8% agarose gels. The gels were treated with short wavelength UV illumination or with dilute acid to nick the DNA, and the gels were base denatured, neutralized and transferred to nitrocellulose paper (MANIATIS, FRITSCH and SAMBROOK 1982). DNA fragments from wild-type lambda phage cut with Hind111 were run in parallel as size standards.

Blots of poly(A) RNA: Whole larvae were homogenized in the presence of 5 M guanidinium isothiocyanate (MANIATIS, FRITSCH and SAMBROOK 1982), cesium chloride was added and the mixture was spun at 110,000 X g for 24-48 hr. The gradients were fractionated, and the RNA band was located by running aliquots of the fractions on an agarose gel. The peak fractions were pooled, and the RNA was precipitated with ethanol. Poly(A)+ RNA was selected by passing the total RNA over oligo(dT) cellulose (type 3, Collaborative Research) (MANIATIS, FRITSCH and SAMBROOK 1982). RNA was size fractionated on agarose gels in the presence of 5 mM methylmercury hydroxide

DNA CHANGES IN ROSY MUTANTS 77 1

and was blotted to diazobenzyloxymethyl (DBM) paper. Alternatively, the RNA was fractionated on formaldehyde agarose gels and was transferred to nitrocellulose. Both procedures are described in MANIATIS, FRITSCH and SAMBROOK (1982).

Construction of genomic libraries: DNA from mutant flies, prepared as above, was partially digested with EcoRI, BamHI or SalI, and the fragments were separated by size on sucrose gradients. Fragments of 15-25 kb were selected and ligated into the lambda vectors Sep 6 (MANIATIS, FRITSCH and SAMBROOK 1982) cut with EcoR1, EMBL4 (FRIS- CHAUF et al. 1983) cut with BamHI, or BFlOl (NORMA NEFF, unpublished results) cut with M I . The ligation mixtures were packaged in vitro (HOHN 1979), plated and screened by the method of BENTON and DAVIS (1977). Recombinant phage from the mutant libraries were compared with phage from the Canton-S walk by restriction mapping and by electron microscopy of heteroduplex molecules (DAVIS, SIMON and DAVIDSON 197 1).

RESULTS

Molecular mapping of landmarks near rosy: DNA segments spanning the rosy region were isolated as part of a chromosomal walk in the 87D-E region, as reported (BENDER, SPIERER and HOGNESS 1983). The region of the chro- mosomal walk was saturated for lethal complementation groups, and the com- plementation groups were ordered using a series of overlapping deficiencies (HILLIKER et al. 1980). The deficiency endpoints were defined on the DNA of the walk, so that the complementation groups were roughly localized on the DNA map (SPIERER et al. 1983). A restriction map of the 60-kb region around the rosy locus is shown in Figure 1, with several rearrangement breakpoints marked.

The rosy locus is flanked by the complementation groups 1S12, on the left, and pic , on the right. The IS12 and rosy loci have not been separated by a deficiency, but their relative order is established by recombinational mapping (CHOVNICK et al. 1976). These two loci are bounded on the left by the endpoint of Df(3R)lG2’ (which removes complementation groups to the left of 1S12), and on the right by Df(3R)126d (which removes complementation groups to the right of rosy). D f ( 3 R ) r ~ ~ ~ removes lS12 and rosy, but not pic . These deficiencies limit the lSl2-rosy region to about 30 kb.

The pic locus is further defined by three rearrangements associated with lethal pic mutations. A revertant of Deformed, Dfd+RX16, is pic- , and it is asso- ciated with a transposition of the 87A-D region into the Deformed locus at 84AB (HAZELRIGG and KAUFMAN 1983). The fusion fragment at the 87D/ 84AB junction was isolated and mapped (SCOTT et al . 1983); the breakpoint is within the 0.8-kb EcoRI fragment at -152 kb (Figure 1). Another pic- rear- rangement, ryp” 14’, fuses 87D with the fourth chromosome (RUSHLOW and CHOVNICK 1984). This fusion fragment has also been recloned, and the break- point also falls within the 0.8-kb EcoRI fragment at -152 kb (RUSHLOW, BENDER and CHOVNICK 1984). A third pic- allele, Cbx Ubx2’g88B, isolated as a revertant of Cbx (BENDER et al. 1983), has an inversion from 87D to 89E. Its breakpoint was mapped by Southern blots to the 4.2-kb EcoRI fragment at -148 to -152. A single breakpoint defines the lS12 locus, ryps11136, associated with an inversion to third chromosome heterochromatin (RUSHLOW and CHOV- NICK 1984). This fusion fragment has also been recloned, and the breakpoint

772 B. COTi E T AL.

TABLE 1

List of rosy alleles examined by genomic blotting

ry allele no. Backnround Mutagen Reference Lesion

1

2

4 5 6 7

8 9

I7 20 21 23 24 26 40 41 48 54

56 57 58 59 60

62 64

7

77316

I 02 1 03 1 05 1 06

110 20 I 203 204 205 206 20 7 208 209 210

Unknown

Unknown Unknown

+O +O +O +O

+O +O

Unknown Unknown Unknown

+O +O +O +O +O +O +O

Unknown Unknown Unknown Unknown Unknown

Unknown Unknown

Unknown

+1 +1 +1 +1

+1 +2 +2 +2 +2 +2 +2 +2 +2 +2

Spon.

Spon. Spon. X-ray X-ray X-ray X-ray

X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray

X-ray X-ray X-ray X-ray X-ray

X-ray X-ray

HD

Gamma Gamma Gamma Gamma

Gamma Gamma Gamma Gamma Gamma EMS EMS EMS EMS EMS

1

1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1

1 1

2

3 3 3 3

3 4 4 4 4 5 5 5 5 5

Deletion of approximately 0.1 kb, including

Insertion of B104 element at +3.2 kb Insertion of B104 element at +2.2 kb Apparent point Apparent point (missing SstI site at -1.0 kb) Apparent point Deletion of approximately 0.6 kb between

Apparent point Apparent point Apparent point Apparent point Apparent point Apparent point Apparent point Apparent point Apparent point Apparent point Apparent point Inversion to 3R heterochromatin, break be-

Apparent point Apparent point Apparent point Apparent point Deletion of 1.1 kb between +0.95 and +2.6

Apparent point, missing SstI site at +0.5 kb Inversion to 64E, break between +1.5 and

3.6 kb with deletion including +1.9 to +3.0 kb

Insertion of 5 kb (copia?) between +1.9 and +2.6 kb

Apparent point Apparent point Apparent point Insertion of at least 5 kb between +1.5 and

Apparent point Apparent point Apparent point Apparent point Apparent point Apparent point Apparent point Apparent point Apparent point Apparent point

SstI site at +0.95 kb

+2.9 and +4.2 kb

tween +1.9 and +2.6 kb

kb

+2.6 kb

DNA CHANGES IN ROSY MUTANTS

TABLE l-Contind

773

ry allele no. Background Mutagen Reference Lesion

211 213 214 218 219 220 222 223 224 225 226 227 228 30 1 402 405 407 50 1 502 503 506

601 602 604 605

606 607 608 609 610 611 612 613

1002 1003 1009

1012 1202 1302 1401 1901 2101

+2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +3 +4 +4 +4 +5 +5 +5 +5

+6 +6 +6 +6

+6 +6 +6 +6 +6 +6 +6 +6

+10 +10 +10

+10 +12 +13 +14 +19 +20

EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS Gamma EMS EMS Gamma Gamma Gamma Gamma

EMS EMS EMS EMS

EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS X-ray

X-ray Gamma Gamma EMS Spon. HD

5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 4 4 4 4

5 5 5 5

5 5 5 5 5 5 5 5 5 5

Apparent point, leaky allele Apparent point, leaky allele Apparent point, leaky allele Apparent point, leaky allele Apparent point, leaky allele Apparent point Apparent point, leaky allele Apparent point, leaky allele Apparent point, leaky allele Apparent point, leaky allele Apparent point, leaky allele Apparent point, leaky allele Apparent point, leaky allele Insertion of 7.6 kb at +0.5 Apparent point Apparent point Apparent point, leaky allele Apparent point Apparent point Apparent point Deletion of 3.4 kb between + 1 . 1 and +5.0

Apparent point Apparent point Apparent point Apparent point, new SstI site at +1.7 or +4.1

Apparent point Apparent point Apparent point Apparent point Apparent point, leaky allele Apparent point, leaky allele Apparent point, leaky allele Apparent point, leaky allele Apparent point Apparent point Deletion of 1.0 kb between +0.95 and +2.6

Apparent point Apparent point Apparent point Apparent point Apparent point Insertion of cobia at +0.3 kb

kb

kb

kb

The background chromosomes designated q + O through ry+*O are homozygous third chromo- somes in the Storrs collection. EMS, treatment with ethyl methanesulfonate; Spon., spontaneous mutant; HD, hybrid dysgenesis (PXM). The references are ( 1 ) LINDSLEY and GRELL 1968; (2) M. GREEN, personal communication; (3) MCCARRON, GELBART and CHOVNICK 1974; (4) GELBART et al. 1974; (5) GELBART, MCCARRON and CHOVNICK, 1976.

774

I

B. COT6 E T AL.

I 1 I I I I I

4 ,,, .\\\\\ -b 4 Df r yX W I 26d W """"" D /

HSC

ps1149 s-sal I ps11136 +Rx16 +I

CbxUbx2198813

t B - m H I H - Hi, d 1 1 1 R - - R I

(I s12 - ) (pic- 1

FIGURE 1.-Restriction map of the rosy region. The thin horizontal line represents the DNA map of the Canton3 wild type; it is marked in kilobase coordinates according to the map of ENDER, SPIERER and HOGNESS (1983). The thicker horizontal bars represent DNA regions re- moved in various deficiencies; the striped extensions of these bars represent the limits of uncer- tainty of the deficiency endpoints. Vertical arrows mark rearrangement breakpoints; the Cbx ujx 2 19888 breakpoint is mapped with the uncertainty shown by the striped horizontal bar. The block labeled HSC marks the location of the heat-shock cognate (CRAIG, ICNOLIA and MANSEAU 1983).

falls between the Hind111 site at -174 kb and the Sal1 site at -174.5 kb (Figures 1 and 3) (RUSHLOW, BENDER and CHOVNICK 1984). Figure 1 also shows, to the left of pic, the position of the heat shock cognate defined by CRAIG, IGNOLIA and MANSEAU ( 1 983).

Molecular mapping of rosy mutations: When the deficiency mapping limited the rosy region to 30 kb, we began to look at rosy mutations in this region. We first ran Southern blots of genomic DNA from spontaneous rosy mutations, since we expected some of these to be caused by insertions of mobile elements. The spontaneous mutations ry2, ry3, ry"y6 and ry2"l did show apparent inser- tions, all within a 4.6-kb EcoRI fragment just to the right of the 1S22 break- point (-1 66.5 to -1 7 1 kb). Genomic libraries were subsequently constructed from three of these mutants, and the rosy region was recovered so that the insertions could be identified. Figure 2 shows the restriction maps of the in- sertions; both ry2 and ry3 have insertions of an element named B104 or roo (SCHERER et al. 1982). The identification was confirmed by electron microscopy of heteroduplex molecules between the rosy mutant clones and a previously isolated B 104 copy (on lambda-bDm 1970, BENDER, SPIERER and HOGNESS 1983). The B104 copy in ry3 has a deletion of about 0.5 kb relative to the canonical copy, and only a fragment of the B104 in ry' was recovered (Figure 2). The ry2'" mutation has an insertion of the copia element, as confirmed by heteroduplexes with lambda-w"5.9, containing the copia insertion of the W"

mutation (BINGHAM, LEVIS and RUBIN 1981). The ry2'"' allele was recovered

DNA CHANGES IN ROSY MUTANTS 775

-3.0 -2.0 -1.0 0 +1.0 +2.0 +3.0 +4.0 +5.0 4 . 0 +7.0 +8.0 I I I I I I I I I I I I

I I 1 I I I I I I I I I 1 I 1 1 1 I 111 1 1 1 1 I I I S H A G G T G E X R E T G T T EBA A HGT ARTS R R E

110 sites in l?gnea lor B. H. R

m

U

no dhg for A. B, T, X S G R

s - S I T - S s t l x - N u l

no 61185 lor B. S kk d

FIGURE 2.-Restriction maps of cloned insertion mutations. T h e horizontal line at the top represents restriction map of the wild-type rosy region. The kb scale is marked in distance from the EcoRI site in the middle of the locus, which is at -171 kb in the map of Figure 1 . T h e DNA to the right of +4.7 kb has not been mapped with all enzymes. T h e four mutant clones are drawn below. Restriction sites are drawn only for the inserted DNA; for each clone, the enzymes used for mapping were only the enzymes with sites marked or noted as having no sites. Only a fragment of the B104 element was recovered from ry2, but the identity of the fragment was verified by heteroduplex mapping, and the total size of the insertion was measured on blots of genomic DNA.

from an experiment that used P-M dysgenesis (KIDWELL, KIDWELL and SVED 1977) to induce rosy mutations (CLARK et al. 1986). The ry2101 allele was mapped relative to other rosy alleles, and from those crosses a rj+ convertant (or spontaneous revertant) was recovered. This convertant looks identical to the background chromosomes used for the dysgenic cross, as if the copia ele- ment was completely removed. Subsequent recombination experiments by CLARK et al. (1986) have shown the same DNA change with three other ryz1O1 convertants, and comparable changes in convertants of the ry3 insertion. They also saw that convertants of two alleles associated with small deletions, ry6’ (see below) and ry5”, were coincident with a recovery of the wild-type DNA map. These changes associated with conversion or reversion prove that the insertions or deletions are at the site of their respective mutations or are within the distance of a conversion interval.

The ry”Y6 mutation was produced by the M R dysgenesis system (GREEN 1977), which is probably equivalent to the P-M system. It also appears to have

776 B. COT)? ET AL.

a copia insertion, as judged by the map predicted from genomic blots. This allele has not yet been recloned.

A fifth mutation, ry301, appeared to have an insertion; it was isolated in a mutagenesis screen using ethyl methanesulfonate (EMS), but since it was the only mutation recovered from a large collection of treated flies, it is quite possibly due to a spontaneous event (GELBART, MCCARRON and CHOVNICK 1976). The ry301 element was also recloned and mapped, as indicated in Figure 2. We found an insertion of 7.2 kb, not obviously homologous to previously described mobile elements. The insertion sequence appears to be repeated 10- 20 times in the wild-type Canton-S genome, as judged by genomic blots and in situ hybridizations. Three homologous copies were recloned from a Canton- S library. The three new copies appeared identical to the ryS0’ copy by electron microscopy of heteroduplex molecules. The new copies had some polymor- phisms in restriction maps; two copies lacked the Sal1 sites at either end of the element, and there were minor length differences in the EcoRI fragments at either end of the element. We have named this element “Calypso.”

The coincidence of the mobile element insertion sites suggested where we should look for lesions in additional rosy alleles. We have examined a large number of alleles, induced with various mutagens, by Southern blotting. We have used as probe a subclone containing the 8.1-kb Sal1 fragment (-166 to -174 in Figure l), or phage clones including this Sal1 fragment. The results are summarized in Table 1, and the lesions we found are mapped in Figure 3. There are two X-ray-induced inversions associated with rosy null mutations; both have breaks in the region defined by the mobile element insertions. Several cytologically normal mutations induced by X-rays or gamma rays have small deletions or insertions, again in the same region.

Genetic mapping of rosy mutations: Several of the alleles with insertions or deletions have been positioned on the genetic fine-structure map in earlier studies (reviewed in HILLIKER and CHOVNICK 1981). These include the inser- tion mutants ry2 and ryIos and the deletion mutants ry’ and ry’. We can now align the genetic and molecular maps, as diagramed in Figure 3. The ry406 mutation, positioned earlier on the genetic map, was found to exhibit 100% co-conversion with a polymorphism for the PvuII site marked by a star in Figure 3. The restriction site and the mutant lesion are therefore assumed to be very close. The IS12 locus can also be roughly positioned by the breakpoint of the PSI I136 inversion. The EcoRI site near the middle of the Sal fragment falls near the left end of the structural element; we define this site as 0.0 kb for designating map coordinates of lesions in Table 1 and Figures 2 and 3. A preliminary version of this mapping suggested that the rosy locus was wholly contained within the 8.1-kb Sal1 fragment of Figure 3, and this prediction was confirmed when this Sal fragment was used in the transformation system of RUBIN and SPRADLING (1982). SPRADLING and RUBIN (1983) have more re- cently used the 7.3-kb Hind111 fragment (-3.2 to +4.1 kb in Figure 3) in transformation experiments. It is also sufficient to rescue completely the rosy mutant phenotype. Rosy RNA: We were confident from the analysis above that most of the

DNA CHANGES IN ROSY MUTANTS 777 Control

Element Structural Element I 1 9

i1005 id097 402 201 8 a6 41 I I 1 I I I I

/ 2 ,O.OOicM ,

* -3 -2 - 1 0 +1 +2 +3 +4 +5 I I I I I I I I I

U BBA AU ffiTAATS R T G BXRBUCTGTP T S HAPG G

I I ! ! ! I

+ In psl1136

FIGURE 3.-Correlation of genetic and molecular maps. The top horizontal line represents the rosy fine structure genetic map; the lower horizontal line shows the DNA restriction map of the 8.1-kb Sal1 fragment (-174 to -166 of Figure 1). The coordinates are as in Figure 2. Below the restriction map are shown the lesions in various rosy mutations. Open triangles indicate mobile element insertions; open horizontal bars indicate deletions of the indicated sizes. The thin lines extending from the ends of each deletion bar represent the uncertainty in the position of the deletion. The ry64 inversion appears to have a net deletion at the break in the rosy locus. The ? I o 6 allele is associated with an apparent insertion at the position shown; the insertion size is unknown. The ry'O6 allele is an apparent point mutation, but it has been shown to co-convert with the starred PvuII site at +0.2 kb. The lines between the genetic and molecular maps show the correlation in positions for the mutations which have been mapped by both methods.

XDH coding sequence is within the 4.6-kb EcoRI fragment (from 0.0 to +4.6 kb in Figure 2). We isolated total RNA from late third instar larvae, a devel- opmental time when XDH enzyme activity is high (CHOVNICK et al. 1977). The RNA was fractionated on oligo(dT) cellulose into poly(A) plus and minus frac- tions; both fractions were size separated on methyl-mercury agarose gels, and the gels were blotted to derivitized paper. The 4.6-kb EcoRI fragment was labeled by nick translation and was used to probe the blot. A single band was labeled in the poly(A)+ lane only (Figure 4). We included as size markers polio viral RNA (7.4 kb), human 28s and 18s ribosomal RNAs (5.3 and 2.1 kb) and bacterial 23s ribosomal RNA (3.27 kb). The rosy RNA ran slightly slower than 2% RNA, but the marker RNAs did not migrate in direct proportion of the log of their molecular weights. We suspect 28s RNA runs anomalously fast, due to secondary structure not completely denatured by methyl mercury. If we rely on the polio and 23s RNAs for calibration, the rosy RNA migrates at approximately 4.5 kb. The XDH monomer peptide is approximately

778 B. CoTk ET AL.

POLY (A) + -

Polio-

20s-

23 S-

18 S-

FIGURE 4.-RNA blot hybridized with a probe from the rosy region. Poly(A)+ and poly(A)- RNA fractions were separated on a methylmercury agarose gel. The gel was blotted to DBM paper, and the paper was hybridized to a nick-translated probe from the 4.6-kb EcoRI fragment (0.0-4.6 kb in Figure 3). Unlabeled RNAs were run in the same gel as standards; their positions are shown. The rosy RNA appears to be about 4.5-kb long by comparison to these standards.

150,000 daltons in molecular mass (EDWARDS, CANDIDO and CHOVNICK 1977), which would require 4.1 kb of RNA coding sequence. An RNA of about 4.5 kb has also been observed by COVINGTON, FLEENOR and DEVLIN (1984), RUSH- LOW, BENDER and CHOVNICK (1984) and CLARK et al. (1984); all studies used the same 4.6-kb EcoRI fragment as probe.

We have also used probes flanking the 4.6-kb fragment against similar blots of larval RNA. A 7.7-kb fragment extending leftward (from the EcoRI site at 0.0, leftward to an artificial EcoRI site in the phage 2844 (BENDER, SPIERER and HOCNESS 1983)) labels two bands in the poly(A)+ lane. One faint band comigrates with the 4.5-kb RNA described above, and the other heavy band runs at about 3.3 kb (not shown). To the right, we have not checked the 0.6- kb EcoRI fragment, but we have used as probe the next 2.4-kb EcoRI fragment (-165.7 to -163.3 kb in Figure 1). It labels strongly one poly(A)+ band at about 1.5 kb (not shown). Thus, the 4.5-kb rosy RNA has some small homology to sequences left of 0.0 kb in Figure 3. We do not see any homology to the right of the EcoRI site, at position +5.2 kb, in the coordinates of Figures 2 and 3.

DNA CHANGES IN ROSY MUTANTS 779

T o determine the orientation of the rosy RNA, the RNA blots were repeated with strand-specific probes. A fragment mapping between +1.0 and +3.0 kb (Figure 3) was cloned into an M13 vector in both orientations. Complementary DNA was synthesized off the two single-stranded templates, using a sequencing primer and the Klenow fragment of DNA polymerase I. The probe copied from right to left (5 ’ to 3’) on the DNA map hybridized to the 4.5-kb RNA; the other probe gave no signal (data not shown). Thus, the rosy RNA is transcribed from left to right (5 ’ to 3’) on the map of Figure 3.

DISCUSSION

Location of rosy: Chromosome walking (BENDER, SPIERER and HOGNESS 1983) and chromosome microdissection (SCHALENGHE et al. 198 1) have been used to recover the DNA of many loci in Drosophila, but the identification of the desired gene within the collected DNA has frequently been difficult. The most efficient strategy is to locate rearrangement breakpoints associated with mutations in the gene; in situ hybridizations can be used to locate the hreak- point within the DNA walk. We have only one euchromatic rearrangement associated with a rosy mutant, ( ~ y ~ ~ ) , and the cytological change associated with this mutation was unknown when we first looked for rosy. We were fortunate to have the extensive series of deficiencies in the rosy region which separate the various complementation groups (HILLIKER et al. 1980). The molecular mapping of these deficiency endpoints limited the DNA region potentially coding for rosy to about 30 kb.

When the potential DNA region is small, the obvious strategy is to look for DNA changes in different mutations. This task was simplified for rosy because so many mutations are available. Most of the rosy mutations are induced on defined background chromosomes; therefore, our search for lesions was not often confused by polymorphism for restriction sites or mobile element inser- tions. The lesions of 13 rosy mutations are indeed clustered to a region of about 4 kb, which is the expected minimum size of the rosy locus. Formal proof of the DNA localization is based on the analysis of gene conversion events of rosy alleles associated with DNA changes. The localization was con- firmed by the successful transformation of rosy mutant flies with this DNA.

Correlation of genetic and molecular maps: The alignment of the genetic and molecular maps is surprisingly good, given the small recombination dis- tances involved and the possibilities for abnormal recombination near large insertions or deletions. The effects of such DNA changes on recombination are considered in more detail elsewhere (CLARK et al. 1986).

The alignment of the genetic map predicts that the rosy protein coding region spans about 4 kb (-0.5 to +3.5 kb in Figure 3). A DNA region of 4 kb would be required to code for the XDH monomer of 150,000 daltons. We must be cautious about this correlation; modification of the XDH protein could cause anomalous migration on SDS polyacrylamide gels, and the mutant sites at the ends of the structural region could be far from the ends of the XDH protein. But in any case, the XDH coding region does not appear to be interrupted by large or numerous introns. The 7.3-kb Hind111 fragment (-3.2

780 B. COT&, et al.

to +4.1 kb in Figure 3) seems to contain the entire locus in transformation experiments; therefore, the locus seems fairly compact. There is, at most, only about 3 kb for introns, nontranslated transcript and adjacent control regions.

The correlation of genetic and molecular maps also locates the control re- gion of the rosy locus. Allelic differences mapping to the control region affect the level of XDH mRNA (COVINGTON, FLEENOR and DEVLIN 1984; CLARK et al. 1984) and, in one case, affect the tissue distribution of the mRNA (CLARK et al. 1984). Mutations which lower the amount of XDH protein have recently been induced in the control region (M. MCCARRON, C. LOVE and A. CHOV- NICK, unpublished results). The mRNA for XDH has been oriented and roughly positioned by the blots described here, and the control region is at the 5' end of the mRNA. Thus, we expect the control region differences to affect the initiation of transcription. DNA sequencing studies are under way to define the base changes in the control variants and mutants.

Mutant lesions: The catalog of mutations in Table 1 provides an unusually large database for the molecular effects of various mutagenic procedures in Drosophila.

Spontaneous mutations in rosy are usually associated with insertions of mobile repetitive elements (4 of 6 mutations). The same conclusion can be drawn from studies of spontaneous mutations at several other Drosophila loci, includ- ing scute (CAMPUZANO et al. 1985), white (ZACHAR and BINGHAM 1982), Notch (KIDD, LOCKETT and YOUNG 1983), Antennupedia (SCOTT et al. 1983) and bithorax (BENDER et al. 1983). Two of the five mobile element insertions in rosy are copies of the B104 element; this element seems particularly frequent in spontaneous mutations at other loci. Two of the spontaneous rosy alleles were produced by hybrid dysgenesis; both appear to be insertions of the copia element. The P-M or MR class of hybrid dysgenesis is most frequently associ- ated with movement of the P element, but dysgenesis-induced copia insertions at the white locus have also been observed (RUBIN, KIDWELL and BINCHAM 1982).

Radiation-induced alleles are often apparent point mutations; only 7 of 4 1 alleles examined had rearrangements or small deletions. (Our genomic blotting procedures would overlook deletions or insertions of less than about 100 base pairs.) The frequency of radiation-induced deletions is underestimated by this tabulation, since larger deletions that affect adjacent complementation groups were not included in our survey. In the initial group of 75 X-ray-induced rosy alleles (LINDSLEY and GRELL 1968), 22 were initially identified as cytological rearrangements or large deficiencies. Assuming that about 30% of radiation- induced alleles are such large aberrations and that about 17% of the remainder have deficiencies or rearrangements detectable on blots, then about 58% of the total number are apparent points.

We examined 36 EMS-induced alleles, and only one had an obvious change on our blots. This allele, ry301, is probably a spontaneous event, as explained above. Thus, most EMS-induced rosy alleles, if not all, are apparent point mutations.

We have examined a comparable number of mutations in the bithorax com-

DNA CHANGES IN ROSY MUTANTS 78 1

plex (BENDER et al. 1983; KARCH et al. 1985). These results have not been so carefully tabulated, but the frequencies of different classes of mutant lesions are clearly different. Almost all of the radiation-induced alleles are deletions or rearrangements, and for some of the phenotypic classes, most of the EMS- induced alleles are also deletions or rearrangements. The lack of apparent point mutations may reflect a target size effect; the bithorax complex is spread over 300 kb, but perhaps only a small fraction of the DNA in the complex is protein coding. It is not clear which locus should be considered more repre- sentative for studies of mutagenesis. The average lethal complementation groups are spaced at intervals of about 40 kb (JUDD, SHEN and KAUFMAN 1972; HILLIKER et al. 1980). If this represents the average size of a comple- mentation group, then the rosy and bithorax loci represent examples on the small and large extremes. But since the rosy locus is mostly coding region, it should be sensitive to most DNA changes and, thus, should better reveal the sequence changes of mutagenic protocols.

We have received help in identification and mapping of several rosy mutations from STEVEN CLARK and MARGARET MCCARRON. We are grateful to KAREN WEPSIC for careful maintenance of rosy stocks. This work was supported by grants from the National institutes of Health to W.B. and A.C.

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