Dominant Defects in Drosophila Eye Pigmentation Resulting From … · 1998. 11. 19. · Dominant Defects in Drosophila Eye Pigmentation Resulting From a Euchromatin-Heterochromatin

Copyright 1998 by the Genetics Society of America

Dominant Defects in Drosophila Eye Pigmentation Resulting From aEuchromatin-Heterochromatin Fusion Gene

Yikang S. Rong and Kent G. Golic

Department of Biology, University of Utah, Salt Lake City, Utah 84112

Manuscript received April 14, 1998Accepted for publication September 14, 1998

ABSTRACTWe have isolated a dominant mutation, pugilist Dominant (pug D), that causes variegated reductions in pteridine

and ommochrome pigmentation of the Drosophila eye. The effect of pug D on pteridine pigmentation ismost dramatic: the only remaining pigment consists of a thin ring of pigment around the periphery ofthe eye with a few scattered spots in the center. The pug D mutation disrupts a gene that encodes aDrosophila homolog of the trifunctional enzyme methylenetetrahydrofolate dehydrogenase (MTHFD;E.C.1.5.1.5, E.C.3.5.4.9, E.C.6.3.4.3). This enzyme produces a cofactor that is utilized in purine biosynthesis.Because pteridines are derived from GTP, the pigment defect may result from an impairment in theproduction of purines. The mutant allele consists of a portion of the MTHFD coding region fused to z1kb of highly repetitive DNA. Transcription and translation of both parts are required for the phenotype.The repetitive DNA consists of z140 nearly perfect repeats of the sequence AGAGAGA, a significantcomponent of centric heterochromatin. The unusual nature of the protein produced by this gene maybe responsible for its dominance. The repetitive DNA may also account for the variegated aspect ofthe phenotype. It may promote occasional association of the pug D locus with centric heterochromatin,accompanied by inactivation of pug D, in a manner similar to the proposed mode of action for brown Dominant.

THE discovery of the white-eyed mutant by Morgan the synthesis of pteridines (for examples and discussionssee Nash and Henderson 1982; Johnstone et al. 1985;marked the advent of Drosophila as a genetic model

organism. Since then, dozens of eye pigment mutants Henikoff et al. 1986). However, these mutations aretypically not cell lethal. If cells can externally acquirehave been isolated in Drosophila melanogaster (Lindsley

and Zimm 1992). The existence of so many easily recog- the nucleotides needed for viability, then it is not clearwhy they cannot acquire the nucleotides needed fornized variants in eye color fostered the development

of biochemical genetics (Beadle and Ephrussi 1935, normal pigment synthesis. Finally, many mutations thatprimarily affect one pigment can also alter levels of1936) and significantly furthered the analysis of pigment

biochemistry (for reviews see Phillips and Forrest pigment in the other family, even though they appearto be synthesized by independent pathways. Thus, the1980; Summers et al. 1982; Kayser 1985).interrelations of pigment biosynthesis remain somewhatThe dull red color of a wild-type Drosophila eye resultsmysterious.from the combination of two families of pigment mole-

The eye of Drosophila has also proven to be an excel-cules: the pteridines, which are bright red in color, andlent system for developmental biology, especially forthe ommochromes, which are brown in color. Thesestudies of cell differentiation and cell-cell communica-pigments are deposited in membrane-bounded, pro-tions. An eye consists of z800 identical repeated struc-tein-containing pigment granules within the pigmenttures called ommatidia, which can amplify develop-cells of the eye and, to a lesser extent, the photoreceptormental defects in an ommatidium several hundredfold.cells (Shoup 1966; Stark and Sapp 1988). PteridinesMost studies of eye development have focused on theare synthesized from the precursor GTP, and ommo-construction of the ommatidia, which make up the ma-chromes are synthesized from tryptophan. However, ourjority of the eye. Much less attention has been devoted toknowledge of pigment synthesis and deposition is in-uncovering the unique developmental features definingcomplete. We do not know the complete reactionthe periphery of the eye (for examples see Wolff andsequences of the pigment pathways or the chemicalReady 1991; Hay et al. 1994). One would expect thatcomponents of the pigment granules. A number of mu-a gene differentially affecting the periphery and thetations affecting purine metabolism cause a reduction inmiddle of the eye would assume a ring pattern of expres-sion. A limited number of mutations that result in aring pattern of eye pigmentation have been identified.

Corresponding author: Kent Golic, Department of Biology, 201 Biologywhite halo is an allele of white that produces an eye withBldg., University of Utah, Salt Lake City, UT 84112.

E-mail: [email protected] a normally pigmented peripheral ring surrounding a

Genetics 150: 1551–1566 (December 1998)

1552 Y. S. Rong and K. G. Golic

Drosophila stock center at Bloomington, Indiana. Df(3R)mbc-lightly pigmented center (Judd 1975; Peterson et al.R1/TM3, Sb flies (Df(3R)mbc-R1 5 Df(3R)95A5-7;95D6-11)1994). The mutations burgundy (a purine auxotroph),were kindly provided by E. Rushton, University of Utah (Rush-

doughnut, lozenge spectacle, and some white transgene inser- ton et al. 1995). Flies with the genotype se Ly dn/LVM weretions show ring patterns or partial rings (arcs) of pig- provided by the Drosophila stock center at Bowling Green,

Ohio.mentation (D. Nash, personal communication; Pat-The original inversion of pug D is associated with the homozy-terson and Muller 1930; Wright 1946; Johnstone

gous lethal mutation, Stubble (Sb). To make homozygous pug Det al. 1985; Rubin et al. 1985; Delattre et al. 1995; Gubb

flies, we screened for double recombinants within the inver-et al. 1997). The cause of the ring patterns has not been sion that crossed Sb off the inversion. Virgin females with theidentified in any of these cases. genotype v/Basc; 1/S 2Cyo; pug D Sb/1 were mated to v; ry

males, with 15–20 of both males and females in a bottle.We have isolated a dominant mutation, pugilist Dominant

They were transferred to new bottles at 5-day intervals. Out(pugD), that differentially affects pigmentation betweenof z30,000 F1 progeny screened, 2 males with the genotypethe margin and the middle of the eye. The pugD muta-v; 1/S 2Cyo; pug D/ry were obtained. pug D homozygous stocks

tion reduces ommochromes and virtually eliminates were made from these 2 males. Only one stock was used forpteridines in the middle of the eye, while preserving future experiments.

Screening for pug D revertants: X-ray mutagenesis was carriednormal pigmentation at the eye margin. Thus, whenout in a Torrex 120D X-ray machine. N-ethyl-N-nitrosoureaommochrome synthesis is blocked by the vermilion or(ENU) mutagenesis was performed as described for EMS bycinnabar mutations, a pugD/1 eye shows a striking ringGrigliatti (1986), except that the neutralization solution

of red pigment around the eye margin. The name pugi- for ENU was 1 m NaOH. Males of the genotype v; pug D culist was inspired by the similarity to a boxer’s black eye. kar 2 Sb/TM3, or v; pug D cu kar 2 were aged for 3–5 days. They

were then either irradiated with 4000 rads or fed a sugarThere is also a variegated aspect of the phenotype: thewater/ENU solution overnight. The mutagenized males werereduction in ommochromes is highly variable, and acrossed to v; ry or C(1)DX, y f/Y female virgins, with 15–20small and variable number of cells in the center of themales and 30–40 females in a bottle. After 5 days, the males

eye show pteridine pigmentation. were discarded and the females transferred to new bottles forThe mutation that causes pugD lies within a gene that further collection. F1 progenies were identified as potential

encodes the trifunctional enzyme, NADP-dependent revertants if they carried the pug D chromosome 3 and showeduniform eye pigmentation.methylenetetrahydrofolate dehydrogenase-methenyl-

Cytology of polytene chromosomes: Salivary gland polytenetetrahydrofolate cyclohydrolase-formyltetrahydrofolatechromosomes were prepared as described by Lefevre (1976).synthetase. This enzyme is referred to as MTHFD, or For determining the breakpoints of the pug D inversion,

C1-THF synthase. We use the former designation pug D/1 larvae were used. For cytology of pug D revertants,throughout this article. MTHFD catalyzes interconver- chromosomes were from revertant/1 larvae. For in situ hybrid-

ization, chromosomes from larvae homozygous for pug D weresion of three derivatives of tetrahydrofolate to provideused. The chromosomes were prepared as described bycofactors for de novo purine biosynthesis (for reviewsPardue (1986). Hybridization and detection were performedsee Benkovic 1980; Jones and Fink 1982; Henikoff using the GENIUS system from Boehringer Mannheim (India-

1987; Appling 1991). This enzyme is encoded by ADE3 napolis). Chromosomes were examined with brightfield andin the yeast Saccharomyces cerevisiae, and mutations in phase-contrast optics.

Southern blot analyses and colony hybridization screens:that gene give rise to purine auxotrophy (Jones 1977).Fly DNA was purified as described by Golic and LindquistIn pugD, a 1-kb piece of highly repetitive DNA is fused(1989). Southern analyses and colony screens were performedto a portion of the coding region of MTHFD. The repeti-as described by Maniatis et al. (1982). Nylon membranes

tive segment consists of z140 iterations of the short were from Boehringer Mannheim. Hybridization probes weresequence AGAGAGA. This sequence is found in high made and hybridization was detected using the GENIUS sys-copy number in centric heterochromatin and Y chromo- tem and CSPD (Boehringer Mannheim) as a chemilumines-

cence substrate. A subclone that spanned one of the two inver-some heterochromatin in D. melanogaster (Bonaccorsision breakpoints was identified by the fact that the sizes ofand Lohe 1991; Lohe et al. 1993; for reviews see Gattithe restriction fragments to which it hybridized were differentand Pimpinelli 1992; Lohe and Hilliker 1995). These in pug 1 and pug D DNA (data not shown).

repeats are at least partly responsible for the dominant Cloning and sequencing: Standard plasmid DNA manipula-pugD phenotype. They may also be the cause of the tions were performed as described by Maniatis et al. (1982).

DNA of P1 genomic clones was prepared by using the QIAGENvariegated aspect of the pugD phenotype. The repetitivePlasmid Midi Kit (QIAGEN Inc., Chatsworth, CA) accordingDNA may be responsible for position-effect variegationto the supplied protocol. P1 clones DS01137 and DS02445(Spofford 1976) that occasionally inactivates the domi- were subcloned into the cloning vector pBluescript II KS (1)

nant mutation. (Bluescript) from Stratagene (La Jolla, CA). For the sequenceof primers used in PCR analyses and sequencing, see Table1. For the approximate locations of these primers on therestriction map, see Figure 1.MATERIALS AND METHODS

The 5-kb HindIII fragment containing the pug D breakpointwas cloned directly from genomic DNA. About 200 mg ofFly stocks: Mutations and chromosomes not described here

are described by Lindsley and Zimm (1992). All flies were pug D genomic DNA was digested with EcoRI, XhoI, and HindIIIrestriction enzymes. The EcoRI and XhoI digests were to reduceraised at 258 on standard cornmeal medium. Df(3R)cu Sb/TM6

flies (Df(3R)cu 5 Df(3R)86C1-2;86D8) were obtained from the the heterogeneity of the 5-kb HindIII fragments because the

1553A Dominant Fusion Gene

homologous to portions of the DNA clones. These were synthe-TABLE 1sized by the Nucleotide Synthesis Core Facility at the University

Primers for PCR and sequencing of Utah. These additional primers were Dist39, DistX59,ProxH2, ProxH3, ProxH59, ProxB3, ProxK1, Rab739, 39pug-

Name Sequences Bst, and MTH39.The BLAST program was used to search for homologous

1. Dist39 59-TCACGCTCGAATGTTATGCA-39 sequences for the MTHFD and rab7 genes. Sequences de-2. DistX59 59-TGGTGGAATCGTGATTGGGA-39 scribed in this article have been deposited into GenBank un-3. ProxH2 59-TGAGCAAGCAAATTGGCGGA-39 der accession numbers AF079459, AF080444, AF080445,4. ProxH3 59-CTTGCACACGGTTAACGAAGGT-39 AF082097, and AF082098.5. ProxB3 59-AGCTGAGTCTTAACGAGCCT-39 Plasmid construction: DNA fragments were purified from6. PROXK1 59-ACTGACCAAGAAGAGCTGGA-39 agarose gels (when necessary) using the Geneclean system

from Bio101.7. ProxK2 59-CACTGGACATGCGAATGGAT-39The 1-kb AGAGAGA repeats from pug D are unstable in regu-8. Rab759 59-TCCGGACGTAAGAAATCCCT-39

lar bacterial cloning strains. Bacterial strains we have tried are9. Rab739 59-GCACTGACAGTTGTCAGGAT-39TOP10 from Invitrogen (Carlsbad, CA), SURE from Strata-10. Test4 59-ACCTTCGTTAACCGTGTGCA-39gene, JM109 from Promega (Madison, WI), and DH5a. Plas-11. Test1 59-CTGTTCTGTTGCAAAAGCCA-39mids that carried these repeats spontaneously generated DNA12. MTH39 59-GGCAATCAATTCCGATTGCTCT-39clones with shorter repeats during culture growth. We found13. LongPCR 59-TGGTGACTGCCAATACGCTA-39that growing cells at 308 instead of 378 tended to stabilize the14. 59pug-K 59-ACAGCAGAGCCGGTACCAAGArepeats. All pug D clones were based on the original genomicTGAGTGGGGCCAAGAT-39p2-1 clone that carries the pug D inversion junction (described15. 39pug-Bst 59-ACGATCTTGCTGCGTCCCAGAA-39 above) and on subclones obtained from the P1 clones that16. ProxH59 59-CCAAATTGCGTATGCCAGCA-39 cover this region.

17. ATPup 59-GATCTTGGTCACCACTCATCTTGA-39 Construction of the 14-kb pug D transgene: The plasmid p2-1 wascut with BamHI and religated. This generated p10-25-15 withPrimer locations are indicated in Figure 1.a 3.1-kb HindIII-BamHI insert. By a series of cloning steps, weadded a 3.9-kb Sal I-HindIII fragment, which was derived fromP1 DS01137, to the left of the 3.1-kb HindIII-BamHI clone.5-kb pug D junction contains no EcoRI or XhoI sites (resultsWe also added a 7-kb BamHI fragment, which was derivedfrom Southern blot analyses). Digested DNA was phenol-chlo-from P1 DS02445, to the right of this HindIII-BamHI fragment.roform extracted and run on an agarose gel. DNA of z5 kbThe correct orientation of the BamHI insert was verified byin size was cut out of the gel and purified by using the Gen-PCR, using primers Rab759 and Rab739. The plasmid p11-27-eclean Kit (Bio101 Inc., Vista, CA). DNA was then cloned18 gave rise to the expected 700-bp PCR product. p11-27-into the HindIII site of Bluescript. Colonies were screened by18 has the 14-kb insert including all three of the potentialhybridization for clones that contained the DNA fragmentcomponents of pug D (Figure 7). This 14-kb Sal I-BamHI insertspanning the proximal breakpoint of the pug D inversion. Thewas cloned as a Sal I-NotI fragment into the transformationidentified clone was labeled p2-1.vector pYC1.8, which generated pP[v1, pug D].DNA clones were sequenced by the Sequencing Facility at

Construction of the pug D transgene with part of rab7 deleted (athe University of Utah. Primers that flank the polylinker sitesBamHI deletion): The plasmid p11-7-7 lacks the 7-kb BamHIof Bluescript were used for most sequencing and were pro-fragment containing the N-terminal two-thirds of rab7 (Figurevided by the Sequencing Facility (T3, T7, M13 reverse, and7). The 7-kb Sal I-BamHI insert from p11-7-7 was cloned as aM13 forward). Additional sequencing was done using primersSalI-NotI fragment into pYC1.8 to produce pP[v1, pug(Bam D)].

Construction of the pug D transgene with a KpnI deletion: p11-27-18 with the 14-kb transgene was cut with KpnI and religated.This generated p11-27-18(Kpn D) with a 10-kb insert (Figure7). The 10-kb KpnI-BamHI insert was cloned into transforma-tion vector pw8, giving rise to pP[w8, pug(Kpn D)].

Construction of the transgene with only the GAGA repeats: Theoriginal clone p2-1, which has the 5-kb HindIII pug D junction,was cut with BamHI and religated. This deleted 2 kb of DNAand produced p1-6-7 (same as p10-25-15). p1-6-7 was cut withClaI and religated. This deleted the start and promoter ofMTHFD and left only z200 bp of MTHFD sequences in theplasmid p1-9-9. The 2.2-kb KpnI-BamHI insert from p1-9-9 wascloned into pw8 to produce pP[w8, GAGA].

Construction of the 3.4-kb KpnI-BamHI pug D transgene: A 3.4-kbEcoRI-BamHI fragment was isolated from the plasmid pP[w8,pug(KpnD)]. This piece is cloned into pHSS6 (Seifert et al.1986). This generates pHSSpug D. A NotI fragment carrying thetransgene from pHSSpug D was cloned into the unique NotIFigure 1.—Molecular maps of relevant portions of P1 clonessite in pP[X97]. This gave rise to the transformation constructDS01137 and DS02445 from wild-type D. melanogaster. BoxespP[X97, pug D]. This construct allows FLP-mediated gene tar-represent coding regions with direction of transcription indi-geting of the pug D gene (Golic et al. 1997; see discussion).cated by arrows. Vertical arrows mark the inversion break-

Construction of the wild-type MTHFD transgene: p7-21-18 is anpoints. Small arrows show the direction (59 to 39) and approxi-EcoRI subclone of P1 DS01137. A 9-kb KpnI-BamHI fragmentmate location of the primers in Table 1, with a numberfrom p7-21-18 contains the entire MTHFD1 gene. This KpnI-assigned to each primer. Restriction enzymes: B, BamHI; H,

HindIII; K, KpnI; R, EcoRI; S, SalI; X, XhoI. BamHI insert was cloned into pw8 to produce pP[w8, MTHFD].


Construction of the transgene with stop codons upstream of the (v1) (Golic et al. 1997), and pw8H (w1). The marker genesfor the P elements are shown in parentheses. Insertions wereGAGA repeats: The construct pP[X97, pug D] has a unique BstEII

site 59 of the GAGA repeats with a recognition sequence of 59- mapped by segregation from dominantly marked chromo-somes.GGTGACC-39. The construct was cut with BstEII to completion

and the ends were filled by the Klenow fragment of DNA Characterization of the ENU-induced pug D revertant(pugDrv18): To detect small deletion(s) possibly associated withpolymerase I from Boehringer Mannheim. The treated plas-

mid was religated. This gave rise to the plasmid pP[X97, the revertant, PCR analyses were performed. DNA was purifiedfrom pug Drv18/Df(3R)cu flies, pug D homozygotes, and wild-typepug(Bst2)] in which the sequence 59-GGTGACGTGACC-39 was

generated at the former BstEII site, with TGA stop codons in flies to test DNA of the 86C region. The DNA was used astemplates in PCR analyses with primers Test4 and ProxK2.two of three reading frames. The sequence changes in pP[X97,

pug(Bst2)] were verified by sequencing. Fragments of 1.9 kb were amplified from all three DNA sam-ples. PCR with primers Test4 and ProxH59 amplified a 0.9-kbConstruction of a transgene that allows translation of the GAGA

repeats and minimal MTHFD sequences: PCR with the primer DNA fragment from all three DNA templates. DNA was puri-fied from pug Drv18/Df(3R)mbc-R1 flies, pug D homozygotes, andpair ProxK1 and ATGup generated a 1-kb fragment from pug D.

In ATGup, a BstEII site was introduced just downstream of wild-type flies to test DNA of the 95D region. The DNA wasused as templates in PCR with primers Test1 and LongPCR.the ATG codon. The 1-kb fragment was cut with KpnI and

BstEII and cloned into pP[X97, pug D], replacing the 1.2-kb All three templates gave rise to a 2.7-kb fragment. PCR withprimers Test1 and Rab759 amplified a 1-kb fragment from allKpnI-BstEII genomic fragment. This generated the construct

pP[X97, pug(MTH2)]. In this construct codons 2–134 of pug D three DNA templates.To demonstrate that the wild-type MTHFD transgene (pug 1)were deleted. The remaining gene consisted of 44 codons

derived from MTHFD along with the GAGA repeats, and could can rescue the mutant phenotype of pugDrv18/Df(3R)cu flies,the following crosses were performed. Female virgin flies ofbe expressed from the MTHFD promoter. The sequence

changes were verified by sequencing. w1118 P[w8, MTHFD]11A, which carry a pug1 transgene withina P element inserted on X, were mated to males of pug Drv18/Construction of a pug D transgene under the control of the heat

shock protein 70 promoter (hsp70): The Drosophila MTHFD pro- Df(3R)cu. As Df(3R)cu/1 flies have a dominant Minute pheno-type (short and thin bristles), Minute1 male progeny have thetein fails to show extensive amino acid homology to MTHFD

from other organisms for the 10–20 amino acids (aa) at the genotype of w1118 P[w8, MTHFD]11A; pug Drv18/1. They weremated to virgin females of pug Drv18/Df(3R)cu. As Df(3R)cu car-very N terminus. Therefore, the translational start site was

predicted as followed. The sequence of the 3.4-kb KpnI-BamHI ries the recessive cu mutation, and the original pug Drv18 chromo-some also carries cu and a linked recessive lethal mutation,fragment was examined by computer for promoter and mRNA

splice site predictions. These programs are available at the pug Drv18/Df(3R)cu flies have curled wings and pug Drv18/pug Drv18

flies are dead. Male progeny that were pug Drv18/Df(3R)cu didweb site of the Berkeley Drosophila Genome Project (http://fruitfly.berkeley.edu/). The first ATG codon lies 125 bp down- not carry the pug1 transgene on X. They all showed the reces-

sive eye pigment phenotype. Female progeny that werestream of the predicted transcription start site. We predictedthat this ATG codon is the translational start for MTHFD. pugDrv18/Df(3R)cu were heterozygous for the pug1 insertion on X.

To generate flies homozygous for pug Drv18, females that wereAdditional supporting evidences are as follows: (1) The se-quence TATCAAGATG matches the Drosophila initiator ATG pug D/pug Drv18 were generated. Homozygous stocks were made

from recombinants that had crossed off the lethal mutationconsensus: TAAC/AAAA/CATG (Cavener 1987) at 8 of 10positions. (2) This ATG is in-frame with the codons that show on the pugDrv18 chromosome.

Heat shock experiments: Because animals with a single copyextensive homology to other MTHFD proteins. This was veri-fied by sequencing a putative cDNA clone. We constructed a of the construct pP[w8, hspug D] show subtle pigment defects

after heat shock, we used animals with multiple copies of theputative cDNA clone of pug D based on these predictions.The pug D cDNA clone was made by splicing together cDNA gene to increase expression of pug D after heat shock. The

hspug D transformants are marked by a hypomorphic white genesequences and genomic sequences. The 380-bp cDNA frag-ment from the conceptual translational start site to the HpaI (w hs). Flies with one copy of w hs usually have orange or yellow

eye color, whereas flies with two copies have red eyes. On thesite 95 bp upstream of the GAGA repeats was cloned from acDNA library. The rest of pug D cDNA sequences (downstream basis of this phenotype, recombinants were made so that one

chromosome 3 harbored two hspug D transgenes. The hspug Dof the HpaI site) were derived from the genomic sequencesof pug D. Since no introns were predicted between the HpaI insertions that were used in the heat shock experiments are

as follows, with chromosomal locations in parentheses: 7A (X),site and the stop codons of pug D, we predict that our clonewould have the correct cDNA sequence. The pNB40 Drosoph- 8A (III), and 9A (III). The flies with the genotype w1 P[w8,

hspug D]7A were made by crossing virgin females of w1118 P[w8,ila embryonic cDNA library from N. Brown (University ofCambridge) was used. DNA from the whole library was used hspug D]7A/1 to wild-type males. The progeny were heat

shocked at early to mid-pupal stage. Male recombinants thatas PCR template, with primers 59pug-K and 39pug-Bst. In59pug-K, a KpnI site was introduced 59 of the ATG. The PCR show pigment defects (see results) should have the geno-

type of w1P[w8, hspug D]. Two such males were recovered.generated a 530-bp fragment. It was cut with KpnI and HpaI.We replaced the KpnI-HpaI fragment in the pugD genomic Flies that are w1 P[w8, hspug D]7A; P[w8, hspug D]8A P[w8,

hspug D]9A/TM3 were heat shocked to generate the eyes shownclone with this 380-bp fragment. The 2.8-kb KpnI-BamHI frag-ment, which contains the pug D cDNA and its 39-untranslated in Figure 9.

To determine the timing of pug D action, flies were allowedregion, was cloned into pw8H from K. Basler (Basler andHafen 1989). This resulted in pP[w8, hspug D] that contains a to lay eggs until pupae with black wings were present in the

vials. Parents were then removed and the vials were heathsp70-driven pug D gene. The sequence in the putative pug D

cDNA was verified by sequencing. shocked in a water bath at 388 for 1 hr. The adults that eclosedwere examined for pigment defects on the day that theyDrosophila transformation: All DNA constructs were intro-

duced into the genome by standard P-element transformation eclosed.Images: Images were obtained and processed as described(Rubin and Spradling 1982). The P-element vectors used

for the experiments described here were pw8 (w1) (Klemenz (Golic 1994; Ahmad and Golic 1996). For Figures 2, 3, 8,and 9, the heads from 3-day-old flies were used.et al. 1987), pYC1.8 (v1) (Fridell and Searles 1991), pP[X97]


RESULTS

Discovery of pugD and basic phenotypes: The domi-nant mutation pugD was discovered in a screen for X-ray-induced chromosomal rearrangements that causedposition-effect variegation of a white transgene (whs) lo-cated on chromosome 2 (Ahmad and Golic 1996). Inone case, when a fly with variegated eye pigmentationwas outcrossed to flies with the w1118 null mutation, someof its progeny showed uniformly pigmented eyes. Inother words, the genetic element responsible for caus-ing variegation segregated from the whs transgene. Themutation that caused the variegation was mapped tochromosome 3.

We wished to determine whether this mutation pro-duced variegation by a specific interaction with the whs

transgene, or whether variegation also occurred in w1

flies. Flies with the mutation were crossed to flies withdifferent insertions of the same transgene and to w1

flies. Variegation of eye pigment was observed in allcases (Figure 2). Because the whs transgene and the w1

gene have different 59 regulatory sequences (Klemenzet al. 1987), it is unlikely that this mutation is exertingits effect through transcriptional regulation of the whitegene.

Many mutations that affect eye pigmentation havedefects primarily in one of the two major pigment path-ways. These pathways can be knocked out independentlyby mutations in the vermilion (v) and the brown (bw)genes. We placed the pugD mutation (as pugD/1) inseparate backgrounds of v and bw to determine whetherit affected one or both pigments. Homozygous bw fliesproduce only ommochrome pigment, and pugD causesa variegated reduction in ommochrome pigmentation(Figure 2). A much more dramatic effect is seen in fliesthat can only synthesize pteridines because they carrythe v mutation. In a v background, pugD almost com-pletely eliminates pteridines in the middle of the eye(Figure 2). However, the periphery of the eye remainsalmost completely pigmented. The result is a strikingring of pigment surrounding a center that is almostcompletely white. When we tilt the eye to look at theeye margin at an angle that is perpendicular to the eyesurface, it can be seen that the peripheral pigmentationis confined to the very edge of the eye (except for theoccasional spots) and appears to be external to all om-matidia (Figure 3). There is also a variegated aspectof the pteridine pigmentation in pugD: small and in-frequent red spots can be seen in the middle of a v;pugD/1 eye (Figures 2 and 3). A small portion of thepugD flies have concave dents in the eye as if the overalleye structure is weak (not shown). Figure 2.—Phenotypes of pug D. The appearance of the eyes

of pug1 and pug D flies in different genetic backgrounds isAlthough the pugD mutation is dominant, homozy-indicated. The pug genotype is indicated at the top of eachgous pugD flies survive (see materials and methods).column. Eye color mutations carried by the flies are indicatedThe phenotype of homozygotes is almost identical to at the left of each row. The flies indicated as w hs carry the

that of heterozygotes, with only a slight lessening of w1118 null mutation and one copy of a hypomorphic whitetransgene. Anterior is to the right.pigmentation. It is likely that this reduction in pigment


Figure 3.—The periph-eral ring of pteridine pig-mentation. Eyes were takenfrom v; pug D/1 flies. Occa-sional red spots in the mid-dle are also visible.

is attributable to homozygosity for the recessive karmoi- and experiences variegated inactivation, producing thered sectors.sin2 mutation that the pugD chromosome carries. Some

pugD homozygotes also have slightly rough eyes (not Because the aneuploid segregants of this transposi-tion survive, we were able to test the effect of the pugDshown).

Mapping the pugD mutation: The pugD mutation is mutation in flies with two copies of pug1 gene. In thesev/Dp(3;Y), pugD ; pug1/pug1 flies, the pugD phenotypeassociated with an inversion on the right arm of chromo-

some 3 (3R), with breakpoints at polytene chromosome is still visible and essentially identical to the phenotypeof v; 1/Tp(3;Y), pugD flies. Therefore, the pugD pheno-bands 86C3-4 and 95D1-6. A simple inversion involves

two breakpoints and creates two new DNA junctions. type cannot be suppressed by a second copy of pug1,providing further evidence that the phenotype does notTherefore pugD could be located at either junction. It

is also possible that pugD is unrelated to the inversion, result from a deficiency of the wild-type product.Cloning and sequencing of the pugD junction: Wild-and their occurrence together may be merely coinci-

dental. type P1 genomic clones derived from the 86C and 95Dregions of chromosome 3 were obtained from the Dro-To map pugD, we screened for X-ray-induced re-

vertants of pugD. Because deficiencies of either the 86C sophila Genome Center. Polytene chromosomes of pugD

homozygotes were hybridized in situ with labeled probesor 95D regions, carried in Df(3R)cu/1 and Df(3R)mbc-R1/1 flies, respectively, do not produce eye pigment made from the P1 clones. If a P1 clone spans one of

the two breakpoints of the inversion, it should generatephenotypes, pugD is not likely to be the result of haplo-insufficiency. Therefore we expected that the dominant two hybridization signals on chromosome 3R from pugD

homozygotes. We found two clones that fulfill this condi-phenotype could be reverted by deleting the pugD gene.Seventeen phenotypic revertants were recovered tion (one example is shown in Figure 5; for restriction

maps see Figure 1).among z70,000 F1 progeny of irradiated males (halfof which received the pugD chromosome). Lines wereestablished from 10 of the revertants, and their polytenechromosomes were studied to determine the nature ofthe reversion. In 9 out of 10 cases, we found a smalldeletion of the proximal inversion junction. The tenthrevertant was associated with a T(Y;3), with the chromo-some 3 breakpoint at the proximal junction of the inver-sion. Figure 4 summarizes the cytology of the revertants.We conclude that pugD is at or close to the proximalinversion junction.

We also recovered one male in which pugD variegatesas a result of insertional translocation of the pugD regioninto the heterochromatic Y chromosome. The eyes of Figure 4.—Cytology of X-ray-induced pug D revertants (rv).

The top horizontal line represents the region of the proximalv; 1/Tp(3;Y), pugD males show red sectors in the middleinversion junction in pug D, with numbered and lettered divi-of the eye on an otherwise white background. The pe-sions of the chromosome shown. The inversion junction isripheral ring of pigment is visible in the white portionsindicated by a thick vertical line and an arrowhead. The thin

of the eye (not shown). This appears to be a typical case horizontal lines represent the portion of the chromosomeof position-effect variegation in which the euchromatic that remains in each revertant. The extent of each deficiency

is marked by two thin vertical lines.pugD gene has been placed close to heterochromatin


Figure 6.—Genomic structure of the pug D inversion. (A)The thick line represents the normal chromosome 3R. Theblack circle represents the centromere. Close-up views areprovided for 86C and 95D regions. Thin horizontal lines in theFigure 5.—Polytene chromosome in situ hybridization with close-ups represent DNA from the regions. Boxes representthe P1 clone DS01137. Chromosomes were taken from pug D

putative transcription units, with directions of transcriptionhomozygous larvae. DS01137 covers 86C3-6 on a wild-type indicated by arrows. Vertical arrowheads mark the inversionchromosome 3R. It generates two hybridization signals (arrow- breakpoints. (B) The proximal inversion junction in pug D withheads) on a pug D chromosome. The other inversion break- centromere to the left. Hatched box represents the GAGApoint lies in 95D1-6 region. Cytogenetic map coordinates close repeats.to the signals are indicated by arrows.

fragment predicted by a simple breakage-and-fusionThese two P1s were further subcloned and Southern event. To determine the nature of the extra DNA, we

blots were used to look for restriction fragment length cloned the 5-kb piece directly from genomic DNA ofpolymorphism by hybridizing digested DNA from pug1

pugD flies (see materials and methods). A 1-kb pieceand pugD flies with probes from either the whole P1 of highly repetitive DNA has been inserted at the proxi-clone or individual subclones. The breakpoints were mal inversion junction, accounting for the increasedthus mapped to individual subclones. The pugD junction size of the HindIII fragment. This DNA is not normallyis located within a 5-kb HindIII fragment. The DNA present in the vicinity of the MTHFD or rab7-homolo-surrounding this junction was sequenced to determine gous genes. The repetitive DNA consists of z140 unitsthe genetic architecture of the region. of AGAGAGA repeat (GAGA repeats) with occasional

Figures 6 and 7 depict the genomic structure of the slight variations. The same repeats have been identifiedpugD junction. At chromosome region 86C, the inver- as a major component of Drosophila heterochromatinsion breakpoint lies within an open reading frame of DNA (for references see the Introduction).a gene that is highly homologous (z60% amino acid In summary, the pugD junction consists of the N-termi-identity) to genes from a number of organisms that nal one-fifth of a gene that appears to encode MTHFD,encode the trifunctional MTHFD enzyme. This MTHFD- 1 kb of AGAGAGA repeats fused to this gene, and ahomologous gene is different from that found by Price rab7-homologous gene 400 bp distal to the junctionand Laughon (1993) at 85C. The gene they reported (Figure 7).appeared to encode the mitochondrial version of the Transformation of the pugD gene: To identify pugD,enzyme. The gene at 86C appears to encode the cyto- DNA from the proximal inversion junction was trans-plasmic form. In chromosome region 95D, an open formed into pug1 flies. A 14-kb DNA clone was con-reading frame z400 bp away from the breakpoint en- structed by splicing together wild-type genomic sub-codes a protein with z90% amino acid identity to the clones of the region and the 5-kb HindIII fragment thatrab7 gene from a variety of other organisms. Satoh et contains the pugD junction (Figure 7). This was placed inal. (1997) have identified a fragment in Drosophila with a P-element vector for germline transformation. Fifteenz91% amino acid identity to rab7 genes from other independent transformants were isolated. All trans-organisms. As their sequences were not published, we formants showed pigment defects in a wild-type back-could not compare the rab7 we identified with their ground (not shown), similar to the phenotype shownfragment. in the top right of Figure 2. Because the P-element

The sequence of the distal breakpoint (non-pug) vector carried a v1 gene as a transformation marker, itshowed a perfect rejoining of the inversion breaks. How- was not possible to examine the phenotypes of trans-ever, the HindIII fragment that contains the proximal formants in a v background. However, the cinnabar (cn)

mutation also eliminates ommochrome pigments, andinversion breakpoint is 1 kb larger than the 4-kb HindIII


Figure 7.—Results oftransformation experiments.At the top, boxes representcoding regions with direc-tion of transcription indi-cated by arrows. The shadedboxes below represent theDNA fragments used fortransformations. The phe-notypes of transformantsare indicated at the right.Restriction enzymes: B,BamHI; C, ClaI; H, HindIII;K, KpnI; S, SalI; X, XhoI.

a cn; pugD/1 fly has the same pattern of pigmentation up only in young flies. We believe that the center of theeye is less pigmented than the periphery. Because it hasas a v; pugD/1 fly (not shown). We crossed the trans-

formants into a cn background and found that the origi- less pigment, it is less effective at reflecting light to theobserver and therefore appears darker.1 This recessivenal pugD phenotype was reproduced (Figure 8). Thus,

pugD is contained within this 14-kb segment. The trans- phenotype was rescued by a P-element insertion carryingpug1 (the wild-type MTHFD), thus identifying a defectformed pugD gene also caused a variegated reduction

in ommochrome pigmentation, similar to the original in pug as the cause of this recessive phenotype. Wegenerated pugDrv18 homozygotes (see materials andpugD mutation (not shown).

Because two genes are present in this fragment of methods), and these flies show the same recessive eyecolor phenotype as pugDrv18/Df(3R)cu flies. This suggestsDNA, we subcloned and transformed flies with portions

of the 14-kb DNA fragment to pinpoint the responsible that pugDrv18 is likely to be a null allele of MTHFD.We also studied the effect of pug-null on ommo-gene. A smaller, 7-kb SalI-BamHI fragment that contains

only the C-terminal one third of rab7 is still capable of chrome and pteridine levels separately. A bw; pugDrv18/Df(3R)cu eye does not show a reduction in pigmentationgenerating the pugD phenotype (Figure 7). We conclude

that rab7 is not the cause of pugD. Furthermore, DNA when visually compared with a bw eye (not shown).This suggests that pug-null does not affect ommochromebetween the SalI and KpnI sites is not necessary for the

phenotype, because a 10-kb KpnI-BamHI fragment can pigmentation. A very weak eye color defect can be seenin a v; pugDrv18/Df(3R)cu eye. Eyes from some of the veryalso reproduce pugD (Figure 7). The pugD gene is entirely

contained within the smaller 3.4-kb KpnI-BamHI frag- young flies show a lighter eye center than the periphery.Therefore we believe that the pug-null mutation slightlyment. This was verified by transformation (Figure 7).

Finally, a construct that lacked most of the remaining reduces pteridines in the eye center. Moreover, thiseye color defect is very similar to phenotypes of otherMTHFD sequences, including the start codon and up-

stream sequences, was also transformed (the 2.2-kb ClaI- mutations that affect purine de novo synthesis: it reducespteridine pigmentation initially, but the phenotypeBamHI fragment in Figure 7). None of the 13 trans-

formants showed the pugD phenotype. Therefore, pugD wanes as flies age (Johnstone et al. 1985; Keizer et al.1989; Tiong and Nash 1990).is a mutation in a gene that appears to encode the

enzyme MTHFD, and it consists of the DNA that codes A mutation that had a phenotype similar to the reces-sive pug phenotype was previously mapped in the vicinityfor the MTHFD N terminus fused to AGAGAGA repeats.

Null mutations of pug have a recessive eye color phe- of the pug gene (Wright 1946). The doughnut (dn)mutation was located at map position 50 on chromo-notype: In other experiments, a pugD revertant allele

(pugDrv18) was obtained by treatment with the chemical some 3. This region corresponds approximately to thepolytene chromosome region 86C, where pug is located.mutagen ENU. Cytological analyses of polytene chromo-

somes (not shown), PCR (see materials and meth- To test for allelism we obtained a dn stock from theMid-America Drosophila stock center. This stock wasods), and Southern blot analyses (not shown) revealed

that it has no detectable deletion at pugD. When therevertant allele was heterozygous with Df(3R)cu, whichdeletes the whole MTHFD-containing region of 86C, we 1 A similar phenomenon can be observed in flies that have small

and infrequent white clones in an otherwise white1 (red) background—observed a recessive phenotype slightly reminiscent ofthese white clones appear to be much darker than the surroundingpugD. In pug-null flies that are otherwise wild type, theeye tissue. Due to the subtlety of the phenotype, we were unable to

center of the eye is dully pigmented, with a bright ring produce a photograph in which the phenotype is apparent. However,the phenotype is identifiable by eye under the light microscope.toward the periphery. This eye color phenotype shows


Figure 8.—Phenotypesof pug D transformants. Thisfigure shows a sample of thephenotypic variation ob-served with independent in-sertions of the 14-kb S-Btransgene (Figure 7). Allflies were homozygous forthe recessive cn mutationand heterozygous for a pug D

insertion.

reported to carry the dn allele heterozygous with a bal- GAGA repeats from 1 kb to z300 bp, and restrictionmapping indicates that this is the only change. The firstancer chromosome. In crosses of the putative dn stock

to flies with deletions of pugD (Figure 4) and to 178 codons should be translated with this transgene,but it did not produce the pug phenotype. This stronglyDf(3R)cu/1 flies, none of the dn/Df flies showed an

eye color phenotype. This would normally lead to the suggests that translation of these 178 codons is not suffi-cient for the phenotype—translation of a long stretchconclusion that pug and dn are not allelic. However,

animals with the putative dn-bearing chromosome did of GAGA repeats (.300 bases) is also needed to producethe phenotype. However, we cannot at this time excludenot survive as homozygotes, so it was not possible to

verify the presence of the mutant dn allele. Therefore, the alternative explanation that the pugS mRNA is desta-bilized by the loss of 700 bp of repeats, and this causeswe cannot with certainty state that pugD and dn are not

alleles of the same gene. reversion of the pug phenotype.Another possible cause for the failure of both pP[X97,Translation of the GAGA repeats is necessary for

the dominant phenotype: By DNA sequencing, no stop pug(Bst2)] and pP[X97, pugS] constructs to regeneratethe pug phenotype is that the chromosomal positionscodons were found upstream of the GAGA repeats or

within the sequenced portion of the repeats (about 700 of the insertion do not allow sufficient expression ofpugD. To address this question, we used the FLP-medi-bp). Stop codons are present in all three reading frames

immediately downstream of the GAGA repeats. There- ated DNA mobilization technique (Golic et al. 1997)to target various transgenes to specific chromosomalfore, it is likely that the 1-kb repeats are translated in

pugD. To test this hypothesis, we made a construct, sites. At one particular site, a pugD transgene with thefull-length GAGA repeats and no stop codons upstreampP[X97, pug(Bst2)], in which the unique BstEII site was

cut and filled. This treatment introduces stop codons of the repeats (the 3.4-kb K-B fragment of Figure 7)generated an eye color phenotype essentially identicalin two of the three reading frames. It also caused a 11

frame shift in the MTHFD coding frame. The new frame to the original pug phenotype. However, when we tar-geted to the same chromosomal site, either the pugDstops 16 codons downstream without reaching the

GAGA repeats. This altered pugD gene is predicted to transgene with a premature stop codon (as in pP[X97,pug(Bst2)]), or the transgene with shorter repeats (as incode for a short peptide of 153 amino acids (aa), in

which 137 of them are from the N terminus of MTHFD, pP[X97, pugS]), the pug phenotype was not produced.These experiments eliminate position effects as theand the extra 16 aa are the result of the 11 frame shift.

Six independent transformants of pP[X97, pug(Bst2)] cause for the failure of both pP[X97, pug(Bst2)] andpP[X97, pugS] to produce the pug phenotype. There-were recovered: none showed any pigment defects even

when homozygous for the transgene. fore, the DNA sequence changes introduced in vitroknocked out the pugD gene. The predicted pugD proteinIn the pugD protein, there are 178 codons upstream

of the GAGA repeats. Only the first 137 would be trans- would consist of the N-terminal 178 aa from MTHFDand a C-terminal z350 aa in which the sequencelated in pP[X97, pug(Bst2)]. One might still argue that

synthesis of all 178 is necessary for the phenotype. How- SLLSSLF is repeated z50 times with occasional substitu-tions of C, V, and P (data not shown).ever, a transgene with shortened repeats failed to re-

produce the dominant phenotype. Because the GAGA We also wished to determine if translation of therepeats alone would produce the pug phenotype. Therepeats are somewhat unstable in regular bacterial

cloning strains, we recovered a spontaneous derivative construct pP[X97, pug(MTH2)] should allow translationof the full-length GAGA repeats plus 44 codons ofof pP[X97, pugD] with an internal deletion, which

we named pP[X97, pugS]. The deletion shortened the MTHFD (see materials and methods). None of the


ing a ring of pteridine pigment. Examples of ring pat-terns of pigmentation are rare, and none are wellunderstood. Thus it seemed that much could be learnedby identifying and characterizing the mutation thatcaused these phenotypes. Genetic and molecular meth-ods have been employed to achieve this goal.

The nature of the pugD mutation: The pug phenotypedoes not result from a simple change in gene dosage.When one copy of pug1 is deleted, flies do not exhibitthe pug phenotype, and pugD is not suppressed by threecopies of pug1 (not shown). Thus, haplo-insufficiencyis ruled out as the cause. It is equally unlikely that thepugD rearrangement causes overexpression of pug1 onthe homolog (possibly by some sort of transvection ef-

Figure 9.—Phenotype of flies carrying the construct pP[w8, fect, e.g., Geyer et al. 1990) and that this overexpressionhspug D]. Eyes are from otherwise wild-type males that carried produces the pug phenotype. Two extra copies of athree copies of the hspug D transgene (for the genotype see pug1 transgene do not produce the pug phenotype (notmaterials and methods). Flies were heat shocked during

shown), and simple deletions of the pugD breakpointearly to middle pupal stage.that leave pug1 on the homolog intact revert the pheno-type. Further, pugD hemizygotes still exhibit the pug

three transformants with this construct show any pig- phenotype. The pug phenotype is caused by the novelment loss. fusion gene present at the pugD inversion breakpoint.

Developmental timing of the pugD effect: To deter- P elements that carry this fusion gene confer the pugmine when in development the pugD gene exerts its phenotype, and, when the coding sequence of this fu-effect on pigmentation, we placed the predicted pugD sion gene is placed under control of the hsp70 promoter,cDNA under the control of the hsp70 promoter (see heat shocks can partially reproduce the pug phenotype.materials and methods). We then heat-shocked ani- The pugD mutation lies within a gene that, based onmals with the hspugD construct at different develop- sequence similarity, encodes the enzyme MTHFD. Themental stages to induce pugD expression. Flies with a activities of this enzyme are essential for de novo purinesingle hspugD gene show pigment defects when heat- synthesis. The intimate relationship between purine me-shocked at early to middle pupal stage, exhibiting occa- tabolism and pteridine synthesis is apparent from thesional small spots that lack pigment (not shown). fact that pteridines are synthesized from GTP. Many

To increase the level of hspugD expression, we gener- mutations that affect de novo purine synthesis also reduceated flies with three copies of this transgene. These flies the level of pteridines in the eye (Johnstone et al. 1985;showed much greater loss of pigment after heat shock Tiong et al. 1989; Tiong and Nash 1990; Clark 1994).(Figure 9). They were used to determine the timing of Thus, it is possible that pugD causes loss of pteridinepugD action. Animals were given a single 1-hr heat shock pigment by interfering with purine biosynthesis.at 388. Flies that eclosed during the third day after heat The pugD allele is a fusion gene in which the N-termi-shock showed pigment loss. Thus, the most sensitive nal one-fifth of the MTHFD coding region (178 codons)period for pugD expression is 3 days before eclosion or is joined, within the coding region, to 1 kb of highlyduring the second day of pupal development. The eye repetitive DNA. The fusion gene was created by anpigments are first visible in wild-type flies around 48 X-ray-induced chromosomal inversion, with the repeti-hr after puparium formation (Phillips and Forrest tive DNA (presumably originating from centric hetero-1980). Therefore, pugD acts near the time when eye chromatin) captured between the normal euchromaticpigments are first made. sequences of the proximal breakpoint. Transcription

and translation of the repeats are apparently necessaryto produce the dominant phenotype, because insertion

DISCUSSIONof a stop codon in the mRNA just upstream of therepeats abolishes the dominant phenotype. The repeti-We have discovered an eye color mutation in Drosoph-

ila melanogaster with two unusual characteristics. First, tive DNA consists of repeats of the sequence AGAGAGA,with occasional slight variations. This part of the genethe pugD mutation causes a dominant and variegated

reduction in ommochromes and pteridines throughout would encode a sequence of the amino acids SLLSSLF,repeated z50 times with occasional substitutions of C,the eye, but null alleles of this gene are not dominant.

Before this discovery, there existed only a few examples V, and P. Because of the high leucine content, thisregion of the protein has the potential to form leucineof such a dominant variegating eye color mutation (for

example, bwD). Second, pteridine pigmentation is seem- zipper motifs, and to thereby promote homotypic pro-tein-protein interactions (reviewed in Vinson et al. 1989;ingly unaffected around the periphery of the eye, leav-


Busch and Sassone-Corsi 1990). Stop codons are pres- ducing pteridine synthesis. The mutant protein mayinteract with the wild-type MTHFD protein or otherent in all three reading frames a short distance down-

stream of the repetitive DNA. Thus, this repetitive pep- members of a purine synthesis protein complex, and bythis route, poison the complex. This model predictstide stretch should constitute two-thirds of the mutant

protein, at the C terminus. that extra copies of pug1 should suppress pugD, but theydo not. If the pugD peptide were acting as a toxic subunit,The trifunctional MTHFD enzyme generates 10-for-

myltetrahydrofolate as a cofactor for two methyltransfer- pug1 subunits should compete for its location in amultimeric assembly. Thus, we do not favor this model.ases in purine biosynthesis [for reviews of folate-depen-

dent enzymes see Benkovic (1980) and Appling (1991); However, the mutant pugD gene may be expressed at amuch higher level than the normal level of the pug1for reviews of purine synthesis see Jones and Fink

(1982) and Henikoff (1987)]. MTHFD contains two gene so that the mutant protein is in great excess tothe wild-type MTHFD, both in pugD/1 heterozygotesfunctional domains. The dehydrogenase and cyclohy-

drolase activities reside in the N-terminal one-third, and in pugD transformants. If true, a manyfold increasein the amount of MTHFD may be necessary to generatewhile the synthetase activity is associated with the

C-terminal two-thirds (Paukert et al. 1977; Tan and an observable suppression of the pug phenotype. There-fore, we cannot completely discard this model. However,MacKenzie 1977). The MTHFD protein appears to

form homodimers and has been suggested to participate we can rule out the hypothesis that pugD affects pigmen-tation by an interaction with the wild-type MTHFD pro-in the formation of a complex of enzymes for purine

biosynthesis in yeast, with both domains of the protein tein, because pugD/pugD and pugD/Df flies both exhibitthe pug phenotype, but no pug1 gene is present.involved in interactions with other members of the com-

plex (Appling 1991; West et al. 1996). A third model supposes that the N-terminal remnantof MTHFD synthesized by pugD may irreversibly bindThe mechanism of pugD action: One hypothesis for

pteridine elimination in pugD is that the mutant pugD and sequester its tetrahydrofolate substrate. If this sub-strate is present in a limiting concentration, it may beprotein disrupts pigment granules. In Drosophila, om-

mochromes and pteridines are present in membrane- reduced to a level that cannot support the pug1-medi-ated synthesis of the cofactor used to make purines andbounded protein-rich pigment granules (Shoup 1966).

Many enzymatic activities for pteridine biosynthesis have thus prevent pteridine synthesis. This model predictsthat the phenotype of pug-null flies with respect to pteri-been shown to be closely associated with granules (Dor-

sett et al. 1979; Hearl and Jacobson 1984). Purified dine pigmentation should be the same as that of pugD,or more severe than pugD, and this was not observed.granules are able to produce drosopterin, a major com-

ponent of the eye pteridines. These results led to the Although pugDrv18/Df flies exhibit a phenotype that isslightly reminiscent of pugD, it is much less severe thansuggestion that pigment granules contain all enzymes

for drosopterin synthesis. The mutant pugD protein the pugD phenotype. This objection might be removedif there was another enzyme capable of providing themight also be located in pigment granules. The C termi-

nus of pugD is expected to be hydrophobic and it might tetrahydrofolate cofactor for purine synthesis. This istrue in the yeast S. cerevisiae. The MTD1 gene in yeastdisrupt pigment granule structure or stability. It has

been suggested that the white protein is located across encodes a cytoplasmic NAD-dependent methylenetet-rahydrofolate dehydrogenase that can provide 10-for-the membrane of pigment granules (Tearle 1991;

Montell et al. 1992). The pugD protein could disrupt myltetrahydrofolate for purine synthesis (West et al.1993, 1996). This enzyme is monofunctional and showsboth pteridine and ommochrome pigmentation by in-

terfering with the function of the white protein in pig- some amino acid similarities to the trifunctionalMTHFD. In Drosophila, there might be an enzyme withment granules. The pugD protein might also bind the

pterin ring of the pteridines, as the pterin ring is a part similar activity so that some level of cofactors couldbe maintained to support purine synthesis even in theof the tetrahydrofolate molecule [for chemical struc-

tures of pteridines and folates see Temple and Mont- absence of the MTHFD proteins. Null alleles of genesin purine de novo synthesis behave as lethals or extremegomery 1984; Pfleiderer 1993]. It may then disrupt

normal pigment synthesis either by binding and seques- semilethals, with characteristic late pupal death (Tionget al. 1989; Tiong and Nash 1990). Genetic and molecu-tering pigment precursors, or by interfering with other

enzymes in the pteridine pathway. This model, although lar analyses suggest that the pugDrv18 mutation is likely anull. However, pugDrv18/Df(3R)cu flies survive very well,simple, does not take into account the normal function

of MTHFD in purine synthesis, nor does it connect the indicating functional redundancy in providing the es-sential folate cofactor for purine synthesis. In pug-nullphenotypes of the null and dominant alleles of pug.

Therefore, we do not favor this model. flies it is likely that de novo purine synthesis still occurs,though at a reduced level, and this allows for near-A second model for pugD action is based more directly

on the supposed role of pug1 in purine synthesis. The normal pteridine synthesis.This theory can explain why the dominant defect ispugD protein might act as a toxic subunit in a multimeric

assembly, crippling purine biosynthesis, and thereby re- more extreme than the recessive defect: the dominant


mutation cripples both modes of synthesis for the pu-rine pathway cofactor, while the recessive allele affectsonly one. However, it still seems that viability should bereduced by the dominant mutation if it causes a severedefect in purine synthesis. This conceptual difficultycould be overcome if the expression of pugD were limitedto the eye. Flies with pugD do sometimes exhibit pheno-types that could be attributed to an impairment of differ-entiation or to cell lethality owing to a defect in purinesynthesis in the eye. Some pugD/1 flies seem to havestructurally weak eyes, and occasionally homozygoteshave rough eyes (not shown). If these defects were ex-tended throughout the body, lethality might result. Inflies with the hspugD transgenes we did observe somedisruption of body patterns after heat shocks, possiblyas a result of cell death induced by pugD throughoutthe body.

The last two models, which propose that the defectin pteridine pigmentation in pugD stems from a defectin purine synthesis, can also be invoked to account forthe ring pigmentation of pugD.

Patterned pteridine pigmentation in pugD: One effectof pugD is to eliminate pteridine pigment from the cen-ter, but not the margin, of the eye. This pattern mightalso be a simple consequence of a defect in purinesynthesis. If the failure to make pteridines in pugD iscaused by purine deficiency, then externally suppliedpurines should suppress the phenotype. Although Dro-sophila can utilize dietary purine, the eye pigmentationprocess occurs in the pupal stage when absorption of

Figure 10.—A model for the pug D pigment ring. Frontalexogenous nutrients is blocked by the pupal case. There-(A and B) or lateral (C and D) views of the phenotypes of vfore, an increased demand for purines (for example,(A and C) and v; pug D/1 (B and D) are diagrammed. A

to make pigment) would have to be met either by an sectional view of one eye is provided for v and v; pug D/1 (Eincreased rate of de novo purine synthesis or by purine and F, respectively). The plane of section is indicated with

arrows in C and D. In E and F the bundles of retinula cellsuptake from neighboring cells or hemolymph. In fact,and their overlying lenses are indicated as cones. The pigmentit has been shown that externally supplied guanine de-cells lie between adjacent bundles of retinula cells. In a pug1

rivatives are actively taken up by the eye, and the trans-eye (E) purine uptake occurs at the periphery of the eye

ported guanine compounds are converted to pteridines (arrows), and purine uptake combined with endogenous syn-(Montell et al. 1992). It has also been shown that gua- thesis is sufficient to produce pigment in all pigment cells. In

a pug D eye (F), endogenous purine synthesis does not occur.nine derivatives can travel between pigment-producingPurines are still taken up at the edge of the eye, but are usedcells in Drosophila (Sullivan et al. 1979; Montell etto produce pigment in only the outer layer of pigment cellsal. 1992). Because of the hemispherical shape of theand are not transported further toward the center of the eye.

eye, only those cells at the periphery have substantialsurface area in contact with noneye tissue (Figure 10).Perhaps then, only at the eye margin would exogenous pression of a gene are common in Drosophila. For ex-

ample, tissue-specific enhancers have been found forpurines be available to cells of the eye. In a pugD flythese would be the only cells able to make pteridine the white gene (Levis et al. 1985; Pirrotta et al. 1985)

and the yellow gene (Geyer and Corces 1987; Martinpigment, and this would generate the ring of pteridinepigment around the eye. Moreover, if pugD expression et al. 1989). Further, even within an apparently uniform

tissue such as the eye, a variety of patterns of geneis eye specific, purine levels could be reduced in theeye but not in the surrounding tissues. This would create expression can be observed (Sun et al. 1995). Therefore,

it is tempting to imagine that regulatory elements withinan even greater difference in purine concentration thatwould certainly facilitate purine transport into the eye. the pugD fragment carried by the 3.4-kb transgene (Fig-

ure 7) provide for pugD expression in the middle of theAn alternative explanation for the differential pig-mentation in pugD is that the mutant gene product is eye but not at the eye margin. These could be part of

the normal regulatory elements for either the MTHFDpresent throughout the eye, except at the periphery.Regulatory elements that allow spatially controlled ex- or rab7 genes. It should be straightforward to identify the


proposed regulatory elements for pugD by performing eye, we infer that these red sectors are the result of pugD

inactivation.further deletion analysis of the 3.4-kb construct. How-In the original pugD and in the pugD transformants, aever, we cannot rule out the possibility that the AGA-

variegated phenotype is also apparent. Variegation isGAGA repeats themselves could constitute a signal formost visible in a background where ommochrome syn-exclusion of pugD from the eye margin. Because thesethesis is blocked and only pteridines are made. In therepeats must also be translated to produce the pug phe-middle of a v; pugD/1 eye, a small and variable numbernotype it may be difficult to establish any role they haveof pigmented spots appear on an otherwise white back-in regulation.ground (Figures 2 and 3). By analogy, this variegationThe effect of pugD on ommochrome synthesis: Be-may also be caused by gene inactivation.cause pugD has a very strong effect on pteridines, we

We propose that the pugD gene experiences PEV as ahave focused our discussion on explanations for thatresult of the small fragment of centric heterochromatindefect. However, pugD belongs to a class of mutationsthat it carries. It seems unlikely that this small fragmentthat affect both pigments of the Drosophila eye (Phil-of repetitive DNA would be sufficient to cause PEV onlips and Forrest 1980; Ferre et al. 1986). The veryits own. However, it may be sufficient to bring about PEVexistence of this class of mutations points to an inter-by a mechanism similar to that proposed for brownDominant

relation between ommochrome and pteridine synthesis.(bwD). In Drosophila, centric heterochromatin aggre-Different models to explain this interrelation have beengates to form a single chromocenter in the nuclei ofentertained by Summers et al. (1982). The general beliefsalivary gland cells (Heitz 1934), and some studies haveis that the two pathways share common systems for pre-provided evidence that highly repetitive DNA can associ-cursor transport and storage. At the ultrastructural level,ate with homologous sequences at nonallelic sites (Barrit has been suggested that the presence of pteridines inand Ellison 1972; Lee 1975; Yoon and RichardsonDrosophila eyes is needed for normal ommochrome1978). In the bwD mutation, z2 Mb of highly repeatedgranule formation (Shoup 1966; Fuge 1967; SummersDNA derived from centric heterochromatin is insertedet al. 1982; Reaume et al. 1991). Therefore the reductionat the bw locus (Slatis 1955; Henikoff et al. 1995).of ommochromes in pugD may be a secondary defectHenikoff’s group has proposed that bwD, which is lo-caused by the elimination of pteridines in the middlecated near the tip of 2R, can loop back and associateof the eye. However, it seems unlikely that the reductionwith centric heterochromatin. It is thought that thisof ommochromes in pugD is simply a consequence ofectopic association is responsible for inactivation of bw1

the absence of pteridines. If that were so, then bw/bwin a bwD/bw1 heterozygote, by virtue of the fact thatflies should exhibit no more ommochrome pigmenta-homologs experience somatic pairing in Drosophila. As

tion than bw/bw; pugD/1 flies, because the bw mutationa consequence, the bw1 allele is also displaced into the

eliminates pteridines entirely (Ferre et al. 1986). But, vicinity of centric heterochromatin, leading to its inacti-pugD does cause a further reduction in ommochromes vation. This model is strongly supported by extensivein bw/bw flies (Figure 2). Therefore the pugD mutation genetic evidence and by direct cytological demonstra-most likely affects ommochrome pigmentation by some tions of the colocalization of bwD and centric hetero-other route. It is possible that its effect on ommo- chromatin (Talbert et al. 1994; Henikoff et al. 1995;chromes is also a result of actively destabilizing pigment Csink and Henikoff 1996; Dernburg et al. 1996). Simi-granules. However, we consider it equally possible that larly, the variegation that is a normal part of the pugD

the effect on ommochromes is a secondary metabolic phenotype may arise when the gene associates with cen-effect, resulting from interactions between different bio- tric heterochromatin, and its expression is thereby si-synthetic pathways. lenced. However, the silencing of pugD would be in cis

Variegated pigmentation in pugD: In our screen for with the repetitive DNA, rather than in trans as in theX-ray-induced revertants of pugD we recovered several case of bwD.cases of classic position-effect variegation (PEV). PEV If this model for variegation of pugD is correct, thendescribes a phenomenon in which a euchromatic gene we would predict that, as with bwD, the position of pugD

experiences stochastic and clonally heritable inactiva- in the genome would influence the degree of genetion when it is juxtaposed to heterochromatin, usually silencing. Proximity to centric heterochromatin is anby chromosomal rearrangements (for reviews see Heni- important factor in determining the frequency of silenc-koff 1994; Karpen 1994; Weiler and Wakimoto 1995; ing with bwD, as well as with a silenced transgene arrayElgin 1996). The cases of PEV on pugD that we recov- (Talbert et al. 1994; Henikoff et al. 1995; Dorer andered showed large red sectors of pigment in the centers Henikoff 1997). Consistent with this expectation, weof their eyes in a vermilion background. These are all have observed that the phenotype produced by pugD

cases in which further chromosome rearrangement has transgenes can vary somewhat in independently isolatedplaced pugD next to centric heterochromatin or the het- insertions—different insertions show differing deg-erochromatic Y chromosome. Because the effect of pugD rees of pigmentation. This variation could have many

sources. First, the repeated DNA is somewhat unstableis to eliminate pteridine pigment in the middle of the


Clark, D., 1994 Molecular and genetic analyses of Drosophila Prat,in bacterial cells, and the phenotypic variation of insertswhich encodes the first enzyme of de novo purine biosynthesis.

may arise because deletions within the repeats may have Genetics 136: 547–557.Csink, A. K., and S. Henikoff, 1996 Genetic modification of hetero-occurred in bacteria. Second, standard quantitative po-

chromatic association and nuclear organization in Drosophila.sition effects may influence the expression of pugD andNature 381: 529–531.

produce variation in pigmentation. Finally, the variation Delattre, M., D. Anxolabehere and D. Coen, 1995 Prevalence oflocalized rearrangements vs. transpositions among events in-may in fact reflect proximity to centric heterochroma-duced by Drosophila P element transposase on a P transgene.tin. Experiments to sort out these possibilities are un-Genetics 141: 1407–1426.

derway. Dernburg, A. F., K. W. Broman, J. C. Fung, W. F. Marshall, J.Philips et al., 1996 Perturbation of nuclear architecture by long-It can hardly escape notice that the predominant com-distance chromosome interactions. Cell 85: 745–759.ponent of the repetitive DNAs at bwD is the sequence

Dorer, D. R., and S. Henikoff, 1994 Expansions of transgene re-AGAGA, while at pugD it is AGAGAGA. Thus, the repeats cause heterochromatin formation and gene silencing in

Drosophila. Cell 77: 993–1002.peated DNA found at bwD and at pugD are quite similar.Dorer, D. R., and S. Henikoff, 1997 Transgene repeat arrays inter-However, variegation mediated by repetitive DNA is not

act with distant heterochromatin and cause silencing in cis andlimited solely to this sequence, or even to sequences trans. Genetics 147: 1181–1190.

Dorsett, D., J. J. Yim and K. B. Jacobson, 1979 Biosynthesis ofthat are derived from centric heterochromatin (Dorer“Drosopterins” by an enzyme system from Drosophila melanogaster.and Henikoff 1994). Thus, it is probably not a specificBiochemistry 18: 2596–2600.

sequence that is responsible for a variegated phenotype, Elgin, S. C. R., 1996 Heterochromatin and gene regulation in Dro-sophila. Curr. Opin. Genet. Dev. 6: 193–202.but rather it is the repetitive nature of a sequence that

Ferre, J., F. J. Silva, M. D. Real and J. L. Mensua, 1986 Pigmentleads to variegation. It may be an intrinsic tendency inpatterns in mutants affecting the biosynthesis of pteridines and

Drosophila for repeated sequences to be localized to a xanthommatin in Drosophila melanogaster. Biochem. Genet. 24:545–569.heterochromatic compartment that is incompatible

Fridell, Y.-W. C., and L. L. Searles, 1991 vermillion as a smallwith the expression of normally euchromatic genesselectable marker gene for Drosophila transformation. Nucleic

(Wakimoto and Hearn 1990; Dorer and Henikoff Acids Res. 19: 5082.Fuge, H., 1967 Die Pigmentbildung im Auge von Drosophila melano-1997). However, the DNA at pugD does have one feature

gaster und ihre Beeinflussung durch den white1-Locus. Z. Zell-that could distinguish it. If further experiments demon-forsch. Mikrosk. Anat. 83: 468–507.

strate that the pigment variegation apparent in pugD is Gatti, M., and S. Pimpinelli, 1992 Functional elements in Drosoph-ila melanogaster heterochromatin. Annu. Rev. Genet. 26: 239–275.caused by gene silencing, then the repetitive DNA at

Geyer, P. K., and V. G. Corces, 1987 Separate regulatory elementspugD would be, by far, the smallest fragment of DNA yetare responsible for the complex pattern of tissue-specific and

identified that can mediate PEV. developmental transcription of the yellow locus in Drosophila mela-nogaster. Genes Dev. 1: 996–1004.We thank two anonymous reviewers for their critiques of the manu-

Geyer, P. K., M. M. Green and V. G. Corces, 1990 Tissue-specificscript. This work was supported by grant HD28694 from the Nationaltranscriptional enhancers may act in trans on the gene located

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Golic, K. G., 1994 Local transposition of P elements in Drosophilamelanogaster and recombination between duplicated elements us-

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Dominant Defects in Drosophila Eye Pigmentation Resulting From … · 1998. 11. 19. · Dominant Defects in Drosophila Eye Pigmentation Resulting From a Euchromatin-Heterochromatin