Proc. Natl. Acad. Sci. USAVol. 88, pp. 7214-7218, August
1991Genetics
Molecular cloning and analysis of small optic lobes, a
structuralbrain gene of Drosophila melanogaster
(calpain/neuronal degeneratlon/behavior/germ-lne
trnsformation/cystelne-rlch motifs)
S. J. DELANEY*, D. C. HAYWARD*, F. BARLEBENt, K.-F. FISCHBACHt,
AND G. L. GABOR MIKLOS*I*Molecular Neurobiology Group, Research
School of Biological Sciences, The Australian National University,
Canberra, ACT 2601, Australia; and tInstitutffr Biologie III,
Albert-Ludwigs-Universitat, Freiburg D-7800, Federal Republic of
Germany
Communicated by Seymour Benzer, April 29, 1991
ABSTRACT Mutations in the small optic lobes (sot) gene
ofDrosophila melanogaster cause specific cells to degenerate in
thedeveloping optic lobes, resulting in the absence of
certainclasses of columnar neurons. These neuronal defects lead
tospecific alterations in behavioral characteristics,
particularlyduring flight and walking maneuvers. We have isolated
thewild-type sol locus by microcioning and chromosomal walkingand
have established Its genetic and molecular limits. Twomatjor
transcripts of 5.8 and 5.2 kilobases are produced fromthis locus by
alternative splicing and are present throughout theentire life
cycle. Sequence analyses ofcDNAs corresponding tothese two classes
oftranscripts predict two proteins of 1597 and395 amino acids. The
first shows similarity in its carboxyl-terminal region to the
catalytic domain of a vertebrate calcium-activated neutral protease
(calpain), whereas its amino-terminal region contains several
zinc-finger-like repeats of theform WXCX2CXIOIICX2C. The second
predicted protein con-tains only the first two of the
zinc-finger-like repeats and ismissing the calpain domain. By
constructing transgenic fliescarrying a single wild-type copy ofthe
sol gene in a homozygoussol mutant background, we have restored the
normal neuroan-atomical phenotype to individuals that would have
developedmutant brains.
The molecular mechanisms by which neurons die in
eithervertebrates or invertebrates are almost totally unknown.
Thisapplies to naturally programmed cell death as well as
toneuronal degeneration caused by mutation (1-3). The
dem-onstration by Miller and Benzer (4) that nearly 50% of
themonoclonal antibodies made to the adult Drosophila braincross
react specifically to the human brain and the increasingnumber of
genes that are found to be homologous betweenflies and humans mean
that a molecular genetic analysis ofneuronal degeneration in
Drosophila may provide insightsinto basic mechanisms of nervous
system development andinto some human brain diseases. Furthermore,
mutations insuch genes that give rise to behavioral changes as a
result ofspecific alterations in neuronal circuitry are
particularlyinteresting in that they allow the identification of
neuronalcircuits that underpin the behavioral repertoires of an
orga-nism (3, 5-7). One such gene is small optic lobes (sob),
which,when mutated, produces a specific defect in the adult
nervoussystem as the result of neurodegeneration. This cell death
iscorrelated with certain behavioral changes. The molecularcloning
of this gene, which we describe in this report, thusallows us in
this instance to complete the link from moleculesto neuronal
circuitry and behavior.
In contrast to the normal methodologies of mutant isola-tion,
the pioneering work of Heisenberg and Bohl (8) used ahistological
method to isolate structural brain mutants usingaltered brain
morphology as an assay. One such mutant from
this type of screen was small optic lobes, in which
celldegeneration in the pupal optic lobes leads to a nearly
50%6reduction in the cell number of the neuropiles ofthe
medulla,lobula, and lobula plate (9, 10). Among the missing
neuronsin adult flies are many columnar neurons-e.g.,
certainclasses of transmedullary neurons that project to the
lobula(Tm neurons) or to the lobula and lobula plate (TmY
neurons)(10). However, it is important to note the specificity of
thecell types involved in, as well as excluded from, the
neuro-degenerative processes. For example, the numberof columnsin a
mutant medulla is normal, and the lamina and centralbrain appear
unaffected. Thus we are basically dealing witha brain in which the
number of cell types in each of therepetitive columns of the
medulla and lobula complex hasbeen reduced (7). These specific
optic lobe defects leavesome behaviors intact whereas others are
altered. Thus theoptomotor yaw response is qualitatively normal
whereasvisual behaviors concerning landing response and
figureground discrimination are abnormal (10).
In this communication we report the mapping of the solmutation
to a 14-kilobase (kb) region in band 19F4 in one ofthe genetically
well-characterized regions of the Drosophilamelanogaster genome
(11, 12), which has also been micro-dissected and microcloned (13)
and where long chromosomalwalks have been carried out. We describe
the cloning of thegene,§ the characterization of its transcripts,
and the resto-ration to normalcy of the brain morphology of mutant
indi-viduals by germ-line transformation. The unusual structuresof
the two predicted proteins produced by this locus and thesimilarity
of one of these to part of a vertebrate protease arediscussed.
MATERIALS AND METHODSMicrodissection and Chromosomal Walking.
Chromosomal
fragments from polytene divisions 19 and 20 were microdis-sected
and microcloned (13). Microclone inserts weremapped to subdivision
19F by using quantitative Southernblots of deficiency genotypes.
These were then used toisolate genomic clones from various D.
melanogaster A phagelibraries.DNA and RNA Extractions, Blotting,
and Hybridization.
Preparations of genomic, bacteriophage, and plasmid DNAswere
made using standard techniques (14). RNA was ex-tracted from
various developmental stages of the Canton-Sstrain using the method
of Chirgwin et al. (15). Poly(A)+RNA was isolated by affinity
chromatography on oligo(dT)-cellulose columns using a commercial
kit (Pharmacia).Poly(A)+ or total RNA was glyoxylated and
size-fractionatedon 1% agarose gels, transferred to Hybond-N
membranes
tTo whom reprint requests should be addressed.§The sequence
reported in this paper has been deposited in theGenBank data base
(accession no. M64084).
7214
The publication costs of this article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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Proc. Natl. Acad. Sci. USA 88 (1991) 7215
(Amersham), and hybridized to DNA fragments labeled byrandom
priming (16).P-Element Constructs and Germ-Line
Transformations.
The construct pSOL2 (see Fig. 1) consists of a 12.4-kb
DNAfragment cloned into the vector pW8 (17) using a
two-stepprocedure. The construct pSOL3 was made by inserting
an11-kb Not I-Spe I fragment from the sol region into the NotI-Xba
I sites in pW8. Germ-line transformation using theseand other
constructs was carried out as described (18), withsome
modifications (19). Embryos of a white genotype (w')were injected
with a mixture of a pW8-based construct(which carries the white'
gene as marker) and the helperplasmid pUChsirA2-3 (20). The number
of insertions in linesof transformant flies was determined by
Southern blot anal-ysis.The ability of the transforming sequences
to rescue the sol
neuroanatomical phenotype was determined by crossing
fliesheterozygous for single autosomal insertions to flies
homozy-gous for the sol allele KS58 and carrying the
mutationswhite-apricot (Wa) and forked bristles (f). The brain
mor-phologies of the resulting transgenic (red eyed) and
nontrans-genic (white eyed) full-sibling male progeny were
examinedby sectioning adult heads using the collar method (8)
withminor modifications.
Isolation of cDNAs. cDNA clones hybridizing to probesfrom the
sol region were obtained from a Drosophila headcDNA library (21)
and from a pupal cDNA library (22).Fourteen positively hybridizing
bacteriophages were ob-tained from 200,000 plaques with only one
cDNA being fromthe pupal library. Eleven bacteriophage inserts were
sub-cloned into pEMBL8+ (23) for characterization by restric-tion
endonuclease mapping and DNA sequence analysis.DNA Sequence
Analysis. The insert of cDNA AcO.22 (see
Fig. 1) was gel-purified, sonicated, and fragments of 0.3-1.0kb
were cloned into the Sma I site ofM13mplO
(Amersham).Single-stranded bacteriophage DNAs were sequenced
usingSequenase (United States Biochemical) in accordance withthe
manufacturers' instructions. Compressions were re-solved using dITP
in place of dGTP and single-strandedregions were completed using
specific oligonucleotide prim-ers. The partial sequences of other
cDNAs shown in Fig. 1were determined from restriction fragments
subcloned intoM13 bacteriophage or by double-stranded sequencing of
theplasmid subclones using M13 or specific
oligonucleotideprimers.
RESULTS AND DISCUSSIONGenetic Localization and Molecular Cloning
of the sol Gene.
The sol locus has been cytogenetically localized adjacent to
DISTALXS'II
the flightless (fli) and sluggish (sig) complementation groupsin
subdivision 19F of the polytene X chromosome (11).Deletion mapping
further indicates that sol lies between thebreakpoints of the
chromosomal rearrangements 16-129 and2/19B (Fig. 1). In addition,
deficiency 2/19B uncovers thesluggish phenotype but not the sol
phenotype (data notshown). This establishes the gene order in a
distal-proximaldirection as flightless-small optic
lobes-sluggish-cen-tromere.
Minilibraries were constructed from most of divisions 19and 20
to obtain DNA from sol and nearby neurogenic genes(11, 13).
Microclone inserts were mapped to regions contain-ing
complementation groups of interest (fli, sol, and sig) andthese
inserts were then used to initiate chromosomal walks.This yielded a
contiguous stretch of cloned DNA 250 kb longthat will be described
in detail elsewhere. The molecularlocations ofa number
ofchromosomal breakpoints were thenestablished using probes from
this cloned DNA. The intervalbetween the breakpoints of
deficiencies 16-129 and 2/19Bgenetically defines the sol locus
(Fig. 1). Molecular evidencethat this 13.7-kb region contains the
sol gene was obtained byanalyzing the DNA of flies bearing ethyl
methanesulfonate-induced sol mutations on Southern blots. Twelve
indepen-dently derived sol mutations were examined in this way
andone of these, sol'078, was found to have a 588-base-pair
(bp)deletion relative to the parental strain in which the
mutationwas induced (Fig. 1). The remaining 11 alleles gave
noobvious changes in restriction fragment length patterns.
Transgenic Organisms. Two constructs from the sol locus(Fig. 1)
were transferred into the germ line of embryos bystandard
P-element-mediated transformation techniques.Appropriate genetic
crosses subsequently introduced a singleautosomal copy of either
pSOL2 or pSOL3 into mutantsolK5S8 males. Relative to a wild-type
brain (Fig. 2A), theS01K5S8 mutation causes a nearly 50% reduction
in the sizes ofthe neuropiles of the medulla, lobula, and lobula
plates (Fig.2B). However, soIKSS8 males carrying an autosomal copy
ofeither pSOL2 or pSOL3 have optic lobes indistinguishablefrom wild
type (Fig. 2C). This indicates that the sol locus andits
appropriate control regions must lie entirely within the10.3-kb
region that is common to these two constructs.
Transcription of the sol Landscape. Radiolabeled DNAfragments
from the region between the breakpoints of defi-ciencies 16-129 and
2/19B were used to probe Northern blotsof various life cycle stages
and these detected two majortranscripts of 5.8 and 5.2 kb (Fig.
3A). Both transcripts arepresent throughout development, although
the level ofexpression in larvae is less than in other
developmentalstages. This transcription unit is the only one
contained
PROXIMALS RB KS B X BSR Sp X X
II 1111 I I
, i, . r cm. i- Ac0.22
= = cm . r---'" -AcO.32oCD r .. i , .3o CL -l l
FZ < ,,,z^,,,seeZ e e^7777777ZZZZ= pSOL27777,7777 7
777,-,=z11 z r z --- .1 z pSOL3
FIG. 1. Molecular map of the sol locus. The tophorizontal line
is a restriction map of the sol region.Restriction sites are shown
for EcoRI (R), BamHI(B), Sal I (S), Xho I (X), Kpn I (K), and Spe I
(Sp).Solid horizontal boxes show the exon map derivedfor the two
sol transcripts; transcription is from leftto right. The open
horizontal boxes depict the eightlongest cDNA clones used to
determine this exonmap. The extent of genomic DNA sequences
notremoved from the chromosomal deficiencies 16-129 and 2/19B and
the mutant allele sol'0'8 areshown by solid boxes in the lower half
of the figure(the semi-solid ends of the solid boxes indicate
thatthe normal chromosome continues proximally anddistally). The
uncertainties in the positions of thebreakpoints of the chromosomal
deficiencies areshown by open boxes. The hatched boxes denotethe
DNA fragments in the constructs pSOL2 andpSOL3 used for DNA
transformation.
5.8 kb
5.2 kbTranscripts {
cDNAs {
Wild typesol 10782/19B16-129
Transformingsequences
Genetics: Delaney et al.
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Proc. Natl. Acad. Sci. USA 88 (1991) 7217
1
MGTISSVLQWSCTKCNTINPTESLKCFNCGTVRKVFPQQQQQQHRSSSITASWTADDALEQEQAEKGQERDKEKGRAAVA
81
RSEYKHVYKSLLRGCLKRPQRNSQNLPANCVDCEDTRKYIKSSIELYRHFSNPALNRIWVCHACGTDNSSVTWHCLICDT
161
VSYLAPIYKDAIAADRGQDLAGSLGNRGELLAADHSHPHHHHHYLHQELEEQHQHQLHSQHLHKRHLKGRSASGSGSGPG
241
SGSGLRRTQSLSTAIDKSASGRSCHICYANNQSKDIFNLPQIKPAPQLTGIPPVAACSNSRFAIANDTFCRRKQNNNNKN
321
QNHKVVRESGAKRKYNFTITTLSRSAAKDAGHGQMKPLRQVVNLNLNLQQEPQQKSPANPQQLHRTQREPAAVSMNPTQ
401
FTIPRNGVFIAVNEWSEPMASSSSVSSSSNHHHHHHSNSNSNSSGNSNIINNNSSSSSGSNKLYENECVALAQQQLRAAA
481
AQAAQAAATAVAIASSPSAKAMAEPAPTATMPIYAQVNKQHKLKKKQQIASESQTNNNTGSGEIADAVSESLTAGLGTST
561
DGSGEASESESQVEEHSIYAKVWKGPRKATESKIMHDPGSSSRLSGAASAAAGTASAGAIAAGVGAAAASRHDNKTQLGN
641
GSRSKMiWICIKCSYAYNRLWLQTCEMqCEAKAEQQQQQLHLQQQQQQQQQHHHHHHHHLQQQQAEAPRDEPWTCKKCTLVN721
YSTAMACVVCGGSKLKSISSIEDMTLRKGEFWTCSHCTLKNSLHSPVCSACdKSHRQPQLSMAMEAVRERPDGQSYEEQDA
801
AAVGGGGGSAHQSGANEVKAPTALNLPLTSVALPMPMLQLPTSTAAGLRGSRSPSPRMQLLPSLQQQRNSSSSGAIPKRH
881
STGGSIVPRNISIAGLANYNLQQGQGVGSASVVSASGAGSGAGAVGASTSSKKWQCPACTYDNCAASVVCDICSSPRGLA
961
SAVLGEALGRKSVRVALTPADIRQESKLMENLRQLEETEALTKWQNIIQYCRDNSELFVDDSFPPAPKSLYYNPASGAGE
1041
GNPVVQWRRPHEINCDGGAYPPWAVFRTPLPSDICQGVLGNCWLLSALAVLAEREDLVKEVLVTKEICGQGAYQVRLCKD
1121
GKWTTVLVDDLLPCDKRGHLVYSQAKRKQLWVPLIEKAVAKIHGCYEALVSGRAIEGLATLTGAPCESIPLQASSLPMPS
1201
EDELDKDLIWAQLLSSRCVRFLMGASCGGGNMKVDEEEYQQKGLRPRHAYSVLDVKDIQGHRLLKLRNPWGHYSWRGDWS
1281
DDSSLWTDDLRDALMPHGASEGVFWISFEDVLNYFDCIDICKVRSGWNEVRLQGTLQPLCSISCVLLTVLEPTEAEFTLF
1361
QEGQRNSEKSQRSQLDLCVVIFRTRSPAAPEIGRLVEHSKRQVRGFVGCHKMLERDIYLLVCLAFNHWHTGIEDPHQYPQ
1441
CILAIHSSKRLLVEQISPSPHLLADAIISLTLTKGQRHEGREGMTAYYLTKGWAGLVVMVENRHENKWIHVKCDCQESYN
1521
VVSTRGELKTVDSVPPLQRQVIIVLTQLEGSGGFSIAHRLTHRLANSRGLHDWGPPGATHCPPIENVHGLHAPRLIT
FIG. 4. Predicted amino acid sequence of the large sol protein.
The amino acid sequence of the long open reading frame present in
thecomposite sequence of the two overlapping cDNAs AcO.22 and
AcO.32 is shown. The 5562-bp DNA sequence used to derive the amino
acidsequence was determined on both strands with a single
nucleotide being sequenced on average 6.7 times. The open reading
frame is shown fromthe first methionine at nucleotide 264 to a
termination codon at nucleotide 5055. The cysteine-rich motifs in
the amino-terminal halfofthe proteinare boxed as is the region in
the carboxyl-terminal half (heavy box) that shows similarity to the
large subunit of vertebrate calpains. The frameshift introduced by
the use of the short form ofexon 3 results in a protein product
that terminates at amino acid 393 and has two additional
aminoacids. An opa repeat is present between amino acids 673 and
702.
calpain are present in the large sol protein. This suggests
thatthis sol protein has protease activity. In addition,
sequenceidentity at the active sites and the surrounding
sequencesimilarity may reflect similar substrate specificities for
thetwo proteins. The known substrates of vertebrate calpainsare
relatively limited and include regulatory molecules suchas protein
kinase C, which is activated by calpain after limitedproteolysis
(32). Other calpain substrates are components ofthe cytoskeleton
(33). It is possible that the sol protein isrequired for the
modulation or breakdown ofthese regulatoryor structural molecules
during the differentiation of certainclasses of columnar
neurons.The large sol protein is not the Drosophila calpain since
its
predicted size (175 kDa) exceeds that of partially
purifiedDrosophila calpain that is only 83 kDa (34). Furthermore,
theregion of similarity between sol and calpain is restrictedalmost
exactly to the protease domain and we are unable tofind E-F hand
sequences that occur in domain IV of the
vertebrate calpains. These sequences are thought to
beresponsible for the calcium activation of calpains (35). It
thusappears that the large sol product represents a protease
thatmay not be activated by calcium ions. The lesion in the
sol'078allele removes almost all of the DNA that encodes
theputative proteolytic domain present in the large sol protein.It
would therefore seem that the proteolytic domain of thelarge sol
protein is required for sol gene function in the opticlobes. The
function, if any, of the small sol product is,however, still
enigmatic. It may become clearer if mutationscan be found that do
not affect the large sol product butperturb the small one.The
larger predicted protein product of the sol locus is
intriguing in that it is an amalgam of zinc-finger-like
motifsand a putative protease domain. In this sense this sol
proteinwould seem to be a good example of exon shuffling wherebya
protease "'module" has been incorporated into a number ofdifferent
proteins. Whether the zinc-finger-like motifs confer
1000o 1,500
/ . t0
FIG. 5. Dot matrix comparison of the large sol protein and the
large subunit of human calpain. The amino acid sequences were
comparedwith the University of Wisconsin Genetics Computer Group
package programs COMPARE and DOTPLOT (26) with a window of30 and a
stringencyof 15. The four domains present in vertebrate calpains
are indicated. Domain II corresponds to the proteolytic domain.
Genetics: Delaney et al.
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Proc. Nat!. Acad. Sci. USA 88 (1991)
801
LFVDDSFPPAPKSLYYNPASGAGEGNPVvQwaHEINCDGGAYPPWAWVRTPLPSDICQGVLGNCWLLSALAVLAEECal
LFRDEAFPPVPQSLGYKDLGPNSSKTYGIKNKRPTELLSN-PQF----IVDGATRTDICQGALGWLLAAIASLTLNDT8ol
LVKEVLVTKEICGQ---GAYQVRLcKDGKWTVL-VDDLLPCDKRGHLVYSQ-AKRKQLIVPLIEKAVAKIHGCYEALVSGCal
LLBRVVPHQQSFQNGYAGIFHFQL1QFGZWVDVVVDDLLP-IKDGKLVFVBSAEGNEFWSALLflAYAKVNGSYKALSGG
vaol RAIEGIATLTGAPCESIPISSLPMPSRDELDKDLiWAQLLSSRCVRFLMGASC K
EZYQQRGLRPRBAYSV
::11::::11: 1 I::::: :1:1: ::I1:1 ::::I. I 1 111Cal
STSEGFEDFTGGVTENYELRKAPSDL--YQIILKALERGSLLG
-----C-SIDISSVLDNEAITF--KKLVKGHAYSV
Sol
LDVKDI----QGHRLLKLRNPNGHYSNRGDWSDDSSLW--TDDLRDALtPHGASEGVFNISFZDVLNYFDCIDI::::
1 :1 :::11III: :1 1:11 1:11' 1 :1 : :. :1 11:11 I::1:1
Cal
TGAXQVNYRGQWSLIRMRNPIPGEVENTGAWSDSSSENNNVDPYEIRDQLRVLDGKF.USFRDrHWFTRLEI
FIG. 6. Alignment of the regions showing similarity between the
large sol protein and human calpain (Cal). The similar regions were
alignedusing the program FASTA. Matches between identical amino
acids are shown by vertical lines and conservative amino acid
matches are shownby colons. The amino acid residues of the active
site of the proteolytic domain of calpain are indicated by the
arrowheads above the alignedsequences.
DNA-binding ability upon the sol products remains to
bedetermined. They may, for example, play a role in theactivation
of the protease portion of the sol product in amanner comparable to
that of the cysteine-rich motifs inprotein kinase C that, it has
been suggested, are required forphorbol ester (and presumably
diacylglycerol) activation ofthe kinase activity (25).The
characterization of genes involved in the development
of the Drosophila nervous system has to date concentratedon
those molecules that are required in embryonic or reti-nular cell
lineages. Although there are at least a dozen locithat when mutated
lead to an elimination of nerve cells fromthe optic lobes (7, 36,
37), few have been molecularlycharacterized. Ofthese, two (asense
and elav) appear to haveprotein products that are either putative
transcription factorsor are involved in gene regulation (38, 39).
Therefore, thecombination of putative transcriptional activation
domainswith and without a putative proteolytic domain in the
solproducts raises intriguing functional possibilities. The
dem-onstration that our transgenic individuals have fully
restoredbrain circuitry means that the molecular dissection of
thecontribution of the two domains of the sol gene products tothe
sol phenotype and their effects on the embryonic andadult nervous
systems can now be determined. In addition,the generation of
transgenic strains in which the sol gene isectopically expressed
may reveal how altered neuronal cir-cuitry results in altered
behavioral repertoires.
We thank Fiona Hall and Jane Olsen for excellent
technicalassistance, Kevin O'Hare for fly and plasmid stocks, Gert
de Couetfor the initial localization ofthe 16-129 breakpoint,
Elaine Napper forhelp in preparation of this manuscript, Martin
Heisenberg for pro-viding the mutant alleles of sol, and Ute Bauman
for performing anearly screen for transcripts in the sol/slg
region.
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