Copyright 0 1995 by the Genetics Society of America

Molecular and Mutational Analysis of a -Family Member Encoded by the flightless I of Drosophila melanogaster

Heinz Gert de Couet,*’+Keith S. K. Fong,* A. G. Weeds,$ P. J. McLaughlin5 and George L. Gabor Miklost211 *Department of Zoology, and Department of Genetics and Molecular Biology, The University of Hawaii, Honolulu, Hawaii 96822, ’Integrative Biology Laboratory, Research School of Biological Sciences, Australian National University, Canberra City, ACT 2601, Australia, $Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, England, $Department of Biochemistry, University of Edinburgh, Edinburgh EH8 9xD, Scotland and ”TheNeuroscience Institute, San Diego, California 92121 Manuscript received April 10, 1995 Accepted for publication July28, 1995

ABSTRACT Theflightless of Drosophila melanogasterhas been analyzed at the genetic, molecular, ultrastructural and comparative crystallographic levels. The gene encodes a single transcript encoding a con- sisting of a leucine-rich amino terminal half and a carboxyterminal half with high sequence similarity to gelsolin. We determined the genomic sequence of the flightless landscape, the breakpoints of four chromosomal rearrangements, and the molecular lesionsin two lethal andtwo viable alleles of the gene. The two alleles that lead to flight muscle abnormalities encode mutant exhibiting aminoacid replacements within the S1-like domain of their gelsolin-like region. Furthermore, the deduced intron- exon structure of the D. melanogaster gene has been compared with that of the Caenorhabditis elegans homologue. Furthermore,the sequence similaritiesof the flightless protein with gelsolin allow it to be evaluated in the context of the published crystallographicstructure of the S1 domain of gelsolin. Amino acids considered essential for the structural integrity of the core are found to be highly conserved in the predicted flightless protein. Someof the residues considered essential for actin andcalcium binding in gelsolin S1 and villin V1 are also well conserved. These data are discussed in light of the phenotypic characteristics of the mutants and the putative functions of the protein.

HE analysis ofmutations that impair theflight abili- product is also essential for thesurvival ofthe organism T ties of Drosophila melanogaster and their rescue by to adulthood.This lethality can be rescued by germline germline transformation has not only provided a sig- transformation of the appropriate constructs of the ge- nificant foundation for the understandingof early myo- nomic flightless landscape. genesis, myofibrillar assembly, and the mechanics of At the ultrastructural level, several Pightlessmutants, muscle contraction, but has also led to significant in- while undergoing apparently normal embryonic and sights into the neuronal and behavioral basis of motor larval development, nevertheless exhibitabnormal control.Furthermore, the transgenic methodologies flight muscle morphology. The flight muscle fibers of that have been brought to bear on the Drosophila ge- these viable flightless mutants show severelydisrupted Z- nome have allowed rapid dissection of function at the discs and are often associated with “striated bundles” genetic, molecular, and behavioral levels. In this con- that have the structural appearance of Z-band material text our laboratories have subjected one such locus, (KOANAand HOTTA1978; MIKLOSand DE COUET1990). termedflightless-I, located on theXchromosome in sub- The myofibrils of affected muscles fray at their periph- division 19F, to a comprehensive analysis at a number eries, indicating problems during myoblast differentia- of different levels (MIKLOSand DE COUET1990). We tion. Although neither cell degeneration nor cell death have previouslydemonstrated by genetic complementa- is apparent, muscle architecture is abnormal and flies tion analysis that several independently isolated viable are incapable of flight. flightless mutations are all alleles offli-I (MIKLOSand DE At the molecular and cell biological levels,severe COUET1990). Furthermore, theflightless phenotype is disruptions of the flightless transcription unit lead to revealed when a number of lethal mutations are made death during embryogenesis (MIKLOSand DE COUET heterozygous with any of the viable alleles of the locus. 1990).Furthermore, germline clone analysisreveals In addition, individuals heterozygous for overlapping that the embryo does not undergo thenormal processes chromosomal deficiencies uncovering the locus die of cellularization. Although nuclei migrate to their posi- during thepupal stage, indicating thattheflightlessgene tions in the egg cortex following 13 rounds of mitotic divisions, they lose their position at the periphery be- Corresponding nuthor: Heinz G. de Couet, Department of Zoology, The University of Hawaii at Mama, 2538 The Mall, Honolulu, HI fore cellularization is complete. Abnormal mesodermal 96822. E-mail: [email protected] invagination ensues; defective gastrulation and finally

Genetics 141: 1049-1059 (November, 19%) 1050 Couet H. G. de et al.

death follows (PERRIMONet al. 1989), indicating a ma- or a rearranged togetherwith a Canton-S chro- ternal requirement for the Jlightkss gene product. mosome. Molecular protocols: Genomic DNA from 0.5 to 3 g of The protein product encoded by the jlightless gene adult female flies or fromfrozen heads offlies of appropriate consists of two distinct regions. The aminoterminus car- genotypes, as well as DNA from bacteriophage A and from ries 17 repeats of a leucine-rich motif implicated in plasmids, was purified by standard methods (SAMBROOKet al. macromolecular interactions, while the carboxytermi- 1989). Two D. melanogastergenomic DNA libraries in bacterio- nal half of the molecule shares significant sequence phage A EMBM (a gift from VINCXNZOPIRROT~;~) were screened with subcloned DNA fragments. The libraries con- similarity with the gelsolin-like family of actin severing sisted of size-selected embryonic DNA from either Canton-S and nucleating factors. We have isolated homologues of or Oregon-R strains. Southern blotting, colony hybridization, this gene from both the nematodeCaenorhabditis ekgans plaque-lift techniques, and DNA hybridization were per- and from Homo sa$+ns (CAMPBEL~Let al. 1993).The formed according to SAMBROOKet nl. (1989). DNA sequencing cytogenetic location of thehuman gene is known utilized the chain-termination method (USB Sequenase kit) and a-""S-dATP. Compressions were resolved using dITP and (CHENet al. 1995), and the locus is includedin re- the inclusion of 40% formamide in sequencinggels. Genomic arrangements that lead to clinical phenotypes. DNAwas sequenced on both strands by a combination of Both the existence of lethal alleles of the flightless subcloning fragments of genomic DNA from phage A into gene in D. melanogasterand the high degree of sequence M13mplO phagemids followed by primerdirected second conservation between the worm, fly and human homo- strand synthesis at specific sites using custom synthesized oli- gonucleotides. The analysis of translational products, data- logues are indicators of a potentially central function base screening, and sequence alignments were performed us- of this molecule throughoutthe metazoa. We have ing PCGENE software (IntelliGenetics Inc., Mountain View, therefore begun to subject this locus to athorough CA) and the University of Wisconsin GCG suite of sequence molecular investigation, and we are studying the cell analysis programs. biological aspects of gene dosage effects in transgenic Northern blotting: Total cellular RNA was extracted from developmentally staged whole animals by routine methods organisms, as well as analyzing various mutant alleles (CIIIRGWINet al. 1979). Poly(A)+ RNA was selected from this and making comparisons to similar molecules in other fraction by oligo-dT-cellulose chromatography(Pharmacia phyla. Detailed crystallographic information is now poly A purification kit). Before electrophoresis, RNA samples available for both gelsolin-like domains (MCLAUGHLIN were glyoxylated at 60" for 1 hr and separated through 1% et al. 1993; MAKKus et al. 1994) and for leucine-rich agarose gels in 10 mM phosphate buffer, pH 7.0 at 3 V/cm in a circulatingbuffer tank. The relative amounts of poly (A)' repeat domains (KOBEand DEISENHOFER1993). In ad- RNA in each sample were normalized by hybridization with dition, the in vitro mutagenesis studies of the gelsolin a D. melanogaster adh probe. gene may allow us to put the analysis of D. melanogaster Sequence analysis of flightless mutations: Specific regions mutants into a molecular perspective. of theJlzghtlpsslandscape fromflies carrying the mutantalleles Inthis communication we report the genomic se- jlil'and jZiOz (MIKLOSand IN COUET1990) were amplified as overlapping sections ofthe translated part of the transcription quence,the location ofseveral chromosomal re- unit by the PCR. Oligonucleotide primer pairs of 25-30 nu- arrangements affecting the locus, and the molecular cleotides were used to cover the transcribed region. Thisstrat- lesions of two lethal alleles of the gene.We also examine egy also incorporated all introns, as well as 120 nucleotides the intron/exon boundaries of the gene in an evolu- upstream of the translational start site. The appropriateDNA tionary context. Finally, we present the DNA sequence fragments were amplified by Taq polymerase (Perkin-Elmer, Norwalk, CT) using standard protocols for template denatur- analysis of two viable alleles of the gene. These data ation, annealing, and primerextension. Amplified DNA prod- indicate that aregion of the S1-like domain of the mole- ucts were isolated by agarose gel electrophoresis, purifiedwith cule implicated in the binding of a calcium ion in gel- Gene Clean (BiolOl, LaJolla,CA), and subsequently cloned solin may be responsible for the observed flightless phe- into the pCRII plasmid vector (InVitrogen, San Diego, CA). notype. Inserts of genomic DNA were completely sequenced using the flanking and internalprimers on either strand. Any alter- ations in the DNA from the mutantswere verified by reampli- MATERIALSAND METHODS fication of the respective target DNA using the PCR followed by direct sequencing of the amplified DNA with appropriate Stocks and : Details of melanogaster stocks D. primers or sequencing of the cloned product. and the cytogenetic characteristics of the chromosomal re- arrangements areas previously described (SCHALETand LEFE- VRE 1976; HOMYKand SHEPPARD1977; LEFEVREand WArKINS RESULTS 1976; GREENet al. 1987; MIKLOSet al. 1987; PERRIMONet al. 1989; MIKLOSand DE COUET1990). To avoid strain-specific Molecularand cytogenetic characterization of the polymorphisms, DNAwas extractedfrom specially con- structed stocks in which the rearrangement andlethal-bearing flightless landscape: The various subregions of the base chromosomes were heterozygous with the wild-type chromo- of the Xchromosome were accessedby microdissection somes from which they were derived. Thus, Df(l)GA104, of subdivisions 19 and 20, by construction of a chromo- Df(l)GE263,and l(l)HC183 were placed over the M56i (Am- somal mini-library inbacteriophage X (MIKI~OSet ul. herst) chromosome, whereas 1(1)D44 (EEKENet al. 1985) and Ilf(l)2/19Bwere heterozygous with their parentaly mei-Y" mei- 1988), and by mapping of the single-copy DNA inserts 41'J5chromosome. In cases where the parental chromosomes against three large deficiencies denoted Df(l)B57, were unavailable, heteraygotes consisted of a lethal-bearing Qf(l)S54, and Df(llJC4, which together span the entire abcde fahi ik Im no

uvw X Y z E EE E E E E

5' 73' f/i 17-257 - GA104 - 16-129 IGE26.3 - 044 -I.....+ a198

5.4kb FI(;IW 1 .-(hetic and molecularorganization of the I9EF-20 region of the Xchromosome of I). mduttogm/rr.The extents of the chro~nosomaldeficiencies pcrtinrnt to this study arc shown at the top and are shown relative to the DNA from each of the different genotypes was cleaved genetic complementation gro~~ps(PI.:KRIMoS r/ nl. 1989).The with a variety of restriction endonucleasesto determine tnolecular location of thc breakpoints antl extent of these the approximate location of the lesions (Figure 2). A% deficiencies in relation to the physical map of the locus and well as the breakpoint determinations, we found that the /lig/t~lr.wtranscription unit is shown at the bottom. W,the smallest restriction fragments to which the respectivc cI11.omtr the lethalallele, denoted l(l)D44, representsa defi- soma1 breakpoints were mapped: - - - in chromosome ciency of -400 bp. Furthermore, I!f(1)2/19Bis not only 1(1)1)44, the extent ofa small deliciency antl the approximate deficient for a region outside and more proximal to site of an insertion in 1(1)2/l YIL The entire IT-kb region has the landscape shown here but has an insert of "5.4 kb, I)rcn sequenced and is the srrhjcct of a separate communica- which is ven, probably due to a mobile element (data tion (G. L. G. MIKI.~~unpublished results). IhRI fragments used as probes for the detection of transcripts and for not shown). Theseresults are summarized in the lower breakpoint mapping in Figurr 2 are I;~hclcclU, V, M', X, Y, panel of Figure 1. antl Z. We also examined genomic DNA from a number of other Jliglt/lm mutants, namely the lethals l(I)IV2, region (Figure 1). Finer mapping was achieved for the L(I)HC183, and 1(1)1

Analysis of the genomic sequenceupstream from the the mutations represent transitions from G -+ A. Both 5’ end of several cDNA clones revealed no regions with lead to the same conservative amino acid replacement obvious sequence similarity to eukaryotic promoter con- of Gly -+ Ser. Interestingly, both mutations affect the sensus sites. However,a T/A-rich region upstream from F1 domain of the gelsolin-like portion of the protein, the presumed transcription initiation site may substi- a domain forwhich the three-dimensional structure has tute for the TATA box. now been elucidated in thegelsolin protein (MCLAUGH- We have previously cloned cDNAs of the C. elegans LIN et al. 1993), allowing us to evaluate the possible homologue of the flightless gene (CAMPBELL et al. 1993), effects of these lesions at the functional level in vivo. and fortuitously, this homologue lies within the recently DNA from the D44bearing chromosomedisplayed a released 2.2Megabases of the C. elegans genomic se- restriction fragment with an alteredlength when quencing project (SULSTONet al. 1992; WATERSTONet probed with fragment X that contains the 5’ end of the al. 1992). This has allowed us to compare the genomic Jlzghtless transcription unit, including the leucine-rich organizations of the D. melanogaster and C. elegans genes repeats of the predicted protein (Figures 1, 2 and 4). as well as to place them within the context of the pre- Additional Southern hybridization indicated that the dicted protein structures. lesion resides within the proximal 1 kb of this fragment. While the D. melanogaster gene is only split by three To determine the precise nature of the rearrangement, Molecular Analysis of flzghtless-Z 1053

1 73 145 217 289 361 433 505 1 577 649 C "" 722 3746 -P 19 1021 Ly~pLysAlaHisThrAlaLysGlyLysSerPr~V~lGl~Ph~PheHis~urrgSer~~ly0lyAl~- 794 43 866 67 938 91

FIGURE3.-Nucleotide sequence of the genomic DNA from the flightless region of the Canton-S strain of D. melanogas- terand its conceptual translation corresponding toEcoRI frag- ments X and Y in Figure 1. Corresponding cDNA sequence is shown in bold letters. Translational STOP signals and the polyadenylation consensus sequence are underlined. The se- quence has been deposited in GENBANKunder the accession number U28044 1054 H. G. de Couet et ul.

A.

FIGURE4.-Genomic sequence alterations and polymorphisms of the flightless transcription unit, relative to the wild-type sequence of the Canton- S strain. Intronic sequences are shown in bold ~~ ~ ~ letters. (A) Sequence deleted in deficiency 044. B. The underlined sequence is unique to the 044 deficiency chromosome andmay represent a tar- Jio2 A 2234 GGCGACTGCTACATAGTCCTCMGACCAAOTTCGATGACCTGGGCCTGCTCGATTGGG~TATTCTTTTGG get site duplication of the sequence 5' of the de- 523 GlyAspCysTyrIleValLeuLysThrLysPhespAspLeuGlyLeuLe~spT~Glu11ePhePheTrp ficiency breakpoint. Arrows indicate the first and Ser the last missing base, respectively, in the wild-type C. sequence.Highlighted nucleotides represent part of intron I. Sequence numbering as in Figure $1 f A 3. (B and C) Point mutations found in the viable 2450 GMGTCATCTACATCGAGGGCGGACGCACA~TAC~~CTATACCATAG~GAGA~ATACACATCACC 595 GluValIleTyrIleGluGlyGlyArgThrAlaThrGlyPheTyrThrIleG1uGl~etIleHisIleThr flightless alleles $if3 and $io', respectively, and Ser amino acid replacements occurring as a conse- D. quence. (D) DNA sequence changes within the transcribed region of the flightless gene observed Inn T in strain Oregon-R (OR, shown above Canton-S CCAGACTGCGTCTACAATOTOGTCACCTTGGTOCGRCTG~TC~AGCGAC~C~~T~CCGMCTGACT ProAspCysValTyrAsnValValThrLeuValArgLeuAs~euSerAspAsnGluLeuThrGluLeuThr sequence), as determined by cDNA sequencing. 0 T 1442 GCCGGTGTAGAGC'ITTGGCAGCGTCOAOTCCCCTG~TCTGTCGCGG~TC~CTGGTTGCGCTGCCGGCT Intronic polymorphisms were revealed by se- 259 AlaGlvValGluLeuTrpGlnArgLeuG1uSerLeuAsnLeuSerArgAsnGlnLeuValAlaLeuProAla quence analysis of PCR-amplified genomic DNA from several mutant strains in the genetic hack- ground of Oregon-R. Spaces were manually in- serted in the sequence to provide optimum align- ment of the two sequences where deletions or insertions occurred. Sequence differences were also discovered between cloned Canton-S DNA and DNA amplified byPCR from a laboratory strain. The 7-bp sequence GAAGTAA between position 353 and 360 was only identified in DNA derived from a A phage clone. This insertion was never observed in PCRderived DNA from either Canton-S, Oregon-R, or any of the mutant strains analyzed.

we amplified DNA from this deficiency-bearing chro- at the deletionsite is duplicated twice in the deficiency, mosome by primer-directed PCR. DNA sequencing re- possibly as a consequence of a transposition event in- vealed that nucleotide positions 233-624 are deleted volving target site duplication before imprecise exci- in deficiency D44, which comprise sequences from 20'7 sion, which may have led to the deficiency. bases upstream of the presumed transcription initiation site, including thetranslational start site at position 505, DISCUSSION and 66 bases of the first intron (Figure 4). The defi- ciency is therefore likely to represent a null mutation. The jlightless gene encodes a novel member of the We have found that the 5' flanking sequence AAACC growing superfamily of gelsolin-related proteins, linked to a series of leucine-rich domains. Gelsolins are actin D. melanogaster filament-severing and -capping proteins that are regu- lated by both calcium and phosphoinositol (reviewed

1000 bp by VANDEKERCKHOVE1990; HARTWIGand KWIATKOWSKI 1991; WEEDSand IMACIVER 1993). The gelsolin family of proteins is composed of a series of repeating seg- ments of -130 amino acids, each characterized by its

I pattern of conserved residues. Gelsolins, villin, and ATG C. elegans TAA Pightless contain six domains. Equivalent severing pro- FIGURE5.-Genornic organization of thejightless transcrip- teins from lower eukaryotes, severin from Dictyostelium tion unit of D. melunoguster and that of its C. elegans homo- discoideum and fragmin from Physurum polycephalum, logue. Intron/exon junctions are shown relative to the pro- contain only three segments; inthis they are similar posed domain structure of the flightless protein by A. Numbers above or below the symbols indicate the phase of to certain capping proteins from mammalian tissues, the intron boundaries relative to the nucleotide position in known as Cap G (Yu et al. 1990; PRENDERGASTand ZIFF the affected codon, using the adopted nomenclature. 1991; DABIRIet al. 1992; MISHRAet al. 1994), and an Molecular Analysis of flightless-Z 1055 actin-modulating protein from an earthworm (GIEBING in accordance with the “introns separating domains” et al. 1994). Sequence data are now also available for hypothesis. Similarly, none of the introns found in the quail, a D. melanogastm gene that encodes villina homo- gelsolin-like part of the C. elegans and D. melanogaster logue (~~AHAJAN-MIKLosand COOLEY1994) and a Dro- flightless genes corresponds to each other or to other sophila homologue of gelsolin (HEINTZELMANet al. known members of the gelsolin family. As was pointed 1993; STELLAet al. 1994). out (STOLZFUSet al. 1994), chimeric proteins with a The three-dimensional structures of G1 of gelsolin relatively short evolutionary history are less instructive (MC~UGHLINet al. 1993) andthe equivalent V1of in terms of intron/exon evolution, because exon shuf- villin (MARKUS et al. 1994) have been solved. These show fling mayhave favored chancecorrespondences be- that the characteristic segmental repeat reflects the un- tween protein secondary structure and the distribution derlying apolar core of the structural fold (MCLAUGH- of exons. Despite this qualification, none of the introns LIN et al. 1993), as originally predicted by MATSUDAIRA found in either theC. elegans or D. melanogasterflightlesess andJANMEY (1988). This has been further supportedby genes coincides with domain boundaries of the protein structure analysis ofsegment 2 of severin(SCHNUCHEL et or with recognizable secondary structure motifs. Al- al. 1994). Thestructure of the complex of G1 withactin though predictions relating to the structural location has also provided details of the contact residues at the of introns suffer from the difficulty of defining the interface and insights into the mechanism of filament boundaries of a domain, in the case of leucine-rich severing. Interestingly, two members of the gelsolin repeat motifs, these have been clearly established. family possess additional domains also found in other The number and distribution of introns within the proteins, which convey specific aspects of their physio- flightless homologues in C. elegans and D. melanogaster is logical roles. Villin carries a somewhat inappropriately open to a number of interpretations. First, the precur- named “headpiece” at its carboxyterminal end that is sor that gave rise to the modernflightless gene may have responsible for its actin-bundling activity. arisen very early during metazoan evolution, and its The flightless protein exhibits 17 repeats of a com- original intron/exon structure was not preserved. The mon leucine-rich sequence motif at its amino terminal second interpretation is that introns disrupted the se- end. These repeats appear to be related to those of quence after the morecomplex gene evolved. Gelsolin- porcine ribonuclease inhibitor where they represent a like domains exist in protists (severin, fragmin), but new class of a/P fold (KOBE and DEISENHOFER1993, prokaryotic homologues have yet to be discovered. If 1994). Similar repeats are presentin many types ofpro- theemergence of gelsolin-like proteins,including teins where they mediate homotypic and heterotypic flightless, was coupled to the appearance of actin-based macromolecularinteractions (reviewed by KOBE and contractilestructures in eukaryotic evolution, then DEISENHOFER1994), e.g., in cell-adhesion molecules their presence in prokaryotes seems unlikely. Proteins (KRANTZ and ZIPURSKI 1990; LOPEZ,1994), develop containing leucine-rich repeats occur ubiquitously mentally importantreceptors (KEITH and GAY 1990; among eukaryotes and some prokaryotes (VENKATESAN CHANGet al. 1993) and transmembranereceptors et al. 1991),but their restricted distributionamong (SCHNEIDER andSCHWEIGER 1991). pathogenic prokaryotes suggests acquisition from eu- Intron/exon structure and protein domain organiza- karyotic hosts. tion offlightless: The location of intervening sequences Developmental implications of flightless expression: in coding genes is thought by some to be related to Most of the gene productsinvolved in egg-polarity and the domain structure of the proteins they encode. In thecomponents of the cytoskeleton that provide a addition, the location of some introns is clearly con- framework for their correct distribution in the egg are served over long evolutionary periods of time and is produced by nurse cells, which remain connected to retained even after duplication events. The currentde- the oocyte until the endof oogenesis. Since these nurse bate over the question of whether introns appeared cells produce maternal products, mutations in genes early or late during evolution is therefore fueled by that are expressed in the ovaries can lead to female comparative structural data. Studies on human villin sterility. Not surprisingly, a number of maternally tran- (PRINGAULTet aZ. 1991) concluded that the size and scribed genes are foundto encode cytoskeletal proteins distribution of 14 introns in the regions encoding V1- (reviewed by COOLEYand THEURKAUF1994; KNOWLES V3 and V4-V6 did not support thepostulated hypothe- and COOI.EY1994). Cytoskeletal proteins are also in- sis of duplication from a precursor gene consisting of volved in the complex mechanics of cell division after only three segments, which itself arose by triplication fertilization of the egg. In Drosophila, cellularization of an original single V1-like domain. Reevaluation of of the syncytial blastoderm and migration of the nuclei these data in the context of the known three-dimen- to the cellular periphery takes place after 14 rounds of sional structure of V1 indicates that most of the introns cell division. Several genes are known whose products split the proposed structural domains of the protein. are required at this stage and mutations at these loci Such cases are compatible with the “introns late” hy- are associated with disruptions of the actin cytoskeleton pothesis. Only one intron (outof a total of 14) is located (SCHEIJTERand WIESCHAUS1993). The fzightless gene 1056 H. G. de Couet et al.

26 36 46 56 66

HGS VVEHPEFLKAGKEP GLQIWRVE 75 SEV AST EAQWKGVGQAP GLKIWRIE 77 DGS RVM HPSFANAGRTP GLEIWRIE 98 HVIL LSA QVKGSLNITTP GLQIWRIE 51 CAP G PQSGSPFPASVQDP GLHIWRVE 54 CEELFLI DYS DIFDEDVGSDE GMWVWEIE 544 DROMEFLI DYSKFFEKDDGQLP GLTIWEIE 533

52;

76 86 96 106 116

HGS LRNGNLQY LNGRA VQGF 125 SEV KEGNSLKH LGGAP CQSY 127 DGS KKDKKLSW LNGGP VQDM 148 HVIL KTASSLSY LGSVA VQGN 101 CAP G EEASHL.. LGERP VQGN 100 CEELFLI EASGQLRH LNATC EMND 594 DROMEFLI DDLGLLDW LGARC EQGD 583

553

126 136 146 156

HGS SEV DGS HVIL Cap G CEELFLI DROMEFLI

FIGURE6.-Partial sequence comparison of the G1-like domains of several membersof the gelsolin-like protein family, indicating residues important for actin binding (W) and apolar residuesinvolved in the integrity of the core (0).Numbers on top of the diagram indicate positions in human gelsolin referred to in the text. Corresponding positions in the Drosophila flightless protein are indicated at the bottom of the diagram. Numbering of amino acids is as described (WAY et al. 1992; CAMPBELL et al. 1993; MACLAUCHLIN el al. 1993). Boxed residues represent core residues of gelsolin essential for structural integrity, as identified by crystallographic analysis. The extent of secondary structure motifs identified in gelsolin is indicated on the bottom of the sequence comparison. HGS, human gelsolin; SEV, severin; HVIL, human villin; CAP G, mouse Cap G; CEELFLI, C. ekgansjightkss; DROMEFLI, D. melanoguster flightless protein.

clearly belongs to this category. The maternal contribu- structural integrity of G1 and V1 are conserved in all tion of gene products by these loci can only be assessed equivalent positions in the predicted flightless protein by germlineclone analysis. Maternal wild-type tran- (Figure 6). There are,however, variations in residues of script can be supplied to a homozygous mutant egg by flightless corresponding to those in G1 that form the the nursecells and can rescue the cell-lethal phenotype actin contact. All of the apolarresidues in the hypotheti- of mutant genesfor extended periodswell beyond em- cal F1-actininterface areconserved, but acidic and other bryogenesis. polar residues in the hydrogen-bonding region of the Germline clones homozygous for the lethalalleles of contact and in the calcium-binding sites are different. flightless failto develop into cellularized embryos (PERRI- These includesidechains equivalent to Aspso (Leu ’” in Fl), GIn% (Leu .5.%), GIu97 (Lyss.”s) and hpim -, hPlio MON et al. 1989), indicating that the gene product is already necessary at early embryonic stages and that (kg5” -, &dm). [It should be noted that the residues sufficient transcript or translation product is maternally of actin involved in the gelsolin interface are completely supplied to rescue homozygous mutant embryos into conserved in both flight muscle actin 88F and in direct the laterstages of larvaldevelopment. Theflightkss gene muscle isoforms (data not shown)]. The extentto which may therefore represent a pivotal locus required for these sidechains contribute to high affinity actin binding basic cellular processes. is unclear; with a single exception they are conserved in Structural homology of the flightless protein with gel- V1, but there are moresubstantial changes in segment 1 solin: Despite the overall sequence similarity ofthe con- of both severin and the D. mhnogmkr villin-like quail ceptual flightless gene product with members of the gel- gene product (MAHATAN-MIKLOSand COOLEY1994). solin family, analysis of the predicted sequence in the The substitution of the residue equivalent to context of the known three-dimensional structure of the by a basic charge would be expected to disrupt the gelsolinG1-actin complex and that ofvillin V1 intramolecular calcium site. This is the calcium trapped (MCLAUGHLINet al. 1993; MARKUS et al. 1994) reveals in the G1:actin complex and probably also in the EGTA several distinct differences, the functional significance stable ge1solin:actin complex (WEEDSet al. 1995). The (or lack thereof) of which is not yet known. The hy- presence of this calcium leads to at least 1000-fold en- drophobic core residues believed to be essential for the hancement of affinity of G1 for actin (BRYAN1988) but M olecular Analysis Molecular of Jightless-I 1057

on gelsolin have revealed sites required for actin bind- 65 (G-S).. ing and severing (WAYet al. 1989, 1990, 1992a,b). The generation of a gelsolin knock-out mouse (WITKEet al. 1995) corroborates the view that the protein is nones- sential for the development and maintenance of mam- malian tissues since this homozygous mutant mouse is viable and fertile. The same applies to severin from D. discoideum, which is also dispensable under laboratory conditions (AND& et al. 1989). The analysisof the flightless gene is potentially significant to this field, since the rapidmanipulability of the Drosophila system allows FIGURE 7.-Stereo view of gelsolin segment 1. Solid gray balls functional evaluation of both flightless and the recently indicate conserved residues contacting actin in the complex cloned melanogaster gelsolin et al. (compare with Figure6). Open balls represent charged residues D. (HEINTZELMAN involved in coordinating the intermolecular calcium ionin the 1993). The discovery of an additional member of the gelsolin G1-actin complex,some of which are not conserved in gelsolin family in invertebrates may provide new in- pzghths F1. The equivalent residue of Arg45of gelsolin is Glu sights into gelsolin function. in flzghths, Ghg7is either Arg or Lys in D. melanogaster and Mutationalanalysis: The Jlightless gene produces a C. ekgans -flightless, respectively. Positions 65 and 143 denote single 5.1-kb transcriptduring development. Several equivalent residues inGI residues that have undergone changes from Gly + Ser in Pightless mutations. chromosomal rearrangements truncatingthe transcript or eliminating the transcriptional activity of the gene, such as those analyzed in this study, all have lethal phe- is not required for thesevering activity of G1-3 or G1- notypes. The complex rearrangement 2/19B exhibits 2. The other carboxylate that is coordinated to this an insertion within the 3' region of the gene, which calcium is that of Asp66,which, in G1, also forms a salt may either truncate the gene product or compromise bridge with kg4' in the apolar core of the structure transcriptional efficiency and mRNA stability. The small (Figure 7). The importance of this salt bridge has been deficiency D44 deletes the5' end of the gene, including highlighted in the related position of G2, where muta- the translation initiation site and upstream sequences. tion of the conserved Asp to the corresponding amide The D44 rearrangement was generated by hybrid dys- causes the rare Finnish Familial Amyloidosis (HALTIA et genesis (EEKEN et al. 1985), likely to have mobilized al. 1990; MAURY et al. 1990). The structure analysis of P elements. Our molecular analyses revealed a short G1 offers an explanation for the origin of this disease duplication in the deletion site that is the hallmark of (MCLAUGHLIN et al. 1993). In Jlightless, no salt bridge a transposition event, and the deletion is likely to have could form in this position because the residue equiva- been caused by imprecise excision of a mobile element. lent to kg4' is replaced by Glu. The existence oftissue- and stage-specific mutant The sequence similarity of the flightless protein with phenotypes on the other hand, namely embryonic le- the gelsolin family extends to the G2 domain, which is thality or adult flightlessness, are explicable a number essential for both severing activity and filament side- of ways. First, the product of this gene may be over- binding of gelsolin (Yu et al. 1990; WAY et al. 1992a,b). utilized during morphogenesis of flight muscles and Based on analogy with severing segment 2, this domain mutations affecting the transcriptional efficiency may should contain a similar structural fold, but thereis no lead to flightlessness without affecting other functions information aboutits actin contacts, which are expected of the adult fly. This hypothesis is supported by our to be significantly different from those in G1. Prelimi- finding that a single flightless transgene inserted into nary data indicate that the humanflightless protein is a the heterochromatin of chromosome Nis able to res- phosphoinositide-regulated actin-binding protein that cue the lethal phenotype of two overlapping deficien- lacks severing activity (D. KWIATKOWSKI,personal com- cies uncovering the fliI locus but does not fully restore munication). flight ability (CAMPBELL et al. 1993).The transcriptional Functions of the flightless protein: While the func- activity ofthe transgene in these lines is probably below tional properties of the flightless protein arejust begin- normal, as assessed by the reduced expression of the ning to be evaluated, the function of gelsolin has been eye-color marker gene in the same context. In contrast, probed at differentlevels. Domain-interchange and do- transgene insertions at other chromosomal locations main-deletion experiments have been carried out be- fully restore flight in the lethal alleleJliIw-z(R. MALESZKA, tween gelsolin and villin (FINIDORIet al. 1992) and be- S. D. HANES, R. L. HACKE~T,H. G. DE COUET, G. L. G. tween Cap G and gelsolin (Yu et al. 1991), and their MIKLOS, unpublished results). effects examined in vivo and in vitro. These experiments Point mutations may affect a function exclusively in have revealed which parts of the gross architecture of a particulartissue. In the contextof the presumedactin- these capping proteins contribute to the in vivo activi- binding and -severing properties of the flightless pro- ties. At a finerlevel the in vitro mutagenesis experiments tein, this may include flight-muscle-specific protein-iso- 1058 H. G. de Couet et a1

forms, calcium concentration, or other protein factors BRYAN,J., 1988 Gelsolin has three actin-binding sites. J. Cell Biol. 106: 1553-1562. interacting with either actin or jlightkss. What may be CAMPBEl.l., H. D., T. SCHIMANSKY,C. CIAUIXANOS,N. OZSARM:, A.B. an analogous phenomenon is observed in mutants at USPRZAKet al., 1993 The Drosophila me1anogasterflightl.~-Igene the JliA locus encoding a-actinin (FYRBERGet al. 1990). involved in gastrulation and muscle degeneration encodes gel- solin-like and leucine-rich repeat domains and is consenwl in Null alleles of a-actinin lead to larval lethality, whereas Caenorhabditis rlegans and humans. Proc. Natl. Acad. Sci. USA 90: several viable alleles specifically cause flightlessness. It 1138&11389. is possible that in both cases functionally overlapping (:HANG, Z., B. D. PRICE, S. BOCXHEIM,M. J. BOEDIGIIEIMER,R. SMIIII r/ al., 1993 Molecular and genetic characterization of the Dr0- proteins present in tissues other than indirect flight sophila tartan gene. Dev. Biol. 160: 315-332. muscle compensate for a lossof function caused by (:HEN, K.3, D. NGL~N,J. D. HOHEISEI.,1. G. YOUNG,G. L.. G. Mlm.os mutations in a-actinin or Jlightless. el al., 1995 The l)ro.cophila me1anogarterflightless-lgene (JiI) hu- man homologue maps within the Smith-Magenis microdeletion Now that the three-dimensional structure of the G1 critical region in 17~11.2.Am. J. Hum. Genet. 56 175-182. domain of gelsolin in complex with actin has been CHIRGWN,,J.M., A. E. PRZYBYIA,R. J. MACDON.") and W. J. Rvrrm, solved (MCLAUGHLINet al. 1993), this information can 1979 Isolation of biologically active ribonucleic acid from be more easily placed into a biological context in the sources enriched in ribonuclease. Biochemistry 18: 5294-5299. COOI.EY,I>., and W. E. TIIEIIRKAL~F,1994 Cytoskeletal function dur- Drosophila system. Two viablemutations are located in ing Drosophila oogenesis. Science 266: 590-596. the F1 domain [$io2in which Gly"'" (equivalent to DABIKI, G. A,, C. L. YOL~NGS,J. ROSENBI.OOM and F. S. SoLwnvmi, Gly" of GI) is replaced by Ser, and JliI' in which G1y"O' 1992 Molecular cloning of human macrophage capping pro- tein cDNA. J. Biol. Chem. 267: 16545-16552. (equivalent to Glyi44of G1) is replaced by Ser], and we EEKEN, ,J. C. J., F. H. SOBEI.~,V. HYIANDand A. P. SCIIAI.~~,1985 therefore looked for possible structural implications of Distribution of MR-induced sex-linked recessive lethal mutations these substitutions. Bothglycine residues are highly in I)rosnl,hilrl mrlunogustrr. Mutat. Res. 150: 261-275. FINIDORI,J., E. FKIEDERICH,D. J. KWIATKOWSKIand D. I.OI;VAKII,1992 conserved in the gelsolin family and provide flexibility 171 vivo analysis of functional domains from villin and gelsolin. J. for turns in the structure. 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