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Molecular and Mutational Analysis of a Gelsolin-Family Member Encoded by the Flightless I Gene of Drosophila Melanogaster

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Molecular and Mutational Analysis of a Gelsolin-Family Member Encoded by the Flightless I Gene of Drosophila Melanogaster Copyright 0 1995 by the Genetics Society of America Molecular and Mutational Analysis of a Gelsolin-Family Member Encoded by the flightless I Gene 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 locus of Drosophila melanogasterhas been analyzed at the genetic, molecular, ultrastructural and comparative crystallographic levels. The gene encodes a single transcript encoding a protein 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 proteins 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 chromosome 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,
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