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Review Gene Disruptions Using P Transposable Elements: an Integral Component of the Drosophila Genome Project Allan C

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Review Gene Disruptions Using P Transposable Elements: an Integral Component of the Drosophila Genome Project Allan C Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10824-10830, November 1995 Review Gene disruptions using P transposable elements: An integral component of the Drosophila genome project Allan C. Spradling*, Dianne M. Stern*, Istvan Kisst, John Rootet, Todd Laverty§, and Gerald M. Rubin§ *Howard Hughes Medical Institute Research Laboratories, Camegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210; tInstitute of Genetics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary; tDepartment of Genetics, Cambridge University, Downing Street, Cambridge, CB2 3EH, England; and §Howard Hughes Medical Institute Research Laboratories, Department of Molecular and Cell Biology, University of Califomia, Berkeley, CA 94720-3200 ABSTRACT Biologists require ge- tion). Novel informatics tools are being able from the Bloomington, IN, Drosoph- netic as well as molecular tools to deci- developed to collect, analyze, and distrib- ila stock center, greatly facilitates both pher genomic information and ultimately ute these data. If this vast new store of gene disruption and mutant identification. to understand gene function. The Berke- structural information is to enhance our Investigators interested in a particular se- ley Drosophila Genome Project is address- understanding of human biology, how- quenced ORF can often find a strain from ing these needs with a massive gene dis- ever, it must be accompanied by more the collection in which that ORF is inser- ruption project that uses individual, ge- efficient methods to correlate genes with tionally mutated. Otherwise, insertions netically engineered P transposable functions. Currently, data base searches can usually be obtained close enough to elements to target open reading frames identify similarities to a given query se- the ORF of interest to rapidly generate throughout the Drosophila genome. DNA quence more than a third of the time. the desired mutations. In addition, BDGP flanking the insertions is sequenced, Moreover, these similarities are often as- strains make it much easier to associate thereby placing an extensive series of ge- sociated with biological meaning because the thousands of genes Drosophila genet- netic markers on the physical genomic many of the entries in the public data base icists have defined over the last several map and associating insertions with spe- have been deposited by individuals in- decades with specific transcription units cific open reading frames and genes. In- volved in hypothesis-driven research. With and their protein products. BDGP lines sertions from the collection now lie within the onset of sequencing whole genomes can be identified that either disrupt such or near most Drosophila genes, greatly and large cDNA collections, database sim- genes or otherwise delimit their positions reducing the time required to identify new ilarities will be found more frequently. on the physical map to small molecular mutations and analyze gene functions. However, because future database sub- intervals. Finally, the inserted P elements Information revealed from these studies missions will come primarily from ge- in BDGP lines carry enhancer traps (6) about P element site speciricity is being nome-sequencing efforts which select tar- that can be used to efficiently acquire used to target the remaining open reading gets on the basis of developing complete information about the expression pattern frames. data bases rather than on their biological of disrupted genes. The strains in the function, the frequency of association of a current collection disrupt 20-25% of es- Genetics provides the most powerful ap- query sequence with well-characterized sential genes, provide information on proach available to understand the func- biochemical mechanisms is unlikely to rise their expression patterns, and link the tion of each human gene and to decipher in parallel. genetic, cytogenetic, and physical maps of the role played by the large noncoding If the model organism genome projects the Drosophila genome at "100-kb inter- component of the human genome. Model are to be maximally useful in assigning vals. The approaches made possible by the organisms, such as Drosophila melano- function to human DNA sequences, they BDGP strains are reducing or eliminating gaster, share many genes with humans must be accompanied by genetic studies so long delays in obtaining the tools needed whose sequences and functions have been that not only the sequences of the genes, to study gene function. In this report, we conserved. In addition to myriad similar- but also their biological functions, are discuss the current status of the BDGP ities in cellular structure and function, determined. To facilitate that end, BDGP gene disruption library and report some of humans and Drosophila share pathways has adopted a broad approach that com- the results revealed by this project. for intercellular signaling (1), develop- bines the determination of the genomic mental patterning (2), learning and be- sequence with the development and ap- Origin of the BDGP Gene-Disruption havior (3), as well as tumor formation and plication of methods for large-scale func- Project metastasis (4). The fruit fly provides a tional analysis. In the past, the effort powerful system to study the function of required to disrupt a particular gene or to Transposable elements provide a potent conserved genes since, unlike humans, any identify the ORF responsible for a mutant means of correlating genetic and molecu- open reading frame (ORF) within the strain's interesting properties has varied lar information because they generate a fruit fly genome can be mutated and widely and has frequently slowed simple, reproducible lesion upon insertion subjected to detailed functional analysis progress. Consequently, BDGP has un- that can be detected much more easily within the context of an intact organism. dertaken a gene-disruption project to ad- than damage produced by other mutagens The Berkeley Drosophila Genome dress this rate-limiting step in utilizing (7, 8). In D. melanogaster, the P transpos- Project (BDGP) has as its primary goal to Drosophila to assign function to human able element has been particularly useful map and sequence the "120 Mb of DNA genes. because it moves with high frequency but comprising the euchromatic-i.e., nonre- The BDGP gene-disruption project can be controlled by limiting the availabil- petitive-regions of the four Drosophila consists of a large collection ofDrosophila ity of an element-encoded transposase (9, chromosomes (see for examples ref. 5; W. strains that each contain a single, geneti- 10). Early efforts focused on cloning spe- Kimmerly, K. Stultz, K. Lewis, V. Lustre, cally engineered P transposable element S. Lewis, D. Sun, R. Romero, C. Martin, inserted in a defined genomic region. The Abbreviations: ORF, open reading frame; and M. Palazzolo, personal communica- BDGP strain library, which is freely avail- BDGP, Berkeley Drosophila Genome Project. 10824 Downloaded by guest on September 26, 2021 Review: Spradling et aL Proc. Natl. Acad. Sci. USA 92 (1995) 10825 cific genes by mobilizing large numbers of a comprehensive gene-disruption library. tosomal insertion sites have been resolved; natural P elements (reviewed in ref. 11). As a byproduct, it was realized that the hence, the average distance between ele- The resulting genetic lines were unsuitable strains would prove extremely useful for ments within autosomal regions is about for genetic or phenotypic studies without linking the physical and genetic maps. 85 kb. extensive outcrossing to remove extrane- When the project began in 1993, more We took advantage of the fact that ous P elements and were rarely saved once than 3000 strains in which single P element many genes had independently been mu- flanking DNA had been cloned. P ele- insertions had been associated with reces- tated in more than one line to determine ment-mediated transformation allowed sive lethal or sterile phenotypes were col- how accurately the insertions had been strains containing just one or a few ele- lected from six laboratories (Table 1). The localized. Since most. Drosophila genes ments to be constructed (12). However, gene-disruption library was assembled studied to date are smaller than 50 kb, one only a limited number of strains could be from this starting material, and it contin- would expect that the insertions in allelic generated by microinjecting DNA, and the ues to be expanded today. In their starting lines would map within the same or an insertions could not be targeted into genes state, the lines were of limited use to the adjacent polytene band. Errors in local- of particular interest. research community and too numerous to ization would' show up as allelic lines In 1988, Cooley et al. (13) showed that be maintained in stock centers. Since most whose insertions had been mapped at individual, experimentally modified P el- of the transposon insertion sites were un- more widely separated sites. Complemen- ements can be mobilized in large genetic known, investigators would have to study tation crosses identified 750 lines that fell screens to generate thousands of stable thousands of lines instead of just the few into 180 complementation groups with mutant strains. About 15% of such trans- whose P element insertions lay in a between two and 25 alleles. The average poson insertions disrupted a gene re- genomic region of interest. Furthermore, standard deviation in the map position of quired for viability or fertility,
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