A Physical Map of the X Chromosome of Drosophila Melanogaster: Cosmid Contigs and Sequence Tagged Sites
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Copyright 0 1995 by the Genetics Society of America A Physical Map of the X Chromosome of Drosophila melanogaster: Cosmid Contigs and Sequence Tagged Sites Encarna Madueno, * George Papagiannakis, Georgina Rimmington, x Robert D.C. Saunders, x Charalambos Savakis,t& Inga Siden-Kiamos, George Skavdis, t7 * * Lefteris Spanos, Jenny Trenear,tt Paul Adam,x Michael Ashburner,tt Panayiotis Benos, t3 * * Viacheslav N. Bolshakov, Daren Coulson, tt David M. Glover, x Sieg-un Herrmann,tt Fotis C. Kafatos, t, * Qlristos Louis, t, * * Tamsin Majerus tt and Juan Modolell* *Centra de Biologia Molecular Severo Ochoa, CSIC, Universidad Autonoma de Madrid, 28049 Madrid, Spain, tInstitute of Molecular Biology and Biotechnology, FORTH, Heraklion, Greece, :Department of Anatomy and Physiology and CRC Cell Cycle Genetics Group, University of Dundee, Dundee, Scotland, Division of Medical Sciences, Medical School and **Department of Biology, University of Crete, Heraklion, Greece, ttDepartment of Genetics, University of Cambridge, Cambridge, England, and IfEuropean Molecular Biology Laboratory, Heidelberg, Germany Manuscript received July 15, 1994 Accepted for publication December 21, 1994 ABSTRACT A physical map of the euchromatic X chromosome of Drosophila melanogaster has been constructed by assembling contiguous arrays of cosmids that were selected by screening a library with DNA isolated from microamplified chromosomal divisions. This map, consisting of893 cosmids, covers -64% of the euchromatic part of the chromosome. In addition, 568 sequence tagged sites (STS) , in aggregate representing 120 kb of sequenced DNA, were derived from selected cosmids.Most of these STSs, spaced at an average distance of -35 kb along the euchromatic region of the chromosome, represent DNA tags that can be used as entry points to the fruitfly genome. Furthermore, 42 genes have been placed on the physical map, either through the hybridization of specific probes to the cosmids or through the fact that they were represented among the STSs. These provide a link between the physical and the genetic maps of D. melanogaster. Nine novel genes have been tentatively identified in Drosophilaon the basis of matches between STS sequences and sequences from other species. OSOPHILA melanogaster is, genetically, the best of the basic regulatory mechanisms being discovered in D” known higher eukaryote. Nearly 90 years of study this fly subsequently have been shown by molecular have provided an unparalleled model for the study of means to have parallels in other organisms. biological phenomena as apparently diverse as popula- Drosophila is also a rich mine for evolutionary stud- tion biology and cell death (ASHBURNER 1989). Over ies. There are now 3318 species known in the family. 6000 genes have been identified (FlyBase 1994) and Most of these, it is true, areknown only to taxonomists, -25% of these have been cloned and sequenced, at but many have been the subject of detailed ecological least in part. Novel genetic techniqueshave been devel- and evolutionary study, by both classical methods such oped, such as transposon mutagenesis and enhancer as polytene chromosome banding and by molecular trapping, that detect DNA sequence elements control- methods [see KIUMBAS and POWELL(1992) and POW- ling the tissue and developmental specificity of genes ELL and DE SALLE (1995) for reviews, respectively]. ( O’KANEand GEHRING1987). These techniques allow Given this rich background and following the devel- the identification of the great majority of genes affect- opment of the requisite technology in other organisms ing any specific developmental process, regardless of ( e.g., COULSONet al. 1986) , it was natural for Drosoph- whether or notmutations inthese genes have an identi- ila biologists to consider mapping the entire euchro- fiable phenotype. Together with the increasing use of matic genome of D. melanogasterwith cloned DNAs. In- screens to recover mutations in genes that interact dur- deed, several such projects are underway, using cosmids ing development, these techniques are providing an ( SIDEN-KIAMos et al. 1990) , YACs ( GARZAet al. 1989; exceptionally detailed view of the control of develop- AJIOKAet al. 1991), P1 phage (SMOLLERet al. 1991) mental and cellular phenomena. Encouragingly, many and combining these various vector-insert systems into The order of authors was determined, with minor variation, ac- a “reference library” (HOHEISELet al. 1991; MERRIAM cording to Article 16 of Royal Decree 2223-1984 of the Kingdom of et al. 1991; HARTLand LOZOVSKAYA1992, for review). Spain, which governs the order of presentation of candidates to the Physical maps will enable the cloning of otherwise un- Spanish CiService. cloned genes, will be a prerequisite to whole genome Corresponding author: Michael Ashburner, Department of Genetics, University of Cambridge, Downing St., Cambridge CB2 3EH, UK. sequencing and will be a resource forcomparative stud- E-mail: [email protected] ies of drosophilid genomes. They will also be a model Genetics 139 1631-1647 (April, 1995) 1632 E. Maduedo et al. for the analysis of the genomes of insects of economic (CHURCH andGILBERT 1984) at 37” for 16 hr. Filters were or medical importance (see ZHENG et al. 1991 ) . then washed three times for 30 min in 6X SSC at room temper- One physical map of the D. melanogaster genome is ature and rinsed twice for 20 min in a buffer consisting of 3 M Tetramethylammonium chloride, 50 mM Tris-HC1, pH 8.0, in the form of overlapping cosmid clones and is being 2 mM EDTA, 0.1% SDS. A temperature of 57-58’ was selected assembled by a European consortium of laboratories for the rinses to allow for a 18bplong match for a 20-bp olige ( SIDEN-KIAMOSet al. 1990; KAFATOS et al. 1991 ) . This nucleotide probe (WOODet al. 1985) . Before exposing to film map is being linked to the polytene chromosome map, the filters were briefly rinsed in 6X SSC. a regular pattern of -5000 bands and interbands DNA preparations: For sequencing, cosmid DNA was pre- pared from 4 ml overnight cultures grown at 37” in TB me- (BRIDGES1935), by in situ hybridization. This permits dium containing 12 g/liter Bacto-Tryptone, 24 g/liter yeast the assignment of selected “canonical” cosmids from extract, 4 ml/liter glycerol, 17 mM KHnP04,72 mM K2HP04, sets of overlapping cosmids (contigs) to specific bands, and 30 pg/ml kanamycin. DNA was extracted from cells by or at least to smallchromosomal map subdivisions, and the alkaline lysis procedure ( SAMBROOKet al. 1989) and puri- provides the linkage between the physical and the ge- fied with Wizard columns ( Promega) according to the manu- facturer’s directions. Melds ranged from 1 to 20 pg. For most netic map to a resolution limit of tens of kilobase pairs. preparations they were between 7 and 10 pg. To enhance the physical map and tie it to the ultimate DNA sequencing: Oligonucleotides ( 20-mers ) comple- map, the chromosomal DNA sequence, we have begun mentary to theSP6 and T7 sites were used as primers to obtain to determine a large number of “sequence tagged sites” sequences of the endsof the insert DNA. DNA wassequenced (STSs) (OLSONet al. 1989). These are sequences of using heatdenatured, double-stranded templates and the lin- ear amplification method ( CRAXTON1991) as described in short stretches of DNA located at known positions within the Promega fmol sequencing kit with several modifications. the cosmid inserts. An advantage of the STSsis that In brief, 1-2 pg of template DNA were mixed, in sequencing they make the physical map independent of the actual buffer, with 20 pCi ‘35S]dATP ( 1000 Ci/mmol) , 3 pmol SP6 cosmids that generated themap. We are obtaining STSs or T7 primer and5 U Tag Polymerase, up to a final volume corresponding to the ends of the DNA inserts byse- of 17 pl. Four-microliter aliquots were mixed with the appro- priate deoxy-and dideoxy-nucleotide triphosphates, heated to quencing from the T7 and SP6 promoters adjacent to 95” for 2 min in a model 480 Perkin Elmer Cetus thermal the cloning site within the cosmid vector.The sequences cycler, and subjected to 30 cycles consisting of 30 sec at 95”, obtained have an average length of 211 bp. 30 sec at 42” and 60 sec at 70”. Three microliters of stop buffer Here we report the map of the euchromatic Xchro- were added to each reaction mixture, DNA was denatured by mosome of D. melanogaster, consisting of 893 cosmid incubation at 80” for 2 min and 2-3 p1 of each mixture was electrophoresed in 6% acrylamide gels. Another portion of clones and 568 STSs. This map is anchored to preex- each mixture was electrophoresed in a secondgel. After auto- isting maps by the identification of cosmidsthat include radiography for 4-5 days the gels were read and the se- genes previously identified and sequenced. quences were entered into a MicroVax computer with the help of a digitizing tablet. Consensus sequences were obtained with the University of Wisconsin software package ( DEVEREUX MATERIALSAND METHODS et al. 1984). STSs were named by appending an “S” or “T” Map assembly and in situ hybridization: The map of chro- to the correspondingcosmid name, accordingto whether the mosome Xwas assembled as described by SIDEN-KIAMOSet al. STS was obtained by priming on the SP6 or T7 recognition ( 1990). The cosmid library used in the construction of the site, respectively. map was generated in the Lo& Gvector (GIBSONet al. 1987) , STS analysis: Approximately 75% of the cosmids yielded which contains SP6 and T7 polymerase recognition sequences readablesequences fromboth termini, 14% failed to se- near and at eachside of the BamHI cloning site. The cosmid quence from oneof the two termini and the rest failed from master library ( 18,432 clones, equivalentto 4.6 genomes) was both ends. More than half of the failures (60%) were due to gridded onto new filters using a robotic device to attain a compressions. The remainingwere due toa failureof elonga- density of 9216 clones per filter (see LEHRACHet al.