DROSOPHILA INFORMATION NEWSLETTER Volume 14, April 1994 The Drosophila Information Newsletter has been established with the hope of providing a timely forum for informal communication among Drosophila workers. The Newsletter will be published quarterly and distributed electronically, free of charge. We will try to strike a balance between maximizing the useful information included and keeping the format short; priority will be given to genetic and technical information. Brevity is essential. If a more lengthy communication is felt to be of value, the material should be summarized and an address made available for interested individuals to request more information. Submitted material will be edited for brevity and arranged into each issue. Research reports, lengthy items that cannot be effectively summarized, and material that requires illustration for clarity should be sent directly to Jim Thompson (THOMPSON@AARDVARK.UCS.UOKNOR.EDU) for publication in DIS. Materials appearing in the Newsletter will be reprinted in DIS. Back issues of DIN are available from FlyBase in the directory flybase/news or in News/ when accessing FlyBase with Gopher. Material appearing in the Newsletter may be cited unless specifically noted otherwise. Material for publication should be submitted by e-mail. Figures and photographs cannot be accepted at present. Send technical notes to Carl Thummel and all other material to Kathy Matthews. The e-mail format does not allow special characters to be included in the text. Both superscripts and subscripts have been enclosed in square brackets; the difference should be obvious by context. Bold face, italics, underlining, etc. cannot be retained. Please keep this in mind when preparing submissions. To maintain the original format when printing DIN, use Courier 10cpi font on a standard 8.5" x 11" page with 1" margins. Drosophila Information Newsletter is a trial effort that will only succeed if a broad segment of the community participates. If you have information that would be useful to your colleagues, please take the time to pass it along. The editors: Carl Thummel Kathy Matthews Dept. of Human Genetics Dept. of Biology Eccles Institute - Bldg. 533 Indiana University University of Utah Bloomington, IN 47405 Salt Lake City, UT 84112 812-855-5782; FAX/2577 801-581-2937; FAX/5374 MATTHEWK@INDIANA.EDU CTHUMMEL@HMBGMAIL.MED.UTAH.EDU MATTHEWK@INDIANA.BITNET *** DIN 14 To add your name to the Newsletter distribution list, send one of the following E-mail messages from the account at which you wish to receive DIN. Via Internet -- To: LISTSERV@IUBVM.UCS.INDIANA.EDU Subject: Message: SUB DIS-L Your real name Via Bitnet -- To: LISTSERV@IUBVM Subject: Message: SUB DIS-L Your real name LISTSERV will extract your user name and node from the E-mail header and add you to the list. Use your Internet address if you have one. You will receive confirmation by E-mail if you have successfully signed on to the list. If you are on the list and do not wish to receive DIN, or you want to remove a soon-to- be-defunct address, replace SUB in the above message with UNS. The SUB command can also be used to correct spelling errors in your real name; the new entry will simply replace the old as long as it was sent from the same USERID@NODE address. *** DIN 14 DIN Vol. 14 TABLE OF CONTENTS >Introduction to Drosophila Information Newsletter >How to subscribe to the Newsletter >TABLE OF CONTENTS >ANNOUNCEMENTS >1994 and 1995 US Drosophila Conferences >New book on development available >NIAAA Program Announcement >Bloomington Stock Center news >REQUESTS FOR MATERIALS >Anything in 62F >MATERIALS AVAILABLE >Compilation of Drosophila cDNA and Genomic Libraries >TECHNICAL NOTES >Injecting un-dechorionated eggs under ethanol *** DIN 14 ANNOUNCEMENTS 35th ANNUAL DROSOPHILA RESEARCH CONFERENCE The next US Drosophila Conference will be held in Chicago, Illinois, at the Sheraton Chicago Hotel, 301 East North Water St., April 20-24, 1994. Contact The Genetics Society of America, 9650 Rockville Pike, Bethesda, MD 20814-3998 (301-571-1825) for registration and housing information. The 1995 US conference will be held April 5-9 in Atlanta, Georgia. *** DIN 14 THE DEVELOPMENT OF DROSOPHILA MELANOGASTER Edited by Michael Bate (University of Cambridge) and Alfonso Martinez Arias (University of Cambridge) A monograph in two volumes, this reference work represents the most comprehensive synthesis of Drosophila developmental biology currently available. The text is complemented with a full-color Atlas for bench use, which graphically illustrates the day-by-day development of the Drosophila embryo. Contents: Developmental Genetics of Oogenesis (A. Spradling) Spermatogenesis (M. Fuller) Mitosis and Morphogenesis in the Drosophila Embryo (V. Foe, G. Odell, B. Edgar) Maternal Control of Anterior Development in the Drosophila Embryo (W. Driever) Pole Plasm and the Posterior Group Genes (D. St. Johnson) The Terminal System of Axis Determination in the Drosophila Embryo (F. Sprenger, C. Nusslein-Volhard) Maternal Control of Dorsal-Ventral Polarity and Pattern in the Embryo (R. Chasan, K. Anderson) Gastrulation in Drosophila: Cellular Mechanisms of Morphogenetic Movements (M. Costa, D. Sweeton, E. Wieschaus) Blastoderm Segmentation (M. Pankratz, H. Jackle) Development and Patterning of the Larval Epidermis of Drosophila (A. Martinez Arias) Development of the Drosophila Tracheal System (G. Manning, M. Krasnow) The Terminal Regions of the Body Pattern (G. Jurgens, V. Hartenstein) Imaginal Disc Development (S. Cohen) The Metamorphic Development of the Adult Epidermis (D. Fristrom, J. Fristrom) Hormones and Drosophila Development (L. Riddiford) The Alimentary Canal (H. Skaer) The Mesoderm and Its Derivatives (M. Bate) Early Neurogenesis in Drosophila melanogaster (J. Campos-Ortega) Embryonic Development of the Drosophila Central Nervous System (C. Goodman, C. Doe) The Peripheral Nervous System (Y.N. Jan, L.Y. Jan) Formation of the Adult Nervous System (J. Truman, B. Taylor, T. Awad) Pattern Formation in the Drosophila Retina (T. Wolff, D. Ready) Genetic Dissection of Eye Development in Drosophila (B. Dickson, E. Hafen) The Development of the Optic Lobe (I. Meinertzhagen, T. Hanson) Epilogue (M. Ashburner) Atlas of Drosophila Development (V. Hartenstein) Poster: Drosophila Third Instar Eye Disc (T. Wolff) 1993, 1564 pp., illus. (147 in color), indexes, ISBN 0-87969-423- 8, 2-volume set, cloth; atlas, paper; poster -- $350. Can be ordered directly by e-mail: benirsch@cshl.org, by phone: 1-800- 843-4388 (continental US and Canada), 516-349-1930 (all other locations), by fax: 516-349-1946, or write to: Cold Spring Harbor Laboratory Press, 10 Skyline Dr., Plainview, N.Y. 11803 Personal orders must be prepaid by personal check, credit card, or money order. All checks must be in US dollars and drawn on a US bank. Please $20 for postage. New York residents, add appropriate sales tax. *** DIN 14 NIAAA PROGRAM ANNOUNCEMENT The following is an excerpt from a National Institute on Alcohol Abuse and Alcoholism Program Announcement of particular relevance to Drosophilists. For the complete announcement see the Program Announcement, Genetic Studies in Alcohol Research (PA 93-086). Copies of this and other Program Announcements can be obtained from the National Clearinghouse on Alcohol and Drug Information (NCADI), P.O. Box 2345, Rockville, MD 20852, telephone: 1-800-729-6686. Information on research grants can be obtained from: Robert W. Karp, Ph.D., Director, Genetics Program, Division of Basic Research, National Institute on Alcohol Abuse and Alcoholism, 5600 Fishers Lane, Room 16C-05, Rockville, MD 20857. E-mail: RKARP@WILLCO.NIAAA.NIH.GOV ALCOHOL-RELATED GENETIC STUDIES IN INVERTEBRATES Because of their small size, short generation time, and high fecundity, the fruit fly Drosophila melanogaster and the soil nematode Caenorhabditis elegans lend themselves to large-scale systematic searches of tens to hundreds of thousands of individuals to find single-gene mutations conferring a specific phenotype of interest. For both of these invertebrate species sophisticated genetic and molecular methods are available which facilitate the cloning of genes based either on the phenotypes they confer, or on their known map locations (Ashburner, 1989; Herman and Shaw, 1987; Mello, et al., 1991; Coulson, et al., 1991). The combined power of these methods has led to important contributions to our understanding of development and functioning of the nervous systems of these species. Many of their genes critical for neurotransmission and central nervous system development (e.g., those encoding neurotransmitter biosynthetic enzymes and receptors, protein kinases, adenyl cyclases, G proteins, ion channel subunits, cell adhesion proteins, transcription factors) have homologues which function critically in the vertebrate central nervous system as well (Molecular Neurobiology of Drosophila: Cold Spring Harbor Laboratory meeting abstracts, 1991; Chalfie and White, 1988). In both of these species, single-gene mutants have been described which alter sensitivity to volatile anaesthetics (Krishnan and Nash, 1990; Sedensky and Morgan, 1991). Cloning of the mutated genes from these mutants will serve to identify gene products which participate in the physiology of anaesthetic sensitivity. The cloned genes can also be used to isolate mammalian (including human) homologues which will be invaluable for studying the mechanisms of action of anaesthetics in these higher species. This approach may well reveal targets for the action of anaesthetics not yet disclosed by direct genetic or biochemical studies on mammals. Although an approach based on systematic mutant searches of mammals (e.g., mice) would certainly be desirable, the impracticability of rearing a sufficiently large number of individuals renders studies in invertebrates more expedient. The example of volatile anaesthetics demonstrates how an approach based on invertebrate genetic studies provides an otherwise inaccessible entree to the elucidation of the mechanism of action in vertebrates of a drug whose molecular targets have not yet been definitively identified. It would be of great interest to characterize in detail the behavioral and developmental responses of Drosophila and Caenorhabditis to ethanol. If such responses as attraction to, consumption of, sensitivity to, tolerance to, and withdrawal from ethanol, as well as ethanol-induced developmental defects can be demonstrated, then systematic searches for single-gene mutations affecting these responses can greatly facilitate the elucidation of the entire chain of physiological events mediating these responses. Cloning of the mutant invertebrate genes discovered by these searches could then lead to cloning of homologous mammalian genes with important functions in responses to ethanol. It is difficult to predict in advance which (if any) invertebrate ethanol-related behaviors will prove relevant to human alcoholism. An objective test for true homology (based on shared underlying genetic or physiological mechanisms), as opposed to analogy (superficial behavioral similarity), is therefore essential for guiding this line of research. Such a test can be accomplished post hoc by testing human homologues of the invertebrate genes for linkage to alcoholism in human pedigrees. Areas needing further research include: Characterization of behavioral and developmental responses of Drosophila and Caenorhabditis to ethanol. Behavioral responses can include attraction to, consumption of, sedation by, motor impairment by, tolerance to, and withdrawal from ethanol. Systematic searches (using either mutagens or wild populations) for mutants altered in the responses mentioned above. Mapping and cloning of the genes altered in mutants discovered in these screens. Characterization of the products of the cloned genes. Cloning of mammalian (including human) homologues of the cloned invertebrate genes. Testing for linkage of the human homologues to alcoholism in human pedigrees. References __________ Ashburner MA: Drosophila: A Laboratory Handbook. Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 1989 Coulson A, Kozono Y, Lutterbach B, Shownkeen R, Sulston J, Waterston R: YACs and the C. elegans genome. Bioessays 13:413- 417, 1991 Herman RK, Shaw JE: The transposable genetic element Tc1 in the nematode C. elegans. Trends Genet 3:222-225, 1987 Krishnan KS, Nash HA: A genetic study of the anesthetic response: Mutants of Drosophila melanogaster altered in sensitivity to halothane. Proc Nat Acad Sci USA 87:8632-8636, 1990 Mello CC, Kramer JM, Stinchcomb D, Ambros V: Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J 10:3959-3970, 1991 Molecular Neurobiology of Drosophila: Cold Spring Harbor Laboratory meeting abstracts, Sept 25-29, 1991 Sedensky MM, Morgan PG: Genetics of response to volatile anesthetics in Caenorhabditis elegans. Ann NY Acad Sci 625: 524- 531, 1991 Chalfie M, White J: The nervous system, in Wood WB (ed): The Nematode Caenorhabditis elegans. Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 1988, pp 337-395 *** DIN 14 BLOOMINGTON STOCK CENTER NEWS * NEW DEADLINE -- The weekly deadline for stock requests is now 11 AM on Thursday. Large orders and requests that don't include our stock numbers must be received on Wednesday to assure inclusion in the current week's order. * CLOSED FOR THE FLY MEETINGS -- The center will be closed April 18 - 24. Orders received by 11AM on April 14 will be shipped April 18. Orders received between 11AM April 14 and 11AM April 28 will be shipped May 2. We are extremely busy for a few weeks after the fly meeting. Please order only what you need immediately during this period. See you in Chicago. * WRONG BREAKPOINTS -- The breakpoints shown in our stock list for Df(3R)p-XT103 #1962 were not correct. The reported breakpoints for this deficiency are 85A2;85C1-2 (we had the breakpoints for p-XT9 instead of p-XT103). Thanks to Hilary Ellis for catching and reporting this error. There are without doubt others. Typographical and transcription errors in the stock lists will be identified and corrected when stocks are added to the developing relational version of FlyBase. In the meantime, always check the breakpoints in our stock list against the new redbook or the aberrations file in FlyBase. We recommend that you check the cytology yourself before investing significant effort in any aberration stock. * USER SURVEY FOR NIH -- Thanks to everyone who responded to the user survey. At last count the response rate was 64%. 60% of respondents had research grants from NIH in 1993, another 8% had students or post-docs supported by NIH training grants, and 4% of the survey population were NIH employees. Among the 200 NIH research grants held by respondents in 1993, 61% were from NIGMS, 12% from NICHHD, 12% from NINDS, and 10% from NEI. However, on average, groups with NEI grants were heavier users of the center than groups with NIGMS grants - 34% of stocks shipped to survey respondents went to groups with NEI funding, the same proportion that went to NIGMS-funded groups. * hsFLP+FRT COMBINATION STOCKS ARE UNSTABLE -- FRT sites are damaged over time when maintained in stock with hsFLP, even when stocks are kept at low temperature. It is very likely that you will get poor clone production if you use FRTs that have been kept with hsFLP for extended periods. We no longer maintain these combination stocks. Everyone who received hsFLP+FRT stocks from the center before we discarded them was notified of the potential for problems. However, it appears that this information has not always been relayed to all potential users in each lab. If you continue to maintain these stocks in your lab we strongly recommend that you discard them. Three stocks are available from the center that can be used as a source of hsFLP: #6 y w[1118] hsFLP1; Adv/CyO, #7 y w[1118] hsFLP1; Dr[Mio]/TM3, and #279 w[1118]; MKRS, hsFLP3/TM6B. Make the necessary hsFLP+FRT combination genotype only when you are ready to generate clones. *** DIN 14 REQUESTS FOR MATERIALS Dr. Robert E. Nelson, UCLA, Molecular Biology Institute, Room 459, Los Angeles, CA 90024, Tel: 310-825-5267, e-mail: nelson@ewald.mbi.ucla.edu. Looking for stocks with transposable elements, mutations, and/or deletions that map within or around 62F. Thanks, in advance, Bob Nelson *** DIN 14 MATERIALS AVAILABLE Compilation of Drosophila cDNA and Genomic Libraries Carl Thummel, Howard Hughes Medical Institute, 5200 Eccles Institute of Human Genetics, Bldg. 533, Univ. of Utah, Salt Lake City, UT 84112 U.S.A. 801-581-2937, FAX/5374, CTHUMMEL@HMBGMAIL.MED.UTAH.EDU. The following is an update of the listing of Drosophila cDNA and genomic libraries that are currently available and in common use. Please do not request shipment of a library unless you have an immediate use for it - many contributors are concerned about the time and money involved in mailing their libraries. Also, please inquire with local colleagues before requesting a library since many of these libraries are already widely distributed. cDNA LIBRARIES --Nick Brown, Wellcome/CRC Institute, Tennis Court Rd, Cambridge CB2 1QR United Kingdom Phone: 44-223-334128 FAX: 44-223-334089 Email: NB117@MB1.BIO.CAM.AC.UK Vector/Insertion/Complexity/mRNA source pNB40/see ref./3x10[5]/0-4 hr embryo pNB40/see ref./3x10[6]/4-8 hr embryo pNB40/see ref. 3x10[5]/8-12 hr embryo pNB40/see ref./1x10[6]/12-24 hr embryo pNB40/see ref./3x10[6]/imaginal discs The Drosophila strain used is an isogenic second chromosome stock: dp cn bw, from the Gelbart lab. Ron Blackman has made a genomic library from this same strain (see below). The vector is a pUC based plasmid with a SP6 promoter at the 5' end of the cDNA and a T7 promoter at the 3' end of the cDNA. The cloning strategy was directional and designed to maximize the number of full-length cDNAs. A useful diagnostic of full-length cDNAs is a non-coding G nucleotide at the 5' end, after the polyC tract; the origin of this nucleotide is, however, unknown. Reference: Brown, N.H., and F.C. Kafatos (1988) Functional cDNA libraries from Drosophila embryos. J. Mol. Biol. 203: 425-437. --Steve Russell, Dept. of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH United Kingdom Phone: 44-223-337733 FAX: 44-223-333992 Email: sr120@mbfs.bio.cam.ac.uk All libraries were made with RNA isolated from Oregon R strain Vector/Insertion/Complexity/mRNA source NM1149/RI/2x10[6]/Male 3rd instar larvae NM1149/RI/6x10[5]/Female 3rd instar larvae NM1149/Directional: RI-HIII/3x10[6]/Adult male heads NM1149/Directional: RI-HIII/1x10[6]/Adult female heads lambda gt11/RI/3x10[5]/Testes --Charles P. Emerson, Jr. or Mary Beth Davis, Biology Dept., University of Virginia, Charlottesville, Virginia, 22901, USA Phone: 215-728-5283 (Emerson); 215-728-5284 (Davis) FAX: 215-728-2412 Email: emerson@castor.rm.fccc.edu or davis@castor.rm.fccc.edu Vector/Insertion/Complexity/mRNA source/Titer lambda gt10/RI/1x10[6]/late pupae/1x10[10] Blunt-ended cDNA was ligated to EcoRI adaptors, then ligated to EcoRI digested gt10 lambda arms. We have isolated cDNA clones corresponding to MHC isoforms that were lengths of 5940 and 5500 bases. Reference: George, E.L., M.B. Ober, and C.P. Emerson, Jr. (1989) Functional domains of the Drosophila melanogaster muscle myosin heavy-chain isoform are encoded by alternatively spliced exons. Mol. Cell Biol. 9: 2957-2974. --Bruce Hamilton, Whitehead Institute, 9 Cambridge Center, Cambridge, MA 02142, USA Phone: 617-258-5174 FAX: 617-258-6505 Email: hamilton@genome.wi.mit.edu Library name/Vector/Insertion/Complexity/mRNA source Head M/lambda EXLX/ApaI-SacI/1.1x10[7]/Oregon R adult heads Head P/lambda EXLX/ApaI-SacI/9x10[6]/Oregon R adult heads Head 1.2/lambda EXLX/ApaI-SacI/2.7x10[6]/Oregon R adult heads Head 2.0/lambda EXLX/ApaI-SacI/1.2x10[6]/Oregon R adult heads Adult/lambda EXLX/ApaI-SacI/>1x10[6]/Oregon R adults 0-24 mojo/lambda EXLX/ApaI-SacI/3.4x10[6]/Can S, 0-24 hr embryos All libraries were cloned directionally into the ApaI-SacI sites of lambda EXLX, as described in ref. 1, with internal restriction sites protected. Lambda EXLX allows in vivo excision of plasmid DNA using a CRE/loxP site-specific recombination system. This vector also allows regulated expression of the insert DNA as a phage T7 gene 10 N-terminal/cDNA fusion protein, under the control of a T7 RNA polymerase promoter (1). The Head 1.2 library was prepared from cDNAs that were size-selected for molecules 1.2 kb or larger by fractionation through an agarose gel. Head 2.0 contains cDNAs that are 2 kb or larger. The cDNA for the Adult library was not size-fractionated. The Adult and mojo libraries were published in ref. 1. The Head M and Head P libraries are unpublished, but I have asked people who use them to refer to ref. 1, since they were constructed in the same way and in the same vector. The two size-selected libraries, Head 1.2 and Head 2.0 were published in ref. 2, which also describes a rapid screening procedure that is very straightforward. References: 1. Palazzolo et al (1990) Gene 88, 25-36. 2. Hamilton et al (1991) Nucl. Acids Res. 19, 1951-1952 --Tom Kornberg, Department of Biochemistry, University of California, San Francisco, CA 94143 USA Phone: 415-476-8821 FAX: 415-476-3892 Email: tomk@ucsf.cgl.edu Our cDNA libraries were prepared from RNA isolated from Oregon R animals, with the cDNA sequences inserted into the EcoRI site of lambda gt10. Libraries will be shipped by Federal Express. Requests should be accompanied by an appropriate Federal Express Authorization Number. Stage/Library designation/Complexity 0-3 hr embryo/D/300,000 3-12 hr embryo/E/500,000 12-24 hr embryo/F/300,000 1st and 2nd instar/G/200,000 early 3rd instar/H/300,000 late 3rd instar/I/300,000 early pupal/P/300,000 late pupal /Q/300,000 adult male/R/300,000 adult female/S/300,000 Reference: Poole, S., Kauvar, L.M., Drees, B., and Kornberg, T. (1985) The engrailed locus of Drosophila: Structural analysis of an embryonic transcript. Cell 40: 37-43. --John Tamkun, Department of Biology, University of California, Santa Cruz, CA 95064, USA Phone: 408-459-3179 FAX: 408-459-3139 Vector/Insertion/Complexity/mRNA source lambda gt11/EcoRI/>6x10[5]/iso-1, 0-24 hr embryos The iso-1 strain, constructed by Jim Kennison, is isogenic for all four chromosomes. Genomic libraries from this strain are also available. Reference: Tamkun et al. (1986), Cell 46: 271-282. --Pat Hurban and Carl S. Thummel, Dept. of Human Genetics, 5200 Eccles Institute, Bldg. 533, University of Utah, Salt Lake City, Utah, 84112 USA Phone: 801-581-2937 FAX: 801-581-5374 Email: cthummel@hmbgmail.med.utah.edu Vector/Insertion/Complexity/mRNA source lambda ZAPII/RI-XhoI/2x10[7]/larval tissues cultured in vitro with cycloheximide + ecdysone lambda ZAPII/RI-XhoI/3x10[6]/0-24 hr embryos lambda ZAPII/RI/3x10[5]/0-24 hr embryos lambda ZAPII/RI-XhoI/2x10[6]/mid-late third instar larvae lambda ZAPII/RI/2x10[6]/mid-late third instar larvae lambda ZAPII/RI-XhoI/2x10[5]/0-15 hr pupae lambda ZAPII/RI/2x10[6]/0-15 hr pupae All RNA was isolated from Canton S animals. Two cDNA libraries were constructed from each of three stages: embryonic, late larval, and early pupal. One set of libraries was primed from the 3' end using an XhoI-oligo dT primer adapter. The cDNAs were directionally inserted between the RI-XhoI sites of lambda ZAPII, such that XhoI is at the 3' end of the insert and RI is at the 5' end. Most of these cDNAs should contain 3' end sequences. The other libraries were synthesized using random primers and the cDNAs were inserted into the RI site of lambda ZAPII. These libraries should have a better representation of 5' ends. Although the synthesis of these libraries went smoothly, none have yet been tested. We would thus like feedback on the results of any screens. The titers are all approximately 10[10] pfu/ml. Please send a Federal Express number to facilitate shipment. --Peter Tolias, Public Health Research Institute, 455 First Ave., New York, New York, 10016 USA Phone: 212-578-0815 FAX: 212-578-0804 Email: tolias@phri.nyu.edu Vector/Insertion/Complexity/mRNA source lambda gt22A/SalI-NotI/5x10[5]/Canton S ovaries, stages 1-14 The available amplified aliquots of the Tolias ovarian gt22A directional cDNA library were titred at 3 x 10(8) pfu/ml (99% inserts) before freezing. The original complexity of this sample was 500,000 independent clones (99.7% inserts). The 5' end has unique sites for EcoRI and SalI (GAATTCGTCGACCCACGCGTCCG), the 3' end has a unique NotI site. Use a fresh Y1090 O/N grown in LB amp (50 ug/ml), 0.2% maltose and 10 mM MgSO4 as recommended by most protocols. If you want to screen this library by PCR, I suggest that you use a small fraction of this aliqout to reamplify and use only reamplified samples for PCR. This library has been widely distributed in the USA, Canada and Europe. To conserve the remaining aliquots, please check oogenesis labs in your area first before requesting it. If it is not available, please send us a Federal Express number to facilitate shipment. When you receive the library, divide it into 50 ul aliquots in siliconized microfuge tubes, add one drop of chloroform, store one of the aliqouts at 4 deg C and freeze the rest at -70 deg C. When a frozen aliquot is required, thaw, use and store at 4 deg C but do not freeze again. Reference: Stroumbakis, N.D., Li, Z. and Tolias, P.P. (1994). RNA- and single-stranded DNA-binding (SSB) proteins expressed during Drosophila melanogaster oogenesis: a homolog of bacterial and eukaryotic mitochondrial SSBs. Gene in press. --Kai Zinn, Division of Biology, 216-76, Caltech, Pasadena, CA 91125, USA Phone: 818-356-8352 FAX: 818-449-0679 Email: kai@seqvax.caltech.edu Vector/Insertion/Complexity/mRNA source lambda gt11/EcoRI/1.2x10[6]/Oregon R, 9-12 hr embryos The complexity is an underestimate for larger cDNAs, since it was >5X size-selected for cDNAs larger than 1.8 kb. The complexity could thus be as high as 6x10[6] for these larger inserts. GENOMIC LIBRARIES --Winifred W. Doane, Department of Zoology, Arizona State University, Tempe, Arizona 85287-1501 USA Phone: 602-965-3571 FAX: 602-965-2012 Email: icwwd@asuacad Vector/Insertion/Complexity/DNA source pWE15/BamHI/4x10[4]-1x10[6]/Amy[1,6] mapP[12] strain of D. melanogaster This cosmid vector contains a T3 and T7 promoter on either side of the insertion site, to facilitate the preparation of end-specific probes for chromosomal walking. Reference: Thompson, D.B., and Doane, W.W. (1989) A composite restriction map of the region surrounding the Amylase locus in Drosophila melanogaster. Isozyme Bull. 22: 61-62. --Ron Blackman, Department of Cell and Structural Biology, 505 S. Goodwin Ave., Univ. of Illinois, Urbana, Illinois 61801 USA Phone: 217-333-4459 FAX: 217-244-1648 Email: Ron_Blackman@qms1.life.uiuc.edu Vector/Insertion/Complexity/DNA source lambda EMBL3/BamHI/1x10[6]/Adult Drosophila virilis lambda EMBL3/BamHI/1x10[6]/Embryonic D. melanogaster, see below Both libraries were prepared by MboI partial digestion of the DNA and insertion into the BamHI site of lambda EMBL3. The inserts can be excised by digestion with SalI. Titer is approximately 5x10[9] pfu/ml. The D. melanogaster genomic library is made from animals that are isochromosomal for chromosome 2, dp cn bw. The same strain was used by Nick Brown for his cDNA libraries. --Howard Lipshitz, Division of Biology, 156-29, California Institute of Technology, Pasadena, CA 91125, USA Phone: 818-356-6446 FAX: 818-564-8709 Email: lipshitzh@starbase1.caltech.edu Vector/Insertion/Complexity/DNA source Charon 4/EcoRI/6x10[5]/Canton S embryos This is the original Drosophila genomic library from the Maniatis lab. It has been amplified several times but is still useful for most purposes. Reference: Maniatis et al., The isolation of structural genes from libraries of eucaryotic DNA. Cell 15: 687-701. --Richard W. Padgett, Waksman Institute, Rutgers University, Piscataway, NJ 08855, USA Phone: 908-932-0251 FAX: 908-932-5735 Email: padgett@mbcl.rutgers.edu Vector/Insertion/Complexity/DNA source/Titer lambda DASH II/Sau 3A/5x10[5]/dp cn cl bw/1x10[8] lambda DASH II/Sau 3A/5x10[5]/st e/1x10[8] Libraries were constructed from adult DNA from dp cn cl bw and st e strains. The dp cn cl bw strain is the same one used by N. Brown in constructing his cDNA libraries. The st e strain is the same one used by Wieschaus, Nusslein-Volhard and co-workers in their screens for pattern mutants. Libraries will be sent if the requester provides a Federal Express number. Reference: Finelli, A.L., C. A. Bossie, T. Xie and R. W. Padgett (1994). Antimorphic Alleles of the Drosophila tolloid Gene Contain Amino Acid Substitutions in the Protease Domain, Development, in press. --John Tamkun, Department of Biology, University of California, Santa Cruz, CA 95064, USA Phone: 408-459-3179 FAX: 408-459-3139 Vector/Insertion/Complexity/DNA source lambda EMBL3/BamHI/>5x10[5]/iso-1; see ref. lambda EMBL3/BamHI/>5x10[5]/D. virilis NotBamNot-CoSpeR/BamHI/high/iso-1 The iso-1 strain, constructed by Jim Kennison, is isogenic for all four chromosomes. Two genomic libraries of iso-1 DNA are available, one in a lambda and one in a cosmid vector. Both are in wide use and have been used successfully by many labs. A cDNA library from iso-1 0-24 hr embryos is also available. Reference: Tamkun et al. (1984), PNAS 81: 5140-5144. *** DIN 14 TECHNICAL NOTES INJECTING UN-DECHORIONATED EGGS OF DROSOPHILA MELANOGASTER UNDER ETHANOL S. Bartoszewski and J.B. Gibson, Molecular and Population Genetics Group, Research School of Biological Sciences, The Australian National University, P.O. Box 475, Canberra City, ACT 2601 Australia, E-mail: bartoszewski@rsbs1.anu.edu.au In using a standard method of injecting Drosophila eggs we encountered a serious problem with egg viability. Dechorionated eggs incubated under oil did not develop, while control, un-dechorionated eggs hatched normally. A method for injecting un-dechorionated eggs has been described (Robertson et al., 1988; Cockburn et al., 1991; K. Matthews, personal communication) which has the advantage that difficulties with controlling humidity and preventing desiccation are avoided. On the other hand, un-dechorionated and non-desiccated eggs accept smaller quantities of injected solution. As we first thought that an infection might be responsible for the reduced egg viability we tried immersing un-dechorionated eggs in ethanol. We found that we obtained good viability when un-dechorionated eggs were injected when covered with 100% ethanol instead of oil. As cytoplasm starts leaking after injecting it is precipitated by the ethanol, sealing the puncture. The treatment probably also dehydrates eggs to some extent, so it is possible to inject similar amounts of DNA as were injected into dechorionated and desiccated eggs. Another advantage of using ethanol is that post injection care is much easier. The use of ethanol would not be appropriate if it might affect the phenotype being investigated, e.g. ADH activity (Bijlsma-Meeles, 1979). Below we give a detailed protocol for the technique: 1. With a paintbrush put about 100 eggs into a drop of water on a coverslip stuck to a slide. Mix with a dissecting needle. Suck out most of the water with a pipette and a tissue. Wash the eggs again if too much yeast remains. 2. Place eggs in small groups about 3-4 mm from the end of the coverslip in tiny drops of water. We usually arrange eggs in 10 groups each containing 5 eggs. Using the corner of a rolled tissue, suck out most of the water from a group of eggs, so that the eggs can be moved with a dissecting needle. Arrange the eggs so that they stick together, at an angle of about 45 degrees to the edge of the coverslip. Repeat with other groups of eggs. Using a tissue suck out remnants of water and discard the remaining embryos. 3. Arrange the slide on the microscope stage. Gently, drop ethanol onto the coverslip. Don't add too much ethanol, because if it seeps over the coverslip you have to dry the slide and the coverslip and mount them again. It is better to add more ethanol as it evaporates. Adjust a needle loaded with DNA so that it is close to the embryos. Under pressure adjust the needle so that it just pierces the tip of one of the eggs. Move the needle with the micro-manipulator horizontally in order to break the tip of the needle. This procedure gives a very fine broken end to the needle. The needle we use is more sharply tapering than that used for dechorionated eggs, but a fine tip is essential. Start injecting slightly away from the tip of the egg, which is much harder than the rest of the chorion. Bending the eggs to one side helps to place the tip of the needle in the germ cytoplasm. 4. After injecting all of the eggs, suck out the ethanol and under a dissecting microscope destroy any eggs that are too old. 5. Put the coverslip with the embryos into a vial of a fly food. The coverslip is pushed into the food so that the eggs are in close proximity to the food surface but not in contact with it. A few drops of water are added to the cotton plug to keep the vial humid. The vial does not require any attention until adult flies start to eclose. This method gives very good viability - in a recent experiment, 505 eggs were injected and 136 adult flies were obtained. This is similar to the standard method using dechorionated eggs, but the injecting and post-injection care takes us about half of the time required by the standard method. References: Bijlsma-Meeles, E. 1979, Heredity 42, 79-89; Cockburn, A.F., Meier, H. and Benedict, M.Q. 1991, DIS 70, 240; Robertson, H.M., Preston, C.R., Phillis, R.W., Johnson-Schlitz, D.M., Benz, W.K. and Engels, W.R. 1988, Genetics 118, 461-470. *** DIN 14