DROSOPHILA INFORMATION NEWSLETTER Volume 3, July 1991 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 for publication in DIS. Materials appearing in the Newsletter will be reprinted, in unedited form, in the next issue of DIS. Back issues of DIN are posted on the Indiana fileserver in the directory fly/news. Material appearing in the Newsletter may be cited unless specifically noted otherwise. Material for publication may be submitted in any of the following formats - Macintosh Microsoft Word or MacWrite, MS-DOS WordPerfect, or text/ASCII file. Figures and photographs cannot be accepted at present. Send material, in order of preference, as E-mail (addresses below), on floppy disk, or as laserwriter or typed hard-copy (not bit-mapped). Technical notes should be sent to Carl Thummel, all other material should be sent 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. 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 many of 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@IUBACS.BITNET THUMMEL@MEDSCHOOL.MED.UTAH.EDU MATTHEWK@UCS.INDIANA.EDU *** To add your name to the Newsletter distribution list, send one of the following E-mail messages. Via Bitnet -- To: LISTSERV@IUBVM Subject: Message: SUB DIS-L Your real name Via Internet -- To: LISTSERV@IUBVM.UCS.INDIANA.EDU 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 are on the list and do not wish to receive DIS, or you want to remove a 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 Vol. 3 TABLE OF CONTENTS >Introduction to Drosophila Information Newsletter >How to subscribe to the Newsletter >TABLE OF CONTENTS >ANNOUNCEMENTS >E-mail directory of Drosophila workers >Lefevre slides available >Midwest regional Drosophila conference >REQUESTS FOR MATERIALS >Information for libraries list >Clones for Drosophila genome project >DATABASES/COMPUTING >IUBIO - New FTP address for Drosophila information >TECHNICAL NOTES >Beta-gal activity in semithin epon sections >Double-staining technique for discs *** ANNOUNCEMENTS E-MAIL ADDRESSES OF DROSOPHILA WORKERS John Haynie established, maintains, and periodically distributes a list of e-mail addresses of Drosophila workers. His list formed the basis of the Newsletter distribution list, for which the editors are ever grateful. If you would like to add your name to his list, send a Bitnet message to John at HAYNIE@GENESEO. When the next update of the list appears (any day now) it will be posted on the Indiana fileserver in the directory fly/news (see note from Don Gilbert, this issue). *** LEFEVRE SLIDES AVAILABLE All of the salivary gland polytene chromosome squashes used by George Lefevre in the course of his radiation mutagenesis work on the X chromosome must soon find a new home or be discarded. If you are interested in having these slides, please contact Burke Judd, N.I.E.H.S., P.O. Box 12233, Research Triangle Park, NC 27709. 919-541-4690; FAX/7953. *** MIDWEST DROSOPHILA CONFERENCE The Midwest regional meeting will be held October 11 and 12, 1991 at Allerton Park, IL. If you are not already on the mailing list and would like information on the meeting, contact Hugh Robertson, Dept. of Entomology, U. of Illinois, 505 S. Goodwin, Urbana, IL 61801 (phone: 217-333-0489, FAX/244-3499. HUGH_ROBERTSON@QMSL.LIFE.UIUC.EDU). *** REQUESTS FOR MATERIALS COMPILATION OF DROSOPHILA GENOMIC AND CDNA LIBRARIES. Carl S. Thummel, HHMI, 5200 Eccles Institute, Bldg. 533, U. of Utah, Salt Lake City, Utah, 84112, USA. 801-581-2937, FAX/5374, THUMMEL@MEDSCHOOL.MED.UTAH.EDU. Although a number of Drosophila genomic and cDNA libraries have been constructed, one must depend on word-of-mouth and publications to learn which libraries are currently available and how useful they are for specific experimental purposes. I would like to create a listing of all available Drosophila genomic and cDNA libraries. This compilation should prevent the construction of duplicate libraries as well as facilitate the isolation of Drosophila genes. This listing will include the following categories: genomic (cosmid, phage) and cDNA (regular and expression). Please specify which libraries you have available and send the following additional information (preferably by Email or FAX). 1. Vector 2. Complexity (i.e. total number of clones that are present in the library) 3. Titer (if known) 4. Source of DNA (Drosophila strain; also stage and tissues for cDNA) 5. Your name, address, phone and FAX numbers 6. Any additional information that might be of use. 7. References (if any) Since amplified phage libraries can be safely shipped by regular mail, without wet or dry ice, the distribution of these libraries should entail little expense. Thank you for your help. This should be a valuable resource for the entire Drosophila community. *** REQUEST FOR CLONES Leonard Rabinow[1] and Robert Saunders[2]. [1]Waksman Institute, Rutgers Univ., Piscataway, NJ 08855-0759, USA. 908-932-0091/0092, FAX/5735, RABINOW@BIOVAX. [2]Dept. of Biochemistry, Univ. of Dundee, Dundee DD1 4HN, UK. (0382)23181 ext. 4790, FAX/201063, BI31@UK.AC.DUNDEE.PRIMEB (reverse node order from USA). Most readers of DIS are probably aware of a number of projects in progress with the aim of constructing a physical map of the Drosophila melanogaster genome. A collaboration among the laboratories of F.C. Kafatos, C. Louis, C. Savakis, D.M. Glover, and M. Ashburner is proceeding by fingerprinting cosmids selected with cytological division-specific probes produced by microdissection from polytene chromosomes (Nucl. Acids Res. (1990) 18, 6261-6270). J. Messing, L. Rabinow, W. Sofer and G. Hamm at the Waksman Institute, are initiating a project to produce a restriction map of the genome, using bacteriophage P1 and automated reading of partial restriction digests. Both projects will correlate their developing physical maps with the genetic and cytological maps of Drosophila. To achieve this, we would like to probe our libraries with loci cloned and mapped by members of the Drosophila community. We would greatly appreciate it if members of the fly community willing to contribute clones would send them to either of the above addresses. It would be most convenient if clones could be sent in plasmids (preferably bearing T7, SP6 or T3 RNA polymerase promoters), or M13, to avoid cross-hybridization problems. A few micrograms of a specific oligonucleotide, if available, could also be used. Submissions would be most useful if accompanied by information on the name, identity, and location of the clone, its vector, a restriction map or sequence, and a reference. To coordinate the overall efforts, clones and data will be exchanged among these groups. Submitted clones will be exchanged only between these two groups, unless specifically authorized by the sender. *** DATABASES/COMPUTING IUBIO - BIOLOGY ARCHIVE HAS A NEW ADDRESS Don Gilbert, Dept. of Biology, Indiana Univ., Bloomington, IN 47405. 812-855-7807, FAX/6765, GILBERTD@IUBACS. The IUBIO Archive of biology software and data is an anonymous ftp archive that you will find on the computer with the Internet name of FTP.BIO.INDIANA.EDU. This archive has been located at the computer called IUBIO.BIO.INDIANA.EDU. The archive has been moved temporarily to another computer, and will be moving back to IUBIO and/or to other computer(s) as we upgrade software and hardware. You should always be able to use the name FTP.BIO.INDIANA.EDU to get to the current archive location. I will also post location changes on the Bionet.software Internet news group. The FTP software on the current computer follows the Unix standard, rather than the VMS syntax found at the previous location of IUBIO. Remember that Unix is case sensitive. Please let me know if you have problems with the new archive location. This IUBIO Archive is on the Internet network of computers with the name FTP.BIO.INDIANA.EDU. The actual host computer and Internet number for this archive may change. The Internet address of the current IUBIO archive computer is 129.79.224.50. Mail about this archive or software on it may be directed to: Archive@Bio.Indiana.Edu (preferred Internet address) Gilbert@Cricket.Bio.Indiana.Edu (alternates if pref. is down) Gilbertd@UCS.Indiana.Edu Gilbertd@IUBACS.Bitnet Access to IUBIO -- If your computer system is linked to the Internet, it probably has an FTP program. Each FTP program has it's own peculiarities, but most follow a general syntax: ftp ftp.bio.indiana.edu -- connect to archive computer user: anonymous password: guest or your real user name (preferred) ? or help -- general help for ftp cd subdirectory -- change to subdirectory (Unix) cd .. -- change to superdirectory (Unix) binary -- use full binary transfer ascii -- use text transfer get any.file -- fetch a file from the archive put my.file -- put a file to the archive (only for receive directory) quit, bye -- close the connection An abbreviated directory of the archive (Unix) Archive.doc About this archive (this document) Files Full list of archive files Files.new Most recent archive files biology/ General biology chemistry/ Chemistry fly/ Drosophila stocks and data help/ Help documents molbio/ Molecular biology science/ General sciences util/ Computer and archive utilities ./molbio: align/ Sequence alignment codon/ Codon tables data/ Molecular data evolve/ Evolution and phylogeny ibmpc/ MSDOS software mac/ Macintosh software rnafold/ RNA secondary structure search/ Databank searching *** BOWLING GREEN STOCK CENTER STOCK LIST NOW AVAILABLE ON LINE Ron Woodruff, Dept. of Biological Sciences, Bowling Green State Univ., Bowling Green, OH 43403. 419-372-2631; FAX/2024; WOODRUFF@BGSUOPIE. The Bowling Green State University Mid-America Drosophila melanogaster stock list Version 4/91 can be accessed through Internet as follows: FTP ANDY.BGSU.EDU login: ANONYMOUS password: OWN-USERID cd pub cd Drosophila dir get filename quit *** TECHNICAL NOTES DEMONSTRATION OF BETA-GALACTOSIDASE ACTIVITY IN SEMITHIN EPON SECTIONS OF DROSOPHILA Simone Tix and Karl-Friedrich Fischbach. University Freiburg, Biology III, Schaenzlestr. 1, 7800 Freiburg, Germany. Fax: 49-761-203-2745; E-mail: KFF@SUN1.RUF.UNI-FREIBURG.DE. In our hands, the quality of studies of the expression of lacZ reporter genes in frozen brain sections is mainly limited by the poor tissue conservation. We have therefore developed a method to demonstrate beta-galactosidase activity in glutardialdehyde fixed semithin epon sections of larval, pupal and adult brains counterstained with methylene blue/borax/toluidin blue. The procedure was carried out as follows: Brains were dissected in 1 x PBS, fixed on ice for 30 min in 1% glutardialdehyde in 1 x PBS, washed 3 x 5 min in 1 x PBS and incubated at 37 oC over night in a staining solution containing 25 ul of 8% X-gal in DMSO per ml staining buffer (Simon et al., Cell 40, 805-817 (1985)). The next day, the objects were washed again for 3 x 5 min with 1 x PBS and fixed for 1 h in 6.25% glutardialdehyde in 1 x PBS at RT. They were then washed again 3 x 5 min in 1 x PBS and dehydrated (each step 10 min at RT, in 30%, 50%, 70%, 90% and 2 x in dried 100% ethanol). A 20 minute incubation period in xylene (!) at RT followed. The brains were transferred for 1 h into a 2:1 mixture of xylene and epon 812 at RT and stored over night at 4 oC in a 1:1 mixture of xylene and epon 812. Then the xylene was slowly evaporated for 6-10 h at RT in the hood. The tissues were transferred into fresh epon and stored over night in the fridge. The objects were embedded in fresh epon the next day and polymerized for 12 h at 42 oC and 36 h at 65 oC. Semithin 2 um sections were cut on a Reichert-Jung ultratome. The sections were baked over night on a hot plate at 60 oC and stained with a conventional mixture of 0.05% methylene blue/0.05% borax/0.01% toluidin blue for not longer than 30 s at 60 oC. After washing off the excess stain with aqua dest. the preparations were embedded with DEPEX (Serva). In sections treated with this protocol it is easy to distinguish the turquoise lacZ expressing cells from the methylene blue/borax/toluidin blue stained cells in the background. In our case, beta-galactosidase activity could even be detected in the fine cytoplasmic lamellae of glial cells. It is possible to discriminate many other cell types due to their degree of counterstaining. The results are very much superior to Pyronin G counterstaining, because the methylene blue/borax/ toluidin blue mix reveals many structural details of the lacZ- negative tissue. *** DOUBLE ANTIBODY STAINING OF IMAGINAL DISCS Angela Pattatucci and Thom Kaufman, Dept. of Biology and HHMI, Indiana University, Bloomington, IN 47405, USA. 812-855-7674, FAX/2577, KAUFMAN@IUBACS. Our protocol for fixation and staining of imaginal discs was described in DIN Vol. 1. Two procedures for effective double- staining of discs have been developed. The first method uses both HRP- and AP-conjugated secondary antibodies and is superior for staining cells at the surface of the disc, such as those of the peripodial cell layer. If primary antibodies from different sources (e.g., rabbit and mouse) are available, then carry out the fixation and staining steps described in our previous note through the washes that follow incubation of the tissue in DAB, but include both primary antibodies in the primary incubation mix, and use one HRP-conjugated and one AP-conjugated (we used goat anti-rabbit AP from Boehringer Mannheim at 1:200) secondary antibody in the secondary incubation mix. For primary antibodies from the same source, the complete staining process must be repeated in series for each primary antibody (always do the HRP reaction first). The AP reaction mix consists of three parts (adapted from Van Rooijen et al., 1984, J. Histochem. Cytochem. 32:677-681): Soln. 1 - 20 mg/ml naphthol AS phosphate (Sigma) in N,N-dimethyl formamide; Soln. 2 - 60 mg/ml fast blue BB base (Sigma) in 2N HCl, to which an equal volume of 4% NaNO[2] has been added (filter by passing the soln. through a small cotton plug in a pasteur pipet; Soln. 3 - 85 mM Tris (pH 9.8), 21 mM MgCl[2], 11% DMSO, and 5 mM levamisole. Make the final reaction mix by adding 25 ul of Soln. 1, then 50 ul of Soln. 2, to 3.66 ml of Soln. 3. Incubate discs in the reaction mix in the dark for about 30 min; stop the reaction by drawing off the staining solution and washing the discs in several changes of PBS. DAB produces a reddish-brown color, while fast blue BB base produces a blue color. The two chromogens produce a deep-purple to black color when present in the same cell (Boorsma, 1984, Histochemistry 80:103-106). The second method uses only HRP-conjugated secondaries and works for cells at any position in the disc. This technique adapts staining elements from Matthews et al. (1990, Dev. Biol. 137:171-183) and Graham et al. (1964, J. Histo. Chem. 13:150- 152). Follow the procedure previously described for fixation and staining up to the final reaction mix for HRP. Add the following reaction mix to the discs: 0.05% DAB in Tri-PBS, 0.07% NiCl[2], 0.003% H[2]O[2] (DAB-Ni produces a slate grey to blue color). Monitor the reaction using a dissecting microscope and stop the reaction with 0.1 vol of 5% NaAz. If the whole process must be repeated (i.e., primaries are from the same source), incubate discs for 2 hr in 0.5% NaAz in PBS to kill any remaining HRP activity. Repeat the process but substitute this two part staining reaction mix: Soln. 1 - 6 mg/ml 3-amino-9-ethylcarbazole (AEC) in N,N-dimethyl formamide; Soln. 2 - 5% DMSO, 0.0075% H[2]O[2] in 0.1M NaAc pH 5.2; add 50 ul of Soln. 1 to 950 ul of Soln. 2. Incubate discs in this mixture until sufficient color has developed, usually about 10 min. Stop the reaction by washing the tissue in PBS. AEC produces a bright red color, while Ni- DAB/AEC produces a brown to purple color depending on the relative amounts of the two chromogens that have polymerized in a given cell. ***