DROSOPHILA INFORMATION NEWSLETTER Volume 8, October 1992 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 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 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 *** 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 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 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. 8 TABLE OF CONTENTS >Introduction to Drosophila Information Newsletter >How to subscribe to the Newsletter >TABLE OF CONTENTS >ANNOUNCEMENTS >hb stocks available >REQUESTS FOR MATERIALS >Wild-type strains from northern latitudes >Unstable ring X >Natural orcein >DATABASES/COMPUTING >Umea stock list >New release of Cytological Features Database >GENETIC NOTES >Updates and corrections to the Redbook >TECHNICAL NOTES >Immunostaining of polytene chromosomes >Modified ppac plasmid for transient expression of proteins in transfected tissue culture cells >Plasmid vectors for expressing proteins in tissue culture cells or studying enhancer function *** The Bloomington Stock Center is currently maintaining a large collection of hb alleles or related material. We will be eliminating the majority of these stocks from the collection in the near future. If you would like to receive any of the stocks listed below you should order them as soon as possible, but no later than December 1, 1992. 3137 Dp(3;4;Y)Rg-pbxV, p[p]/p[p]; Dp(3;1)68, y[2]/FM6 1751 hb[2-16] rsd/TM3 1750 hb[2-4] rsd/TM3 1752 hb[5-1] rsd/TM3 1756 hb[Rg-pbx-rv1]/TM1 1757 hb[Rg-pbx-rv2]/TM1 1758 hb[Rg-pbx-rv3]/TM1 2399 In(3R)hb[D]/T(2;3)ap[Xa] 3038 l(3)85A-V1/TM3 (potential deletion) 3039 l(3)85A-V8/TM3 (potential deletion) 2400 Rg-pbx[+RE1]/TM3 3045 Rg-pbx[+RE2]/TM3 3049 Rg-pbx[+RE4]/TM3 3050 Rg-pbx[+RE5]/TM3 3041 Rg-pbx[+RE6] ca e[s]/TM3 3048 Rg-pbx[+RE7]/TM3 3051 Rg-pbx[+RE8]/TM3 3047 Rg-pbx[+RE9]/TM3 3046 Rg-pbx[+RG2]/TM3 3040 Rg-pbx[+RX1] ca e[s]/TM6B 1743 Rg-pbx[b16] rsd/TM3 1741 Rg-pbx[b2] rsd/TM6B 1745 Rg-pbx[b50] rsd/TM6B 1747 Rg-pbx[e21] Ki red e/TM3 1737 Rg-pbx[pr1-4] p roe/TM6B 1759 T(Y;3;4)P96, Rg-pbx/red cv-c sbd *** REQUESTS FOR MATERIALS WILD STRAINS OF DROSOPHILA MELANOGASTER John Ringo, Dept. Zoology, U. of Maine, Orono, ME 04469, USA. 207-581-2556, FAX/2537, RINGO@MAINE. I am seeking wild strains of Drosophila melanogaster captured above the 45th parallel. The higher the latitude the better. I hope to obtain strains from a wide range of geographic locations. Both flies and information about whom to contact directly for flies will be deeply appreciated. *** UNSTABLE RING X Kathy Matthews, Dept. of Biology, Indiana U., Bloomington, IN 47405, USA. 812-855-5782, FAX/2577, MATTHEWK@INDIANA.EDU. The Bloomington Stock Center would like to have a line of R(1)2, w[vC] that is sufficiently unstable to provide a useful number of mosaics, but not so unstable that it is extremely difficult to maintain. If you have such a line, please call me. *** NATURAL ORCEIN Michael Ashburner, Dept. of Genetics, U. of Cambridge, Downing St., Cambridge CB2 3EJ, England. 44-223-333969, FAX/333992, MA11@PHX.CAM.AC.UK. Does anyone know a source for Natural Orcein? Gurr's (that is BDH) now only stock synthetic. If you let me know directly then I will broadcast the information in the next issue of the Newsletter. Thank you. *** DATABASES/COMPUTING EUROPEAN STOCK CENTER AT UMEA STOCK LIST An electronic version of the European Drosophila Stock Center's 1991-1992 stock list is now posted on the Indiana file server in the file Umea.txt. *** CYTOLOGICAL FEATURES DATABASE, VERSION 9207 Sally Amero, Dept. of Molecular and Cellular Biochemistry, Loyola U. Medical Center, 2160 South First Ave., Maywood, IL 60153. 708-216-3365, FAX/8523, YM60SAA@luccpua.bitnet. This is a database of polytene chromosome sites that have been found to bind antibodies to particular Drosophila proteins. The database is maintained by Sally Amero and has been formatted by Michael Ashburner. It is available by anonymous ftp from the Indiana fileserver (ftp.bio.indiana.edu) in the directory fly. The database is kept in two files: Amero.txt is the database itself and Amero.Refs are the references for the database. The file Amero.doc contains information about the organization of Amero.txt. *** GENETIC NOTES UPDATES AND CORRECTIONS TO THE REDBOOK (Editor's note: if you have similar corrections/updates to the Redbook, please send them to KM. A cumulative file of these corrections will be posted on the Indiana fileserver.) (1) mhc, myosin heavy chain -- The draft entry on mhc, published in DIN, volume 68, is accurate, but the entry in the final version of the "Genome of Drosophila melanogaster" includes an appended exon diagram of mhc, page 459, that incorrectly labels the five alternative exons 11's. In our primary publication describing the complete structure of mhc, George, et al. Mol. Cell Biol. 9, 2957-2974 (1989), we designated the order of these exons (5' to 3') to be 11e, 11a, 11b, 11c, 11d. (The diagram on page 459 incorrectly orders these exons 11a, 11b, 11c, 11d, 11e). -- Charles Emerson, emerson@castor.rm.fccc.edu (2) Khc, kinesin heavy chain -- The symbol for the kinesin heavy chain gene, shown on p. 296 as Kin, is Khc. -- Bill Saxton (3) p. 638: "shaven: see svb" should read "shaven: see sv" (4) p. 109: add entry "cel, cell lethal: see l(3)84Ab" -- Ken Howard (5) p. 259: add entry "grh, grainy-head: see Ntf" -- Larry Marsh *** TECHNICAL NOTES IMMUNOSTAINING OF POLYTENE CHROMOSOMES Daniele Zink and Renato Paro, ZMBH, University of Heidelberg, Im Neuenheimer Feld 282, D-6900 Heidelberg, Germany. 49-6221-566878, FAX/565891, bsa210@cvx12.inet.dkfz-heidelberg.de Many people have contacted us regarding our protocol for immunostaining of polytene chromosomes. Our current protocol is listed below, in its entirety. We hope that this will be of use to others who are interested in identifying protein binding sites in the polytene chromosomes. 1. Preparation of 3rd instar larvae: Use bottles with rich medium (i.e. 8 g agar, 18 g dried yeast, 10 g soybean meal, 7 g molasses, 80 g malt extract, 80 g cornmeal and 6.3 ml propionic acid in 1 lt water). Add a large drop of live baker's yeast on top of the dried medium. Let the flies lay eggs to the point where larvae will hatch under uncrowded conditions (< 100 larvae/bottle) and grow larvae at 18[o]C. For salivary gland preparations, use crawling 3rd instar larvae. 2. Chromosome squashes: Dissect two pairs of salivary glands in solution 1. Try to get rid of most of the fat body without separating the two glands. Using a tungsten needle with a hook, transfer the glands to a drop of solution 2 on a siliconized coverslip. Fix the glands homogeneously by moving them with the tungsten needle for 10-30 sec in solution 2 (time needs to be adjusted for each individual antigen). Move glands into a droplet (40 ul) of solution 3 on a coverslip (Corning or equivalent quality, 22x22 mm, not siliconized) and leave them for 2 - 3 min. During this incubation, break up the glands and get rid of remaining chitinous structures of the pharynx using tungsten needles. Lower a poly-L-lysine treated slide onto the coverslip. Under the stereo-microscope, tap the coverslip with a pencil until cells are broken up. Hold the coverslip and spread the chromosomes using the eraser-end of the pencil. Remove excess fixative by pressing slides (coverslip down) onto blotting paper. Examine the preparation under phase contrast. Mark the position of the coverslip. After freezing slides in liquid nitrogen, flick off coverslip with a razor blade. Wash slides two times for 15 min. in PBS, slowly shaking the rack. Proceed with the immunostaining or keep the slides (up to one week) in 100% methanol or in 50% (w/v) ammonium sulfate at 4[o]C. 3. Immunostaining: Stored slides are washed 2 x 15 min. in PBS. Block for 1 hour in blocking solution at room temperature (rt). Add 40 ul affinity-purified primary antibodies (i.e. rabbit polyclonal antibodies; try dilutions in the range from 1:50 to 1:500 in blocking solution) to each slide. Cover with coverslip and incubate for 1 h at rt in a humid chamber. Rinse in PBS. Wash: 15 min in PBS, 300mM NaCl, 0.2% NP40, 0.2% Tween20-80; then 15 min in PBS, 400mM NaCl, 0.2% NP40, 0.2% Tween20-80. If background problems persist, NaCl conc. can be raised to 500mM. Shake rack thoroughly during washing. Rinse in PBS. Add 40 ul diluted secondary antibody (i.e. anti-rabbit IgG (Fc) HRP- conjugate, Promega cat.# W4011, 1:100 dilution) with 2% normal (goat) serum in blocking solution. Cover with coverslip and incubate for 40 min. at rt in humid chamber. Rinse in PBS. Wash exactly as described above for the primary antibody. Rinse in PBS. Add 100 ul 0.5 mg/ml DAB-solution (diaminobenzidine tetrachloride; Sigma # D5637) + 0.01% H[2]O[2] (Merck # 7210). Watch the color develop under phase contrast. Stop reaction by dipping slides in PBS. Wash 10 min. in PBS. 4. Cytology: Stain chromosomes for 10-20 sec. in Giemsa solution (Merck # 9204; 1:130 dilution in 10 mM sodium phosphate buffer pH 6.8). Rinse in distilled water. Mount in 99.5% glycerol and immediately examine the slides under the microscope - the giemsa stain fades within a few hours. Chromosomes can be washed in PBS and restained. For storage, slides can be frozen at -20[o]C. Entelan (EM Science) can be used as a permanent mounting solution. In order to increase the contrast between the signals and the chromomeres (important for black and white photography) DAB precipitates can be enhanced by applying a silver amplification system (Amersham). The enhancement is performed according to the manufacturer's protocol, except that the silver amplification step is shortened to about 1 min. Solutions and reagents: Solution 1: 0.1% Triton X-100 in PBS pH 7.5. Solution 2: 3.7% formaldehyde, 1% Triton X-100, in PBS pH 7.5. The 37% formaldehyde stock solution used to make this solution is prepared as follows: 1.85g paraformaldehyde dissolved in 5 ml water, add 70 ul 1 N KOH, dissolve by boiling). Solution 3: 3.7% formaldehyde, 50% acetic acid. Important: Solutions 2 and 3 should be made fresh every 2 - 3 hours! Blocking solution: 3% BSA, 10% non-fat dry milk, 0.2% NP40, 0.2% Tween20-80 in PBS. Do not worry about the turbidness of the solution. It still works! Preparation of Poly-L-lysine coated slides for chromosome squashes: Start with 100 - 200 slides in racks. Soak slides in a corrosive detergent solution for 2 hrs. Wash under running tap water for 2 hrs. Wash in distilled water, two changes. Dip in 95% ethanol, two changes. Air dry. Dip slides into poly-L-lysine solution (slide adhesive solution, 0.1% w/v in water, Sigma Cat# P 8920). Withdraw rack, solution should wet slides uniformly and stay on slides. Air dry slides. *** PLASMID VECTORS FOR EXPRESSING PROTEINS IN DROSOPHILA TISSUE CULTURE CELLS OR STUDYING ENHANCER FUNCTION Michael Koelle and David Hogness, Dept. of Developmental Biology, Beckman Center, Room B300, Stanford University School of Medicine, Stanford, CA 94305-5427, U.S.A. 415-723-6263, FAX/415-725-7739. Two plasmid vectors for expression of proteins in transfected Drosophila tissue culture cells have been constructed. 1) pMK26 is a vector for protein expression from the actin 5C promoter in transient transfections. It contains unique SalI, HindIII, EcoRV, and PstI sites downstream from the Drosophila actin 5C promoter and upstream from a fragment containing the SV40 splice site and poly(A) addition signals. It was derived from Bluescript+KS and pPac (Krasnow et al., Cell 57: 1031-1043, 1989). 2) pMK33 is a vector for inducible protein expression from the metallothionein promoter. This plasmid contains a bacterial hygromycin-resistence gene driven by the Drosophila copia promoter, allowing the selection of stable transformed cell lines. Unique XhoI, EcoRV, BamHI, and SpeI sites are located downstream from a Drosophila metallothionein promoter and upstream from the Drosophila actin 5C poly(A) addition signal. Open reading frames inserted into this multiple cloning site will be efficiently expressed upon addition of copper to the culture medium. This plasmid contains fragments from pPac, pHSX-MT (Kaufman et al., Cell 59: 359-371, 1989), and pcophyg (Rio et al., Cell 44: 21-32, 1986). We have also constructed a general purpose lacZ reporter plasmid for use in Drosophila and Drosophila tissue culture cells. This plasmid, pMK42, contains a minimal Drosophila Adh distal promoter (from -34 to +53 with respect to the transcription start site) driving a hybrid lacZ gene, consisting of the Ubx untranslated leader and initiation codon, the E. coli lacZ ORF, and SV40 splice and poly(A) addition signals. A unique SalI site is located upstream of the Adh TATA box for the insertion of foreign enhancer elements. pMK42 is a derivative of pD(delta)5'-34 (Heberlein et al., Cell 41: 965-977, 1985) and cP(beta)bxd6.2 (Irvine et al., Development 111: 407-424, 1991). All of these plasmids have been extensively tested (e.g. Koelle et al., Cell 67: 59-77, 1991). Further information and DNA is available upon request. Please direct inquiries to Betty Swyryd at the address and phone number given above. *** MODIFIED pPac PLASMID FOR TRANSIENT EXPRESSION OF PROTEINS IN TRANSFECTED TISSUE CULTURE CELLS Lisa Urness and 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 pPac plasmid was constructed by Krasnow et al. (Cell 57: 1031-1043, 1989) to allow efficient expression of proteins in transfected Drosophila tissue culture cells. pPac contains a single unique BamHI cloning site located between the actin 5C promoter and poly(A) addition signal. To extend the usefulness of this plasmid, we have inserted a polylinker into this BamHI site. The modified vector, which we call pPacPL contains unique BamHI, EcoRV, SpeI, XbaI, KpnI, SacI, NotI, and HpaI restriction sites for the insertion of open reading frames. We will be happy to provide DNA and maps to anyone interested in using this modified vector. ***