DROSOPHILA INFORMATION NEWSLETTER Volume 1, January 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 (see below). Materials appearing in the Newsletter will be reprinted, in unedited form, in the next issue of DIS. 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. We have made obvious substitutions in most cases (e.g. u for micro, B for beta). 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 Univ. of Utah Medical Center Indiana University Salt Lake City, UT 84132 Bloomington, IN 47405 801-581-2937; FAX/5374 812-855-5782; FAX/2577 THUMMEL@MEDSCHOOL.MED.UTAH.EDU MATTHEWK@IUBACS.BITNET 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. 1 TABLE OF CONTENTS >Introduction to Drosophila Information Newsletter >How to subscribe to the Newsletter >TABLE OF CONTENTS >ANNOUNCEMENTS >DIS Vol. 70 - Traditional DIS >32nd Drosophila Research Conference >Corrections to Ashburner Drosophila Handbook and Manual >REQUESTS FOR MATERIALS >Clones needed for Drosophila genome project >DATABASES/COMPUTING >Integrated Drosophila Database >Drosophila genetic maps >Drosophila GENMAP database >IUBIO//Stock center stock lists >GENETIC NOTES >Conventions for the naming of genes and their alleles >TECHNICAL NOTES >Drosophila codon tables >A single base error in the pCaSpeR-Bgal polylinker >P element-mediated transformation of D. melanogaster using purified P element transposase >Targeted tissue-specific expression of genes in Drosophila - A P element expression system that uses the Gal4 activator >Whole mount in situ hybridization to imaginal discs using digoxygenin labeled DNA probes >Antibody staining of imaginal discs >Single-fly DNA preps for PCR; Inverse PCR >Topical insecticide test method for Drosophila >EQUIPMENT >Population bottle update *** ANNOUNCEMENTS DROSOPHILA INFORMATION SERVICE Vol. 70 James Thompson, Dept. of Zoology, Univ. of Oklahoma, Norman, OK 73019. 405-325-4821; FAX/7560. Drosophila Information Service has a long and respected history of promoting communication among geneticists, ecologists, systematists, and molecular biologists who focus upon this species in their research. DIS is a non-profit service to biologists world-wide. As you know, Philip Hedrick has stepped down after many years as editor of DIS. I hope you will join me in thanking him for his efforts. In the interests of maintaining this useful organ of communication for the Drosophila community, I have volunteered to become the next editor of DIS. The format of DIS will remain very similar to those in the past, but some areas will be added or expanded. * Research notes and new mutant sections will continue. * Coverage of techniques will be expanded to include more molecular and cellular techniques. * A group of assistant editors will be invited to help solicit material and develop new areas of coverage. * International representatives will be included to help draw attention to Drosophila activities world-wide. * Information will be provided on accessing and using new sources of information, such as the Drosophila Information Newsletter and computerized stock and clone lists. * Information on Drosophila Research conferences in the U.S. and abroad will be included to help promote better communication among all Drosophila workers. DIS 70 may be ordered by sending a check for $8.00 (U.S. dollars drawn on a U.S. bank, payable to "Drosophila Information Service") per copy to the above address by 1 April 1991. *** 32ND DROSOPHILA RESEARCH CONFERENCE The 1991 Drosophila Research Conference will be held March 20-24 at the Chicago Hilton and Towers, Chicago, Illinois, USA. The Drosophila group at Indiana University, Bloomington, IN is responsible for the program. Deadline for advance registration is January 31, 1991. For further information, contact: Anne Marie Langevin, Meetings Manager, Genetics Society of America, 9650 Rockville Pike, Bethesda, MD 20814; 301-571-1825. *** REQUESTS FOR MATERIALS 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 US]. 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, 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. *** Michael Ashburner, Department of Genetics, Univ. of Cambridge, Cambridge CB2 3EJ, England. 44-223-333969; FAX/333992; MA11@PHX.CAM.AC.UK. The following errors have been found in my Drosophila Handbook and Manual. If any further errors are found please bring them to my attention. Errors are given as page number/line number; lines are lines of text from the top (+) or bottom (-) of the page. Handbook: 22/7+; for "The diploid chromosome number.." read "The haploid chromosome number.." 87/Table 6.9 legend line 5+. after "(Ref. 23)" insert "; (3)" 99/4- for "Drosophlia" read Drosophila" 99/4- for "larval-curicle" read "larval-cuticle" 99/1- for "Drosophlia" read "Drosophila" 100/3+ for "teisseri" read "teissieri" 377/18+ delete "DCOs" 468/6+ for "+4QCO" read "+6QCO" I thank Scott Hawley for spotting this mistake. 539/5- for "e[s]" read "e" 928/left column/3+ the "in press" reference is "Proc. Nat. Acad. Sci. USA 86:6704-6708" 985 Table 32.6 Column headings: these have been badly scrambled, they should read, from left to right, as follows: F2 GD sterility (%) ---------------------------- Cross Developmental F1 GD sterility at emergence 15 days 10 days temperature % at 18 at 28.5 [o]C [o]C [o]C I thank Dr Ronsseray for bringing this to my attention. [Editor's note: these headings had to be compressed from MA's format to stay within the e-mail line length limit; I hope it is still clear - KM] 1061/right column/4- the "in press" reference is "Cell 59:499-509" 1076 Table 35.1 The totals are incorrect. They should read "3026" for the Drosophilidae, "442" for the Steganinae and "2572" for the Drosophilinae. 1088/2- for "Hungarica" read "Hungariae" 1101 section 35/4.5 the correct title for Evenhuis and Okada 1989 is "In Catalog of the Diptera of the Australasian and Oceanian Regions" ed. N.L. Evenhuis, pp. 609-638. 1130/13+ for "Amiota virosa" read "Amanita virosa" 1181 Fig 37.8 legend. For "D. melanogaster (a); D. simulans (b)" read "D. simulans (a); D. melanogaster (b)" I thank Robert Saunders for spotting this error. 1195/2+ for "wolbachia" read "Wolbachia" 1275 left column/29+ delete this line and add ", 924" to line 28+ 1276 left column/1+ for "Protease" read "Propargite" 1299 left column/28+ for "Amanitabisporgera" read "Amanita bisporgera" 1313 left column/35+ for "D. melanogastor" read "D. melanogaster" (in italics) 1313 left column/36+ delete "4" Manual 194/4+ add ")" after "1989" 370/11+ ("Injection buffer for embryos (Spradling 1986; Protocol 136") for "Na phosphate pH 7.8" read "Na phosphate pH 6.8" I thank Helen Epstein for spotting this mistake. *** DATABASES/COMPUTING INTEGRATED DROSOPHILA DATABASES Daniel Hartl, Dept. of Genetics, Washington Univ. School of Medicine, Box 8232, 4566 Scott Ave., St. Louis, MO 63110, USA. 314-362-7076, FAX/7855, HARTL_D@WUMS. On Thursday April 27, 1989, at the 30th Annual Drosophila Research Conference in New Orleans, Kathleen Matthews organized an informal discussion of Drosophila databases that was attended by about fifty individuals, representing various universities, and Delill Nasser, representing the National Science Foundation. The discussion focused on the urgent need to integrate Drosophila data into a common database in view of the rapid rate at which information was accumulating. The group agreed unanimously that a Drosophila Database Workshop including Drosophila geneticists and informatics specialists should be held, and Michael Ashburner and Daniel Hartl agreed to prepare an application requesting support from the National Center for Human Genome Research, since Drosophila is one of the key model organisms. The proposal was funded, and the workshop was held in Bethesda, MD, December 6-7, 1990. Approximately twenty individuals participated in the Drosophila Database Workshop, including experts in various aspects of informatics, individuals presently involved in developing an integrated system for sharing genetic data in C. elegans, and approximately ten Drosophila geneticists variously involved in maintenance of Drosophila Stock Centers; editorial oversight of Drosophila Information Service; compilation of descriptions of mutants, P-element insertions, and chromosome aberrations; global physical and genome mapping; and maintenance and dissemination of specialized databases (e.g. clones). The meeting was also attended by John Wooley and Machi Dilworth, representing the National Science Foundation, and David Benton, representing the National Center for Human Genome Research. The goals of the workshop were to define the next steps toward integration and dissemination of the databases and to identify a small nucleus of individuals willing to undertake detailed planning and implementation of the system. The extensive data that have accumulated over the past 80 years about the genetics and biology of Drosophila is a tremendous resource not only to Drosophila researchers but to researchers working with other organisms. In the past, a small number of individuals have taken it upon themselves to establish and maintain parts of these data for the good of the whole Drosophila community. The individuals who have maintained "Genetic Variations of Drosophila melanogaster", foremost among them Dan Lindsley, serve as a fine example of this tradition. However, complete reliance on the selflessness of individuals may no longer serve the needs of the field. First, the data are accumulating too rapidly for single individuals to stay abreast of without major sacrifice of their own research careers. Secondly, the data consist of several types, including stock lists, descriptions of mutations, lists of artificial chromosome clones, cosmid contigs, DNA sequences, and so on. These databases constitute a rich resource but are not presently integrated because they are maintained in different formats. Thirdly, some of the stalwart volunteers are unable to continue their database maintenance for personal reasons or because of the vastly expanded effort required. Fortunately, there is a long history of cooperation among Drosophila workers that serves to ease many of the problems sometimes encountered in data acquisition, integration and dissemination. Participants in the Drosophila Database Workshop all agreed that the top priority for a Drosophila database would be to develop a system for integrating the existing individual databases. A group consisting of Thomas Kaufman, Michael Ashburner, William Gelbart, Kathleen Matthews, and John Merriam, with the assistance of the National Library of Medicine, represented by James Ostell, agreed to begin the implementation of these efforts with a view to having a system in operation as soon as possible. *** DROSOPHILA GENETIC MAPS Michael Ashburner, Department of Genetics, Univ. of Cambridge, Cambridge CB2 3EJ, England. 44-223-333969; FAX/333992; MA11@PHX.CAM.AC.UK. Version 9012 of the Drosophila genetic database has been released. Changes from the previous release are given in the file MAINDOC.TEXT. Note that Postscript files of this version have not been released. The database can be obtained electronically from three sources: the archive fileserver at the Department of Biology, Indiana University, Bloomington, Indiana and the Netserver at EMBL, Heidelberg. In addition these files are also available on the SEQNET facility to registered users within the United Kingdom. I am very grateful to Don Gilbert at Indiana, Rainer Fuchs at EMBL and Alan Bleasby at Daresbury for making this database available in these ways. The files are: MAINDOC.TEXT - General documentation (this file) FUNCTIONDOC.TEXT - Documentation of FUNCTION file LOCIDOC.TEXT - Documentation of LOCI file MAPDOC.TEXT - Documentation of MAPLOCI file SYNONYMDOC.TEXT - Documentation of SYNONYM file REFSDOC.TEXT - Documentation of REFS file FUNCTION.TEXT - Genes sorted by function LOCI.TEXT - Genes sorted by gene symbols MAPLOCI.TEXT - Genes sorted by genetic map position SYNONYMS.TEXT - Table of synonyms of gene symbols REFS.TEXT - References From Indiana: The files are kept on IUBIO.BIO.INDIANA.EDU and are accessible by FTP (File Transfer Protocol). The files are in the directory [ARCHIVE.FLY.LOCI]. To obtain any of these files use the following commands: >ftp iubio.bio.indiana.edu ftp> user: anonymous ftp> password: guest ftp> cd [archive.fly.loci] ftp> get DOC.TEXT ftp> get LOCI.TEXT ... et cetera ... or ftp> mget *.TEXT (to retrieve all TEXT files) ftp> bye The numeric address of IUBIO is 129.79.1.101, if your nameserver is as archaic as some. From EMBL: These files are available from the Netserver at EMBL, and if you do not have the facility for FTP this is the way to get them. For HELP send an e-mail message to NETSERV@EMBL.BITNET with the text HELP DROSOPHILA [editor's note: HELP, or other command, must be in the body of the message, NOT on the subject line - KM]. For a listing of the files on the EMBL Netserver send the message DIR DROSOPHILA. To obtain a particular file send an e-mail message with the text GET FILENAME to NETSERV@EMBL.BITNET, where FILENAME is one of the filenames listed above. From SEQNET: The SERC SEQNET computing facility at the Daresbury Laboratory is available to researchers in the United Kingdom. This computer (UK.AC.DL.DLVH) can be directly accessed via JANET. For an account write to Dr. Alan Bleasby, SERC Daresbury Laboratory, Warrington WA4 4AD, Cheshire or send e-mail to AJB@UK.AC.DARESBURY.DLVH. These files are kept in a directory called DATA1:[DROSOPHILA]. *** UCLA DROSOPHILA GENMAPS DATABASE John Merriam, Joy Johnsen and Geunbae Lee. Biology Dept., Univ. of California, 815 Hilgard Avenue, Los Angeles, CA 90024-1606, USA. 213-825-2256, FAX/206-3987, IBENAPR@OAC.UCLA.EDU. SUBJECT COVERAGE. The DROSOPHILA GENMAPS DATABASE is an index to sources of information in Drosophila melanogaster genetics. The subject coverage is limited to genetics research done with molecular biology techniques where genetic information has been localized on the cytogenetic map. DOCUMENT TYPES. Sources of information include journal articles, conference reports and personal contacts. DATES OF COVERAGE. The project began in 1983, but contains some 1970's citations. The literature coverage has been reviewed with bibliographic indexing sources for the period 1980-1990. DATABASE SIZE. The database now includes 857 references leading to 2683 pieces of localized genetic information. The project locates sources through bibliographic searches, attending meetings, and soliciting contacts. The current literature review has produced 624 new references awaiting indexing decisions, which will be completed in 1991. ACCESS IN PRINT. The most current published report is: Genetic Maps 1990 5th edition, edited by S.J. O'Brien. Cold Spring Harbor Press. (September 1989 report) SUPPORT & AFFILIATION: The database project is funded by the National Library of Medicine on NIH grant LM04896. THE CURRENT SUMMARY DATABASE REPORT. The ASCII version of the database report is available on the IUBIO Archive for Biology via FTP. Follow the instructions in the Ashburner report above, but substitute the following command for moving into the relevant directory: cd [ARCHIVE.FLY]. The name of the file is Clonelist.txt. See the file Clone.readme for further information about the clone database. To see a list of all files in ARCHIVE.FLY type DIR. *** STOCK CENTER DATABASES//IUBIO ARCHIVE FOR BIOLOGY Kathy Matthews, Indiana Univ. Drosophila Stock Center, Dept. of Biology, Indiana Univ., Bloomington, IN 47405. 812-855-5782, FAX/2577, MATTHEWK@IUBACS. Don Gilbert, an IU Drosophila biologist turned computer whiz, has established the IUBIO Archive for Biology. To quote from Don's document, "The Archive will maintain biology software and data. Molecular biology is the area of concentration. It will include software for Macintosh, VAX-VMS, Unix, MS-DOS and any other important computer operating systems. Access to the archive is via anonymous FTP programs that access computers on the Internet." Instructions for connecting to IUBIO are given in the Ashburner article above. To find out more about IUBIO, see ARCHIVE.DOC in the root directory of IUBIO. Don can be reached at GILBERTD@IUBACS. Data relevant to Drosophila are in the directories ARCHIVE.FLY and ARCHIVE.FLY.LOCI (Ashburner maps only). In addition to the clone lists also described above, ARCHIVE.FLY contains lists of the Drosophila stocks held by the stock center at Indiana. All of the stocks carried at IUDSC are included in one of the three files FLYLIST.TXT, PLIST.TXT, and NEWP.TXT. These files are sorted by stock number so you will need some way to search them. An assortment of files are also posted that contain subsets of stocks from these tables that are sorted in some way to make them more accessible (e.g., DEFICIENCIES.TXT contains all of the deficiency stocks in the collection sorted by breakpoint). I will be posting more subtables soon. See the readme files posted in ARCHIVE.FLY for further information about the stock center lists. *** GENETIC NOTES CONVENTIONS FOR THE NAMING OF GENES AND THEIR ALLELES Dan Lindsley and Georgianna Zimm, Dept. of Biology, Univ. of California, La Jolla, CA 92093, USA. 619-534-3109, FAX/0053. Genetic loci, by which we mean the segments of DNA responsible for single transcription units, are recognized by virtue of (a) mutant phenotypes, (b) polypeptide products, and (c) transcripts. Alternative transcripts and polypeptides may result from a single transcript, and mutant lesions in the same transcription unit may have different and seemingly unrelated phenotypes. Ideally each locus is designated by a single name and symbol. For genes identified by mutations, the designation is a simple noun or adjective that describes an important and obvious phenotype of the mutation; the designation is abbreviated with a one to three-letter symbol (longer in special circumstances) that begins with the same initial letter as the name. If the phenotype is recognized in heterozygotes, the mutation is considered dominant and the designation begins with an upper case letter; if visible only in the homozygous or hemizygous condition, the mutation is considered recessive and begins with a lower case letter. In cases where mutations in the same gene have been recovered and named independently, they are alleles, and it is desirable that all be designated by a single name and symbol and differentiated from one another by superscripts. It is further desirable that the superscripts be short and simple rather than complicated acquisition numbers, which are without meaning to all but the author; information is sacrificed for the sake of brevity. Deficiencies for genes are not alleles and are therefore not symbolized as such. In general the rules of priority determine which of several alternative designations is chosen; exceptions are where the majority of mutations have a phenotype different from that used in first defining the locus, or where the protein produced by the wild-type allele is identified. Other considerations being equal, the name of the protein encoded is considered most descriptive of gene function and therefore the most appropriate designation for a gene. Many genes are known only from their protein product, i.e., from their wild-type product. The wild-type alleles are considered to be dominant and are named after the polypeptide that they encode, using an upper- case initial letter. The practice of using D as the initial letter of the symbol for genes encoding Drosophila homologues of genes originally identified in other species is considered superfluous and therefore avoided. Loci identified only from normal transcripts are temporarily named for the transcript, including in the name the polytene location of the locus, pending identification of the polypeptide product; here too, an upper- case initial letter is used. Genes with similar mutant phenotypes or functions (e.g., lethals, Minutes, Chorion proteins, Heat shock proteins) are designated similarly but differentiated from one another with a specific designator such as polytene location or in some cases of proteins by molecular weight. Members of gene families (e.g., Actin, Tubulin) are distinguished by polytene location. In instances where neither the location nor product is known, arbitrary distinguishing symbols are used. *** TECHNICAL NOTES DROSOPHILA CODON TABLES VERSION 10.0 Michael Ashburner, Dept. of Genetics, Univ. of Cambridge, Downing St., Cambridge, England. (0223)333969, FAX/333992, MA11@UK.AC.CAM.PHX (invert node order from US). These Tables are supplied with the understanding that they can be freely used for research, although if quoted in any publication a suitable acknowledgement (e.g. Michael Ashburner, personal communication) would be appreciated. I will automatically post new versions on the BIOSCI Bulletin Board. These will generally be compiled whenever enough new data warrents the work. I am very happy to include new sequences that have not yet made the Sequence Data Banks, if these can be sent to me by electronic mail with sufficient data for the coding sequences to be extracted. If anyone should need the files of coding sequences that have been used to generate these tables please send me a message. Two series of Tables are included, one for "host" genes and one for orfs carried by transposable elements. For each series you have a codon table, a base composition and the names of the sequences used to compile these [Editor's note: the lengthy names tables have not been included here; if you need this information send me an e-mail message and I will send you the complete file as submitted by the author - KM]. By and large these sequences are taken from the EMBL, GENBANK or DAYHOFF Libraries. However some have been privately communicated to me. All sequences have been checked that they translate but many are incomplete. Hence, for example, the number of sequences is greater than the number of TER codons. The latest versions of the databanks used are EMBL V20.0 and GENBANK V61.0. The "host" gene coding sequences are from a total of 687.482-kb of sequenced DNA. Table 1A: Base composition of "host" genes: T=67608 C=96225 Y=0 Pyrimidine=163833 A=82349 G=94852 R=0 Purine=177201 N=9 Nucleotides=341043 Table 1B: Codons of "host" genes: TTT 1071 TCT 709 TAT 1089 TGT 649 TTC 2719 TCC 2370 TAC 2350 TGC 1790 TTA 351 TCA 674 TAA 109 TGA 40 TTG 1566 TCG 2054 TAG 59 TGG 1104 CTT 777 CCT 729 CAT 1237 CGT 1118 CTC 1417 CCC 2362 CAC 2044 CGC 2051 CTA 705 CCA 1394 CAA 1478 CGA 775 CTG 4341 CCG 1933 CAG 4490 CGG 797 ATT 1658 ACT 893 AAT 2251 AGT 1102 ATC 2963 ACC 2827 AAC 3168 AGC 2238 ATA 675 ACA 947 AAA 1376 AGA 467 ATG 2804 ACG 1522 AAG 4704 AGG 649 GTT 1164 GCT 1710 GAT 3057 GGT 2019 GTC 1747 GCC 4510 GAC 2821 GGC 3735 GTA 533 GCA 1251 GAA 1823 GGA 2376 GTG 3180 GCG 1578 GAG 5074 GGG 502 Total=113676 Table 2A: Codon table TE genes: TTT 475 TCT 197 TAT 340 TGT 164 TTC 309 TCC 189 TAC 327 TGC 197 TTA 430 TCA 280 TAA 10 TGA 2 TTG 326 TCG 140 TAG 2 TGG 217 CTT 311 CCT 165 CAT 276 CGT 130 CTC 210 CCC 168 CAC 262 CGC 114 CTA 296 CCA 378 CAA 553 CGA 177 CTG 232 CCG 131 CAG 298 CGG 83 ATT 571 ACT 301 AAT 755 AGT 264 ATC 300 ACC 252 AAC 532 AGC 248 ATA 540 ACA 481 AAA 1089 AGA 383 ATG 323 ACG 141 AAG 482 AGG 180 GTT 281 GCT 291 GAT 477 GGT 203 GTC 197 GCC 278 GAC 490 GGC 207 GTA 290 GCA 369 GAA 766 GGA 267 GTG 254 GCG 149 GAG 429 GGG 104 Total=19283 Table 2B: Base composition TE genes: T=14151 C=11974 Y=0 Pyrimidine=26125 A=20241 G=11483 R=0 Purine=31724 N=0 Nucleotides=57849 *** A SINGLE BASE ERROR IN THE pCaSpeR-BGAL POLYLINKER. Howard D. Lipshitz[1] and Carl S. Thummel[2], 1- Division of Biology, 156-29, California Institute of Technology, Pasadena, CA 91125, USA. 818-356-6446, FAX/564-8709. 2- HHMI, Dept. of Human Genetics, Univ. of Utah Medical Center, Salt Lake City, UT 84132, USA. 801-581-2937, FAX/581-5374, THUMMEL@MEDSCHOOL.MED.UTAH.EDU. The P element vector, pCaSpeR-Bgal (Thummel, Boulet, and Lipshitz, 1988, Gene 74: 445-456), is being widely used for fly transformation studies. Expression of the B-galactosidase gene in this vector depends upon the insertion of both a promoter sequence and downstream translational start codon, fused in-frame with lacZ. An error is present in the published DNA sequence of the polylinker region, located upstream of the lacZ coding region. The sequence in the middle of the polylinker should read: ...GGT/CGA/CTC/TAG/AGG/ATC/CCC/GGG/CGA... This will not affect the reading frame for EcoRI fusions, but will shift the reading frame for insertions at the BamHI site. Also, this means that insertions into the PstI and XbaI sites will not be useful since there is an in-frame stop codon in the downstream polylinker sequence. Note that this same DNA sequence is present in the pCaSpeR-AUG-Bgal polylinker; however, it will not affect constructions using this vector since it is not within a protein coding region. We apologize for any inconvenience this may have caused. *** P ELEMENT-MEDIATED TRANSFORMATION OF D. MELANOGASTER USING PURIFIED P ELEMENT TRANSPOSASE Paul Kaufman and Donald Rio, Whitehead Institute for Biomedical Research and Dept. of Biology, MIT, Cambridge, MA 02139, USA. 617-258-5242; FAX/5061. We have recently found that a P element transposase- containing chromatographic fraction (the 'TdT 0.3M' fraction, as described in Kaufman, et al., 1989, Cell 59: 359-371) will efficiently promote P element-mediated transformation upon microinjection of Drosophila melanogaster embryos. In our hands, approximately 50% of fertile G[0] injectees yielded transformed progeny, using the standard 11Kb pDm30 rosy vector. The transposase used was diluted to approximately 1.5 ug/ml in our standard chromatography buffer (20 mM Hepes-NaOH, pH 7.6, 20% glycerol, 0.1M KCl, 0.2 mMEDTA, 0.5 mM DTT, 0.05% Nonidet P-40), supplemented with 25 ug/ml BSA, 70 ug/ml PMSF, 0.2 mM Na metabisulfite, and 0.5 ug/ml antipain, leupeptin, pepstatin, chymostatin, and aprotinin. I do not know if all these protease inhibitors are truly necessary. We've found that a molar ratio of 0.3-1.0 transposase molecules per DNA molecule works well; at a ratio of 3.0, many multiple inserts and dimer inserts are observed. Also, I've used a final DNA concentration of 0.25 mg/ml, although we haven't tested others. However, I did all my experiments with the pDm30 vector, and I don't know if the optimal ratio changes with transposon length. I prepare the transposase/DNA mixtures by adding 1 ul of 1 mg/ml DNA (in standard injection buffer) to a 3 ul transposase aliquot on ice; this can either be frozen in liquid nitrogen and stored at -80[o]C or used directly to load a microcapillary for embryo injection. I performed injections as per published protocols, except that I siliconized the microcapillaries (Drummond Scientific; 75 mm long, OD 1.0 mm, ID 0.75mm) before use by treating them with 5% dichlorodimethylsilane in CCl[4], followed by extensive washing with ethanol and drying in a baking oven. I generally load 2 ul of transposase/DNA mixture for a day's injections, immediately quick freeze the remainder in liquid nitrogen, and store it at -80[o]C. Material that remained in the needle at the end of the day was discarded, but I haven't directly tested the stability of the transposase activity at 18[o]C. Although we have sent aliquots of transposase to many groups recently, our resources won't allow us to continue this practice indefinitely. However, the transposase-overproducing cell line, MTDelta2-3 (see ref. above), has been placed in the American Type Culture Collection, #CRL10191. The transposase purification protocol is also described in above reference. We would be pleased to know about your results in terms of the protein/DNA ratio used, efficiency of transformation, or length of plasmid or cosmid used. *** TARGETED TISSUE-SPECIFIC EXPRESSION OF GENES IN DROSOPHILA - A P ELEMENT EXPRESSION SYSTEM THAT USES THE GAL4 ACTIVATOR Andrea Brand and Norbert Perrimon, HHMI, Dept. of Genetics, Harvard Medical School, Boston, MA 02115, USA. 617-732-7581, FAX/7663. We have developed a two-part system for targeting ectopic tissue-specific expression of genes during different stages of Drosophila development. This system depends on the establishment of two independent fly strains - one strain contains a P element that expresses the yeast Gal4 activator protein and the second strain contains the target gene under the control of a Gal4- inducible promoter. Thus, in one strain, the Gal4 activator protein is present but has no target gene to activate, and in the second strain the target gene is silent. When the two lines are crossed, the target gene is turned on only in the progeny of that cross, allowing dominant phenotypes (including lethality) to be conveniently studied. We have expressed Gal4 in Drosophila in two ways. First, using the enhancer detector technique Gal4 can be expressed in a wide variety of patterns in embryos, larvae, and adult flies. We have established a number of these strains and have shown that they direct tissue- and cell- specific transactivation of a Gal4-dependent lacZ gene. Second, to induce expression at different times during development, we have developed fly strains carrying a heat-inducible Gal4 gene, using the hsp70 promoter. Although these flies have background levels of Gal4 expression, the level is significantly increased upon heat-shock. We can provide more information and the following materials to anyone interested in using this expression system. (1) Flies carrying an enhancerless Gal4 for hopping to different sites in the genome. (2) Flies that express Gal4 in a specific tissue, if available. (3) Flies that have a heat-inducible Gal4 gene. (4) pGATB and pGATN: P elements that contain either a BamHI or NotI site upstream from the Gal4 coding region. This vector allows the insertion of tissue-specific promoters for generating a known expression pattern. (5) pUAST: A P element vector for expressing the target gene under the control of Gal4. This vector contains five Gal4 binding sites, the hsp70 minimal promoter (not heat-inducible), a polylinker with six unique restriction sites for inserting the target gene, and the SV40 intron and polyadenylation signal. Please send requests by FAX, if possible (617-732-7663). A manuscript is currently in preparation describing this expression system in more detail. *** WHOLE MOUNT IN SITU HYBRIDIZATION TO IMAGINAL DISCS USING DIGOXYGENIN LABELED DNA PROBES Helmut Kramer and Larry Zipursky, Molecular Biology Institute, UCLA, 741 Circle Dr., Los Angeles, CA 90024-1570, USA. 213-206- 3750, FAX/5272. as modified by: Jim Masucci and Michael Hoffmann, McArdle Labs, Univ. of Wisconsin, 450 N. Randall Ave., Madison, WI 53706, USA. 608-262- 8854, FMHOFF@VMS.MACC.WISC.EDU. We have found that Buffer 5 from BMB's Genius Kit works 5 fold better than Vogel buffer, and random primer from Pharmacia works better than BMB's. Labelling of probes: Buffer 5: 0.5 M Tris pH 7.2; 0.1 M MgCl[2]; 1 mM DTE; 2 mg/ml BSA, 3 mg/ml pdN6. Buffer 6 (=10X nucleotide mix): 1 mM each dATP, dCTP, dGTP; 0.65 mM dTTP; 0.35 mM digoxygenin-11-dUTP. PBS: 140 mM NaCl; 10 mM KPO[4], pH 7.2. Use 100-500 ng DNA cut into pieces of less than 400 bases. 1) Combine: DNA, 1 ul Buffer 5, 4 ul pdN6 (25 mg/ml, Pharmacia; 2 ul may be enough), H[2]O to 10 ul. Boil 10 min, quick chill on ice. 2) Add: 1 ul Buffer 5, 2 ul Buffer 6, 1 ul Klenow, 6 ul H[2]O. Incubate at 16[o]C overnight. 3) Boil 10 min, chill on ice, add: 1 ul Buffer 5, 1 ul Buffer 6, 1 ul Klenow, 7 ul H[2]O. Incubate at 37[o]C for 4 hr. 4) Add: 4 ul 0.5 M EDTA. Heat to 80[o]C for 15 min. 5) Add: 50 ug tRNA, 2 ml PBT (PBS + 0.1% Tween). Put over centricon 10 (Amicon). 6) Add another 2 mls PBT and spin in centricon 10 again. 7) Adjust volume so DNA concentration is 2 ng/ul. 8) Add equal volume of 100% deionized formamide (probe conc.= 1 ng/ul). 9) Store at -20[o]C. For hybridizations, use 40 ng/100 ul hybridization volume for discs or 10 ng/100 ul for embryos. Staining discs: Hyb Buffer: 50% deionized formamide; 5X SSC; 200 ug/ml tRNA; 100 ug/ml sonicated, boiled salmon sperm DNA; 0.1% Tween 20. All steps at room temperature unless noted otherwise. Staining Buffer: 100 mM Tris-HCl; 100 mM NaCl; 50 mM MgCl[2], pH 9.5 (20[o]C). 1) Dissect discs in PBS for 20 min or less before fixing. Remove fat body and internal organs but leave discs attached to cuticle and mouth hooks and place tissue in an eppendorf tube. For everting discs, dissect discs away from all other structures and do all the fixations in nets. Transfer with a treated pasteur pipet to siliconized eppendorf tubes for the hybridizations (everting discs tend to stick to the sides of untreated tubes). Treat pipets by pipetting spit through them and rinsing in distilled water. 2) Fix discs in 4% paraformaldehyde pH 7-7.5 (important!) in PBS for 15-20 min on ice. 3) Fix discs in 4% paraformaldehyde + 0.6% Triton in PBS for 15 min. (room temp). 4) Wash discs 3 times, 5 min each in PBT. 5) Digest with 10 ug/ml proteinase K for 3-5 min. 6) Wash 2 times, 5 min each in PBT + 2 mg/ml glycine. 7) Wash 3 times, 5 min each in PBT. 8) Fix in 4% paraformaldehyde + 0.2% glutaraldehyde in PBS for 15 min. 9) Wash 5 times, 5 min. each in PBT. 10) Wash in 1:1 mix PBT:Hyb buffer 10 min. 11) Wash in Hyb buffer 10 min. 12) Incubate in Hyb buffer at least 1 hr at 48[o]C. 13) Heat denature probe and add to a final concentration of 40 ng/100 ul. 14) Hybridize for 36 hr at 48[o]C in 100 ul Hyb buffer plus probe. 24 hr may be adequate. 15) Wash in Hyb buffer 20 min at 48[o]C. 16) Wash in 1:1 PBT:Hyb buffer 20 min at 48[o]C. 17) Wash in PBT for 10-12 hr at 48[o]C changing buffer about 5 times. 18) Leave on ice overnight (not necessary, but usually convenient). 19) Add 1 ml 1:2000 anti-DIG antibody from Genius Kit which has been preabsorbed for 1 hr against fixed larval heads. Incubate for 1 hr. 20) Wash four times, 2 hr each in PBT. 21) Wash in staining buffer for 15-20 min. 22) Mix 3.5 ul X-phosphate, 4.5 ul NBT (from Genius Kit) per 1 ml staining buffer, add 1 ml to discs and monitor staining. In general, let staining go a little longer than you think you should since it appears fainter under higher magnification. Background determines how long to let it go. I have let staining go overnight for really light signals with varying degrees of success. 23) Stop reaction by washing several times in PBT. 24) Dissect and mount discs in 80% glycerol; seal coverslip with nail polish. Conditions to vary when problems with background occur: 1) Length of fixation - increasing fix time lowers the signal, including background. 2) Amount of probe - less probe, less background. 3) New antibody - it can go bad. *** 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. Fixation and staining protocols that work well for Drosophila embryos are not optimal for imaginal discs. We recover uniformly well preserved discs that stain reliably from the procedure of Glicksman and Brower (1988, Dev. Biol. 127:113-118) modified as follows. 1) Dissect tissues in Tri-PBS (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na[2]HPO[4], 1.8 mM KH[2]PO[4], 0.2% Triton X-100, pH 7.5), gather in a 1.5 ml tube of Tri-PBS on ice (at least 10 animals can be processed in one tube). Tear larvae in half and invert the anterior portion; remove all gut tissue from mutant larvae by cutting at the esophagus just anterior to the proventriculus. Prepare control larvae as above, but leave the proventriculus attached to the esophagus to serve as a marker, and process in the same tube with mutant animals. 2) Replace Tri- PBS with 280 ul PBS, 120 ul 10% paraformaldehyde in PBS, and 500 ul heptane. Shake by hand for 30-45 seconds. 3) Replace first fixative with 520 ul of PBS, 240 ul of 10% paraformaldehyde in PBS, and 40 ul of fresh DMSO (freeze aliquots of a freshly opened bottle; use each frozen aliquot only once); rock for 20 min. 4) Remove second fix and wash twice with methanol. 5) Replace methanol wash with 980 ul methanol and 20 ul of 30% hydrogen peroxide; rock for 30 min. 6) Wash four times in a ml of 0.1% BSA in Tri-PBS, 10 min. each wash. 7) Replace last wash with 452 ul Tri-PBS, 5 ul 10% BSA, and 40 ul normal goat serum (NGS); rock for 30 min. (these volumes work well for primary antibodies used at concentrations of 1:100 or less; adjust as necessary for antibodies requiring higher concentrations). 8) Add primary antibody directly to the tube and rock overnight at room temperature. 9) Wash tissues 5 times in 1 ml 0.1% BSA in Tri-PBS, for 5, 10, 15, 20, and 25 min. respectively. 10) Replace last wash with 445 ul Tri-PBS, 5 ul 10% BSA, and 40 ul NGS, rock for 30 min. 11) For rabbit primary antibodies, add directly to tube 10 ul Fab' goat anti-rabbit HRP conjugate (Protos Immunoresearch) and rock for 90 min. (substitute appropriate secondary antibody for non-rabbit primaries). 12) Wash twice, 10 min. each, in 0.1% BSA in Tri-PBS, followed by three washes in Tri-PBS. 13) Replace last wash with 450 ul Tri-PBS and 50 ul DAB 5 mg/ml in 0.1 M Tris pH 7.5; rock for 5 min. (DAB is a carcinogen - wear gloves and treat all waste as hazardous). Add 5 ul of 0.3% hydrogen peroxide in water. Monitor color development under a dissecting microscope. Stop reaction by removing DAB solution and quickly washing tissue five times with PBS. Dissect discs and mount in Aqua-Polymount (Polysciences). *** SINGLE-FLY DNA PREPS FOR PCR Greg Gloor and William Engels, Dept. of Genetics, Univ. of Wisconsin, Madison, WI 53706, 608-263-2213, FAX/262-2976, WRENGELS@WISCMACC.BITNET. We have developed a simple method for the rapid and reproducible isolation of DNA from single flies for amplification by the polymerase chain reaction (PCR) (Saiki et al, Science 239: 487), and direct sequencing by asymmetric PCR (Gyllensten and Erlich, Proc. Nat. Acad. Sci. 85: 7652). The simplicity of this procedure means that the problem of contamination with other amplified or cloned DNA is greatly reduced. Sufficient DNA is obtained from one fly for a minimum of 50 PCR analyses, and the DNA is stable for at least one month in the refrigerator. A simple modification of this technique allows the isolation of DNA suitable for use in inverse PCR (Ochman et al, Genetics 120: 621-623). These methods substantially reduce the time involved in DNA isolation, and among other uses, allows the PCR to be used to monitor the segregation of an allele for which there is no phenotype or transposition of an unmarked P element (Engels et al. Cell 62: 515-525). A. DNA PREPARATION PROTOCOL: 1. The squishing buffer (SB) is 10 mM Tris-Cl pH 8.2, 1 mM EDTA, 25 mM NaCl, and 200 ug/ml Proteinase K, with the enzyme diluted fresh from a frozen stock each day. 2. Place one fly in a 0.5 ml tube and mash the fly for 5 - 10 seconds with a pipette tip containing 50 ul of SB, without expelling any liquid (sufficient liquid escapes from the tip). Then expel the remaining SB. 3. Incubate at 25-37[o]C (or room temp.) for 20-30 minutes. 4. Inactivate the Proteinase K by heating to 95[o]C for 1-2 minutes. NOTES: This preparation can be stored at 4[o]C for months. We typically use 1 ul of the DNA prep in a 10-15 ul reaction volume. It does not matter if fly parts (wings, bristles, legs) are inadvertantly added to the PCR mixture. Product will typically start to appear after 24-25 cycles, but 28-30 cycles seems to give maximal yield. Increasing the number of flies does not seem to increase the signal significantly, probably due to increasing concentrations of inhibitors. There should be no problem scaling up the number of flies screened if the volume is increased proportionately. B. DNA PREPS FROM MANY INDIVIDUAL FLIES A similar method can be used with 96-well (8 x 12) micro plates to prepare DNA from a large number of individual flies. Up to 80 flies can be tested with a single plate. 1. Place one anesthetized or frozen fly in each well. All rows (A-H) can be utilized, but leave the first and last columns (1 and 12) empty to ensure complete heating in step 3. 2. Add 50 ul of SB to each well and macerate each fly with a toothpick for 5-10 seconds. Then cover the plate with an adhesive-backed strip to prevent evaporation and contamination. Incubate at room temperature as before. 3. Use two standard-size heating blocks (9.5 x 7.5 cm) pre-heated to 95[o]C to inactivate the Proteinase K. Sandwich the micro plate, with its adhesive lid still in place, between the two heating blocks. The lower block should be inverted so that the tube holes are facing downward and the flat surface is touching the bottom of the micro plate. After 2-3 minutes remove the micro plate and set it on the bench top with the upper hot block still on top. This upper block will gradually cool to room temperature, preventing condensation on the underside of the adhesive. NOTES: It is helpful to tape a piece of waxed paper over the open micro plate while the PCR tubes are being set up. That way the DNA samples can be drawn from each well by poking the pipet tip through the waxed paper. This procedure reduces the possibility of contamination and helps to keep track of which wells have been used. C. INVERSE PCR PMSF (phenylmethylsulfonylfluoride) can be used instead of the 95[o]C treatment to inactivate the Proteinase K if the DNA preps are to be used for inverse PCR or other methods that require double-stranded DNA. 1. Add 1 ul of 0.1 M PMSF to the fly prep following step 3 of protocol A above. Then heat the mixture to 65[o]C for 10 - 15 minutes to denature any proteins not inactivated by the proteinase. 2. Add 4 ul of fly supernatant to 16 ul of 1.25X NdeII buffer (125 mM Tris-HCl pH 7.6, 12.5 mM MgCl[2], 188 mM NaCl, 1.25 mM DTT). Add 0.5 ul of NdeII (BRL) and incubate at room temp. for 15 min. Inactivate the enzyme by heating to 65[o]C for 15 min. 3. Take 3 ul of digested DNA and add to 7 ul of ligation mix (5 mM MgCl2, 20 mM DTT, 0.8 mM ATP). Add 0.5 ul T4 DNA ligase (NEB) and incubate at room temperature for 20-30 min. Inactivate the enzyme by heating to 95[o]C for 2-3 min; this also serves to nick the DNA. NOTES: For the first attempt with a new insertion, we recommend using the following conditions: denature at 94[o]C for 45 sec., anneal at 60[o]C for 45 sec., extend at 72[o]C for 4 min; try 30 - 35 cycles. - The restriction enzyme appears to be the most critical component in this protocol. The enzyme must be specific under conditions of very low DNA concentration. Sau3A1, for example, is too promiscuous and digests at several sites in addition to its canonical restriction sequence. The protocol should work with other enzymes. The protocol has been used for a combined inverse/asymmetric PCR procedure to get DNA sequences flanking P element insertions. *** TOPICAL INSECTICIDE TEST METHOD FOR DROSOPHILA. Dave Dapkus, Dept. of Biology, Winona State University, Winona, MN 55987, USA. 507-457-5274. Insecticide tests with Drosophila are notoriously variable, making it difficult to work with insecticide resistance genes. A test method based on the topical application of insecticide with a micropipette is described here. The method is very sensitive and gives reasonably repeatable results. The basis of the test is application of insecticide solutions to test flies with a 0-10 ul adjustable Hamilton pipette (model 84250) equipped with a 2.5 cm blunt-ended needle. In the test procedure each fly is treated individually with a 0.25 ul sample of insecticide solution. Each dose is picked up from a holding vial. As the plunger of the syringe is depressed, a tiny drop forms at the tip of the needle. Touching the drop to the fly causes the solution to spread out over the body of the fly, wetting it thoroughly. Test applications are made under 7x magnification. To avoid possible "wicking" of insecticide solutions into a porous substrate, applications are made on a glass microscope slide. Slides are changed after each group of flies is treated to avoid a possible build-up of insecticide residue. Test results seem to vary with the size and nutritional condition of test flies. Therefore, flies are raised and aged under optimal conditions: they are grown at low density and aged for two or three days on heavily yeasted vials before testing. Female flies are tested since they give less variable results than males. The insecticide used in developing this test was DDT although the method should work well with other non-volatile insecticides. Serial dilutions are made from a 0.1 gm/ml stock solution of DDT in acetone to obtain a logarithmic series of doses in which each dose is 1.14 times as concentrated as the next lower dose. In one series of tests involving the dominant resistance factors on one chromosome of a highly resistant population, 0.59 x 10[-4] to 3.26 x 10[-4] gm/ml solutions covered the entire range of resistance tests. Solutions are kept in ground glass-stoppered volumetric flasks. A sample of each DDT solution is contained in a small 0.5 ml polyethylene centrifuge tube during the test. Batches of 10 females are separated out, aged together, treated at the same time and stored in a common container prior to scoring mortality. This group of flies is stored on a small (5 cm x 1 cm) plastic petri dish (Gellman model 7242). To avoid desiccation stress a 2 cm square of paper towel wetted with 30 ul of tap water is included in each plate. There is little or no mortality in flies treated with acetone and placed in such containers for up to 24 hours. Fly mortality is scored at 16 to 21 hours and the proportion dead on each plate is calculated. Test results vary somewhat from day to day, probably changing as the growth, aging and test conditions vary slightly. To minimize the effects of such fluctuations, strains to be compared are grown side by side in several vials. Several replicate tests are run on small batches of flies in random order. Two different experimental designs are used. In the first, a series of flies are tested at several DDT doses to get a dose response curve. A comparison of two strains by this method is best achieved by testing both strains on the same day from flies grown and aged together. This method has the advantage of being the traditional method of resistance analysis and of having well- established statistical methods for its analysis (Finney, 1971, "Probit Analysis"). However, it is too time consuming when several strains are to be compared. The second method, based on a randomized block design, is more efficient for comparing larger numbers of strains. In this method all strains to be compared are tested at a common dose. Best discrimination is achieved at a dose causing approximately 50% mortality. One useful system is to collect flies, do a "pretest" at various doses after two days aging and do the actual test on the third day using a dose determined by the pretest results. In this method one replicate of each strain is tested in random order in each block. The significance of the differences between the strains can be tested with a two-way analysis of variance. A sufficient number of blocks is tested to achieve the desired power of discrimination. The randomized block design is useful since many test series show significant block effects which can be removed by the blocking procedure. In the statistical analysis it is appropriate to transform such proportion data with an angular transformation (Sokal and Rohlf, 1981, "Biometrics"). The method can be further improved by substituting 1/4N for mortalities of zero and 1-1/4N on plates with 100% mortality, where N is the number of flies on the plate. This test method and experimental design have been in use for over a year with good results. The dose needed to kill the same strain of flies varies, apparently with the flies' condition. However, comparison of two strains at different times gives consistent results as long as the flies have been grown and aged together. Using these methods it has been possible to detect apparent single gene segregations in two resistant populations tested. In these tests, doses that give 50% resistant strain mortality give near 100% control mortality. Four to eight replicate blocks give clear cut discrimination between resistant and sensitive strains. It seems likely that it would be possible to detect considerably smaller differences in resistance. *** EQUIPMENT POPULATION BOTTLE UPDATE. Dave Dapkus, Dept. of Biology, Winona State University, Winona, MN 55987, USA. 507-457-5274. Recent attempts to construct population bottles as described by Dapkus (1978, DIS 53:209) failed when a supplier for the plastic milk bottles could not be found. 250 ml wide mouth nalgene bottles (Scientific Prod.) can be substituted if the connector is modified. Grind the bottle caps down to 4.5 cm with a lath or by hand and bore a 3 cm hole through each cap with a hole saw. Glue caps to each end of a hose connector designed to join 2 inch I.D. polyethylene hose (available at hardware stores) with feathering disc contact cement. Mount in the same relative position by screwing each cap onto a common bottle before gluing. Also drill a 2 cm hole through the top of the hose connector to supply air, control excess humidity and allow sampling of the population; plug with a 3.8 cm foam plug (Carolina Biological Supply). These units are inexpensive to set up and are easier to store and maintain than other population cages. About 60 ml of standard cornmeal/agar medium is placed on a slant in each bottle. Bottles are changed, alternately, every two to three weeks. Each unit maintains a population of a few hundred flies with minimal effort. The units are ideal for teaching. In a short period of time students can study several generations of natural selection. A good method is to establish a mutant population and introduce a few wild type flies. Each lab group can observe the course of selection by repeatedly sampling in their "own" population unit and watching allele frequencies change. ***