DIN Vol. 14

Kathy Matthews matthewk at fly.bio.indiana.edu
Mon Apr 4 11:45:11 EST 1994


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 at 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 at INDIANA.EDU
CTHUMMEL at HMBGMAIL.MED.UTAH.EDU          MATTHEWK at INDIANA.BITNET
***

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***
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

***
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.  
***

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 at 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.

***
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 at AOAA1.SSW.DHHS.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
***

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.
***

REQUESTS FOR MATERIALS

Dr. Robert E. Nelson, UCLA, Molecular Biology Institute, Room
459, Los Angeles, CA 90024, Tel: 310-825-5267,  e-mail:
nelson at 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
***

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 at 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 at 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 at 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 at castor.rm.fccc.edu or
davis at 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 at 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 at 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 at 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 at wombat.phri.nyu.edu

Vector/Insertion/Complexity/mRNA source
lambda gt22A/SalI-NotI/5x10[5]/Canton S ovaries, stages 1-14

This is a cDNA expression library in which the inserts are
directionally cloned.  A SalI site is present at the 5' end and a
NotI site is at the 3' end.

--Kai Zinn, Division of Biology, 216-76, Caltech, Pasadena, CA
91125, USA  Phone: 818-356-8352  FAX: 818-449-0679  Email:
kai at 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 at 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 at 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 at 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 at 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.
***

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 at 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.
***



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