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reins w~s di~ed a~'~ust 3 liters o~ 0.~ n~.l ~TA ~pH S.0), 0.1 Nu~ p~otm wac ka~onated ~ Trito~accEc ~ca-;olya~ g~s, ~d pzot~s ~ gel ~ w~¢ u~ed for i~un~on o~ BALB-c ~ as d~cfiEc~ ~y ~o ct (19~8). F~ions o~ spl~n ~]ls ~d my¢lo~ cogs, production hybfidoma c~ ~, ~d scre~g for positive hybfido~ w~c ~nducted as d~hed by I~ ~d ~# [1986], ~g ~c proc~cs o~ G~6 md ~lst~ {1982}. Epitope mapping and Western blot -na/ys~s Peptide mapp~g e~enta were candu~ted by ~ba~g 120 ~l of EDTA nu~ ~acr in ~e presence of ~creasing conc~a~o~ o~ ~s~ [Boeh~ger ~e~ Bioche~- ~J, d~ 10~ 25, or 50 ~ ~ 125 ~IT~s-HC1 [pH 6.8}, 1% SDS, and 7.5% #ycerol [~ter ~e procaine of Clevdand et ~. 1977} at 37"C for 45 m~. The ~estions were stopped by ~e ad~fion o~ SDS to a final cenc~afion of ~% ~d of ~-mereaptoe~ol to 166 ~, ~e ~pl~ were bored, ~d id~fi~ a~quots w~e loaded onto 1~% SD~polyac~l~ide gds [30% : 0.4%}, as d~ibed by Laemmli 11970}. For Western blot ~yses, proteins were transte=ed from gds to ~troeeHu- lose filters ~d probed as d~eribed by 1~ and Elg~ [1986J. Cla~fied as~tes fl~d diluted I : 20 ~ bloe~ng solution was used as ~e pm~ amibody, ~d l~I-labeled ~ti-mouse see- onda~ ~tibody [F[ab']2] ~ent {~er~ Co~.l was used at a $1ufion o~ 1 : 500 ~ bloe~ b~er. The filters were ~- posed ov~i#t to pr~l~hed Kodak X-Omat film at -80"C ~ ~ ~tensi~g screen. Screening eDNA libraries The Xgtll eDNA expression library, subeloned from the origi- nal kgtl0 library at the EcoR[ sites [M. Philip and D. Bmtlag, pets. eomm.I, was screened with mAb Y1DP., using essentially the procedure of Huynh etal. 11985}, as described by lames and Elgin [19861. However, repeated screening attempts suggested an instability of the Pep clone in this vector, so the primary semen was pex4ormed at a dilution of 9 x 10s plaques on ten 150-ram Luria broth {LBJ/ampieillin plates, using Escher~chia co//strain Y1088. Twenty-five positive plaques from the pri- mary screen were resereened by Western blot analysis of the proteins produced in a high-titer Z-ml minilysate of E. colJ strain Y1088 grown at 37°C in the presence of LPTG for 6 hr {Silhavy et al. 1984}~ three isolates produced positive signals. One phage with a 1.8-kb insert was chosen for fusther analysis. A f~-galaetosidase-PEP fusion protein was expressed in E. co~~ strain ¥1089 lysogenized with the phage insert, tollowing the proeedmes of Huynh etal. [1985]. Following temperature shock and induerion with IPTG, the bacterial cells {from 100-ml eul- turesJ were collected by eentrifugation, resuspended in 10 mr.t Tris-HC1 {pHLS), and frozen in liquid ni~ogem Foz protein analysis, the samples were fraetionated in a 7% SDS-polyacryl- amide gel for Western blot analysis. The 1.8-kb Pep insert was gel-purified by GeneClean IBIO 101l, lalx:led with [ot-s2p]dATP [Kmcrshaxa Corp.I by random priming IFeinbcrg and Vogelstein 1983}, and used to screen a second eDNA library f~om 8- to 12-ht embryos {Bro,~m and Kaf- atos 1958}. One-tenth the complexity of the library [and th~c- f~re 3 × 10" colonies} w~s screened, and seven po,itive clones were isolate& The inserts in the pNB~ vector ranged in size from 1.F to 2.7 kb~ th~ clone c~nt~inin~ the 2.7-kb inse.~, des- ignated p3221, w~s chosen f~r fi~rther analy~s. Salivary glands were disszeted/ram third instar larvae in 45% acetic a~id, and polytene e]zromos~mes were squashed onto subbed microscope slides as described b7 Fardue [1986}. Tie 2.5-kb H_fndl~ fragment item p3221 was isolated b~ GeneClean {BIO 101}, labeled with biotin-UTP (Bethesda Research Labora- toriesl by random priming IFeinherg and Vogels*.ein 19831, and hybridized as described by Paxdue 11986}. The signal was visu- alized using the DNA Deteerion System from Bethesda Re- search Laboratories. Northern blot analyMs The developmental Northern Falter reported by Haynes et aL {1990} was reprobed with the 2.5-kb Hind~I fragment from the Pep eDNA. The fragment was purified from agarose gels by CeneClean (BIO 101} and labeled with [a-zZPIdATP {Amcrsham Corp.} by random priming {Feinbcrg and Vogelstein 1983}. A total of 1 x 109 epm was included in the hybridization~ performed in the solutions recommended by Maniatis etal. {19821. The hy- bridization reaerion was allowed to incubate fur 24 hr at 65"C. The filter was washed twice in Zx SSC, 0.1% SDS~ at room temperature, and twice in 0.2xSSC~ 0.1% SDS, at 60"C. The filter was Mr-dried for 15 min, covered with plastic wrap, and exposed to Kodak X-Omat AR film at - 80°C with an intensi- fying screen. Sequence analysis The insert in p3221 was subeloned in two pieces into the Blue- Script phagemid tot sequence determination. One 2.5-kb Hin- dilI fragment covers the 5' end of the gone, extending into the pNB40 vector, and one 300-bp ~eoRI fragment overlaps ~50 bp of the HindIII fragment and extends to the 2~coRI site 20 bp into the vector. These insert fragments were gel-purified by Gone- Clean {BIO 101) and cloned into the appropriate sites of Blue- Script Plus phagemid IStratagene, La lolla, CA) in E. co//BB4 strain. Each orientation of each insert was isolated. A series of nested deletions in each strand was created using exonuclease nt and SI nuelesse {both from Boehringer Man- nheim Biochemicals} for both the p3~9.1 and kgtll plasmids. Prior to ligadon with T4 DNA ligase {Boehringer Marmheim}, the ends o~ the deletions were filled in using Klenow fragment {Bethesda Research Laboratories} and nucleoside triphosphates. VCS-M13 helper phage for making single-stranded templates, dideoxynucleoddes, primers, and Sequenase enzyme for se- quencing reaerions were all obtained from Stratagene. dATP labeled with ~sS was purchased from Amersham. Several prim- ors were synthesized by the University of Virginia Protein and Nucleic Acid Research Facility. The contiguous sequences were assembled, and consensus sequences weffe computed using the ODB UTIL program {Staden 1980}. The consensus sequence was translated into protein se- quence using the DM program IMount and Conrad 1986}. The PEP protein sequence was compared to Gcnbank using the FASTP program (Pealson and Lippman 1988}. The sequences obtained ~or the Xgtll and the p~l inserts axe identical, ex- cept for the presence of 58 bp on the amino-terminal side oi the Xgtll insert, which is missing from the p32Z1 insert. lmmnnoflnorescenee assays Immun~flu~rescenee anal}~e~ of polytene daznmosomes were c~nducted b7 the meth~:ls of Silver zml Elgin [1977}, as mcdi- GE2~ES ~- DEVF.LOP.M_.E~f 197 40000050

fled by lames and El~in [19S5). PolTte~e chromosomes were squ~h=d in a 45% ~c~d= ~d-3.7% Io~al~)'d= ~t~ ~- lutio~ ~fi~y YID~ wa~ used ~ a n~t hyhddoma ~pe~- t~t~ ~-p~fied goat ~d~odies s~fic ~or the ~rbo~'- te~d dom~ d ~e l~gest sub~t o~ ~A pd~erase H (Fisher et ~. 1989) were ~uted 1 : I~ m bloe~g buffer. ~fi- mo~e F~C-~niugated an6body ~d ~d-goat I~C- eoniugated mfibody were ho~ obt~ed horn ICN ~m~obi- do~eals and ~luted l : 100O ~d I : 3~0, respecfivdy. Yox r~- t~ ~e coverslips were ~pped off the e~omosomes v~th a razor bhde, ~d the s~d~ were washed ~ree ~m~ ~ ~S at room temperature. The c~omosomes w~e photographed ~ou~ a ~i= O~- omat fluoresc~ce ~eroscope using Kodak Tri-X film. ~ctu~ were printed ns~g ~-eon~ast filters on Kod~ Polyeon~ast pap~. Devdopmen~ stages were dete~ed by comp~son o~ ~e paling pa~ems to ~ose in Table 1 of ~hb~ ~d Ber- end~ 11978}. Acknowledgments We are truly indebted to T.C. James, Paul Adler, and Bob Kadner (or guidance and assistance, to ~ae Moon Lee and Amo Green- lea~ [or the provision o( anti-pol II antibodies, to Susan Haynes, Felix Karim, and Carl Thurnmel [or assistance with the North- em blot analyses, to Valerie Dietrich and Jack Diani for tech- nical assistance, and to Keith Yamamoto for suggestions and advice. This work was supported by National Institutes o( Health INIH] grant GM39271 to A.L.B., NIH grant GM31532 to S.C.R.E., and NIH grant 5-S07-RR05431-28, grant IN 149P from the American Cancer Society, and March of Dimes grant 1-1200 to S.A.A.A.L.B. is the recipient o( an American Cancer Society Faculty Keseareh Award. Sequence data described in this paper have been submitted to EMBL/GenBank Data Libraries under accession number X56689. The publication costs of this article were defrayed in part by payment of page charges. 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