Drosophila embryo acid fusion library (RFLY1)    November 1991 

This library is described in Finley et al., 1996, PNAS 93, 3011-3015.

RNA

Wild type Drosophila melanogaster total embryonic RNA was obtained from Susan Abmayr (Maniatis lab) ca. 9/88. I pooled equal quantities of total RNA from 0-4 hr, 4-8 hr, and 8-12 hr embryos and selected poly(A+) RNA on an oligo(dT) cellulose column.

cDNA synthesis

         I made unidirectional cDNA with an EcoRI sticky end at the 5'-end and an XhoI sticky end at the 3'-end using a method similar to the variation of the Gubler and Hoffman technique (Gene 25: 263-269, 1983) described by Huse and Hansen (Stratagene Strategies 1: 1-3, 1988). I synthesized the first strand with Superscript (BRL) using a primer (JG33, see Jenö Gyuris handout) containing an XhoI site 5' to an 18nt poly(dT) tract. The first strand synthesis contained 5me-dCTP instead of dCTP to protect internal XhoI sites from subsequent cleavage with XhoI. I synthesized the second strand with E. coli DNA pol I in the presence of RNaseH, made the ends blunt by treating with Klenow, Mung Bean nuclease, and then Klenow again (unpublished protocol developed by J. Gyuris), and ligated EcoRI adaptors onto them. The sequence of the adaptors is shown below.

      5'AATTCGGCACGAGGCG 3'     (JG31)

           3'GCCGTGCTCCGC 5'     (JG32)

I cut the cDNA with XhoI and size fractionated it using a 1 ml CL-4B column. The three largest fractions, representing cDNA from 0.5 to larger than 4 kb, were pooled and ligated into the unique EcoR1/XhoI sites of plasmid pJG4-5 (ApR, TRP1+, yeast 2um replicator) downstream of the GAL1 promoter, an ATG, and sequences encoding the SV40 nuclear localization signal, a hemagglutinin epitope tag, and the B42 acid patch transcription activating domain (see pJG4-5 handout).

         I transformed E.coli (Stratagene SURE cells: mcrA, L(mcrCB-hsdSMR-mrr)171, endA1, supE44, thi-1, gyrA96, relA1, lac, recB, recJ, sbcC, umuC::Tn5(Kanr), uvrC, [F', proAB, lacI^qZ LM15, Tn10(Tetr)] ) with the ligation mix and plated onto 20 LB amp plates (24cm X 24cm) and grew at 37oC for 24 hours.

         I collected 4.2 X 106 independent transformants by scraping the plates (first cooled to 4oC), and prepared plasmid DNA after overnight growth in 8 liters of LB amp.

         Over 90% of the plasmids of this library (RFLY1) contain cDNA inserts whose sizes range from 0.5 to 2.5 kb (average size about 1 kb). To show that fusion proteins are made I transformed yeast strain EGY48 (see Erica Golemis handout) with the library DNA and did Westerns on lysates from individual transformants using a monoclonal anti-hemagglutinin antibody. I found that at least 25% (9 of 33) of galactose-grown transformants made fusion proteins that were larger than the acid patch-epitope tag moiety alone.

Amplification of the above library. February 1992

         The library was amplified by electroporation of SURE cells with RFLY1 DNA. Transformants were plated on 20 24cm X 24cm LB amp plates. 2.0 X 107 independent transformants were collected as before and plasmid DNA was purified with two consecutive CsCl gradients.

*** NOTE

The primary library may be contaminated with a phage. Approximately 1 in 10⁶ E.coli transformants lyse. This is not a problem provided amplifications are not done in liquid, which might lead to premature lysis of most of the culture. Also for this reason, amplified libraries should not be re-amplified.

Russ Finley                        Brent lab - November 1991

Present Address:

Russell L. Finley Jr.

Center for Molecular Medicine & Genetics                

Wayne State University School of Medicine       

540 East Canfield Avenue                                          

Detroit, Michigan 48202

 http://proteome.wayne.edu/