The following information accompanied stocks donated to the Bloomington Stock Center by Kent Nybakken, Boston Biomedical Research Institute.
P{UAS-cos.N} was generated by PCR amplifying the cos (FBgn0000352) coding sequence plus the terminator codon from a full-length cos cDNA using oligonucleotide primers to introduce in-frame 5' Bam HI and 3' Not I sites. These sites were cut and the fragment was cloned into the Bam HI and Not I sites of pP{UAST}.
P{UAS-cos.S572A} is identical to P{UAS-cos.N} except for the introduction of a mutation by site-directed mutagenesis to change serine 572, which is phosphorylated by fused (fu) protein, to alanine.
P{UAS-cos.1-500.HA}, P{UAS-cos.500-1000.HA} and P{UAS-cos.500-1201.HA} were generated by PCR amplifying the coding sequences for amino acids 1-500, 500-1000 and 500-1201, respectively, from a full-length cos cDNA using oligonucleotide primers to introduce in-frame 5' Bam HI and 3' Not I sites. These sites were cut and the fragments were subcloned into the Bam HI and Not I sites of a vector made by Kent Nybakken containing an in-frame, C-terminal, triple hemagglutinin (HA) tag followed by a stop codon. The triple-HA tagged cos fragments were then excised with Bgl II and Xba I, and directionally cloned into the Bgl II and Xba I sites of pP{UAST}. The final arrangement of features in all three constructs is Bgl II--Eco RV--selected cos codons--Not I--triple HA--stop codon--Sma I--Xba I.
P{UAS-cos.927-935A.HA} was produced using oligo-based mutagenesis to convert T927, S931, T934 and S935 to alanines in the cos cDNA. The remaining steps in its construction were identical to those immediately above.
P{UAS-cos.500-1201.HA}21 is an X chromosome insertion.
P{UAS-cos.N}18, P{UAS-cos.1-500.HA}15, P{UAS-cos.1-500.HA}20, P{UAS-cos.500-1000.HA}6, P{UAS-cos.S572A}44 and P{UAS-cos.927-935A.HA}1 are second chromosome insertions.
P{UAS-cos.500-1201.HA}36 and P{UAS-cos.S572A}37 and are third chromosome insertions.
--
Kevin Cook, Ph.D.
Bloomington Drosophila Stock Center
http://flystocks.bio.indiana.edu
