Subject: PBac Half-P Constructs
Personal communication from: Brian Ring and Dan Garza, Florida State
University, Novartis
To: Bloomington Drosophila Stock Center
Subject: PBac Constructs from the Project 'Genome Wide Dispersal of
Half-P Elements in Drosophila Using PiggyBac'
Dated: 23 May 2003
Background: This is the information needed to curate the two piggyBac
constructs that are present in the set of PBac insertion lines donated to
the Bloomington Stock Center by Brian Ring and Dan Garza. The primary
reference is Brian's dissertation, FBrf0152434 (Chapter 4):
Ring, Brian C., 'Functional Analysis of the Drosophila Genome Using
Transposable Elements'. Florida State University. 2001 pgs. 130-199.
Data on the insertion lines themselves will be submitted separately as a
computer file.
Information communicated:
1) PBac{5HPw+}
This is a 5' half-P construct marked with w+mC. The minimal 5' P-element
cis acting sequences and 8 bp target site duplication derived from P{lacW}
were cloned into the BglII site of Al Handler's pB{Dmw} construct (Handler
and Harrell, 1999, Insect Molec. Biol.  8:449--457 ; FBrf0123037) and
transformed into flies. Handler created pB{Dmw} by cloning the w+mC
allele (4.2-kb EcoRI fragment) into the HpaI site of the original piggyBac
plasmid p3E1.2 (Cary et al., 1989, Transposon mutagenesis of baculoviruses:
analysis of Trichoplusiani transposon IFP2 insertions within the FP-Locus
of nuclear polyhedrosis viruses. Virology 161: 8--17). The white mini-gene
interrupts the piggyBac transposase open reading frame, but otherwise
leaves the piggyBac element intact, with the respective promoters in
opposite orientation (per Handler and Harrell, 1999).
Insertions of this construct used for the screens:
PBac{5HPw+}X7 \- This homozygous viable and fertile X chromosome insertion
was the progenitor of the A series of insertions.
PBac{5HPw+}X10 \- This homozygous viable and fertile X chromosome
insertion was the progenitor of the B series of insertions.
2) PBac{3HPy+}
This is a 3' half-P construct marked with y+mDint. The minimal 3'
P-element cis acting sequences and 8 bp target site duplication derived
from P{lacW} were cloned into the 3' end of the yellow mini-gene (Geyer and
Corces, 1987, Genes Dev. 1: 996--1004; FBrf0046293) in plasmid yellow-BSX,
a generous gift from Pam Geyer, to form the intermediate plasmid p3HPy+.
This DNA was then shuttled into a modified site in p3E1.2 (Cary et al.,
1989, see above) to form pPBac{3HPy+}, which was transformed into flies.
The resulting yellow mini-gene and 3' P-element interrupt the piggyBac
gene and reside in opposite orientation with respect to promoters.
Because intronic sequences necessary for y+ expression in bristles (all
types), tarsal claw and arista are not present in y+mDint, y- flies
transformed with PBac{3HPy+} lack y+ pigmentation in these structures.
Insertions of this construct used for the screens:
PBac{3HPy+}X0 and PBac{3HPy+}X3 \- These homozygous viable and fertile X
chromosome insertion were the progenitors of the C series of insertions.