Supplementary Figure Legends
Figure S1. Construction of the H(hsp/TP5) and H(hsp/TP6) transformation vectors. A
PCR-amplified cassette containing the P element was inserted into a BamHI site (B)
downstream of the Drosophila hsp70 promoter in a transitional plasmid. These PCRamplified cassettes spanned base pairs 38 to 2867 in the canonical P element (O’Hare and
Rubin 1983). The hsp70/P fusions, flanked by NotI (N) sites, were then inserted into a
NotI site in the 5’ portion of a hobo element in the transformation vector pHawN to
produce the H(hsp/TP5) and H(hsp/TP6) constructs. Rectangles in the P elements are
exons or portions of exons, and the darkened regions within them either are untranslated
or are translated out of frame. Bent arrows indicate the direction of transcription from the
P, hsp70 and hobo promoters. This schematic is not drawn to scale.
Figure S2. RT-PCR analysis of expression of fully spliced germ-line mRNA from native
(N) and transgenic (A-C) TP6 elements. The CP transgene, which expresses Ptransposase mRNA in the germ line, was used as a control. Samples with (+) and without
(-) reverse transcription (RT) were analyzed by PCR amplification with primers P∆0/1-d
and P∆2/3-u. This amplification generates a 1442 bp product from fully spliced germ-line
CP mRNA and a 512 bp product from fully spliced germ-line TP6 mRNA. The 702 bp
product detected in the transgene samples probably results from P∆2/3-u binding
downstream of the 2/3 intron in TP6 cDNA and priming DNA synthesis through it.
1
Figure S3. RT-PCR analysis of expression of native (N) and transgenic (A-C) TP6
elements to detect mRNA that has retained the 2/3 intron. The CP transgene, which
expresses P-transposase mRNA in the germ line, was used as a control. Samples with (+)
and without (-) reverse transcription (RT) were analyzed by PCR amplification with
primers P∆0/1-d and P2575-u. This amplification generates a 2039 bp product from CP
mRNA that has lost the 0/1 and 1/2 introns, but that has retained the 2/3 intron., and it
generates an 1109 bp product from TP6 mRNA that has lost the 0/1 intron but that has
retained the 2/3 intron. Products that would indicate loss of the 2/3 intron are not
distinctly visible in this gel; thus, fully spliced cDNA is much less abundant than cDNA
that retains the 2/3 intron.
Figure S4. RT-PCR analysis to compare P-element mRNA levels in flies carrying the CP
transgene and either native (N) or transgenic (A-D) TP5 elements. Samples with (+) and
without (-) reverse transcription (RT) were analyzed by PCR amplification with
appropriate primers. Each experimental set consisted of eight independently isolated
RNA samples, four from flies carrying the native TP5 element and four from flies
carrying a TP5 transgene. These samples were randomly paired to compare P mRNA
levels between native and transgenic genotypes. (A) Amplification using primers Aub-d
and Aub-u to detect aubergine mRNA. (B) Amplification using primers TP5-d and
P∆2/3-u to detect germ-line mRNA from the TP5 elements. The expected product is 471
bp long; the 661 bp product probably arises from P∆2/3-u binding downstream of the 2/3
intron in unspliced TP5 cDNA and priming DNA synthesis through it. (C) Amplification
using primers P∆0/1-d and P∆2/3-u to detect germ-line P-transposase mRNA from the
2
CP transgene. The expected product is 1495 bp long. The 1685 bp product is probably
due to P∆2/3-u binding downstream of the 2/3 intron in partially spliced CP cDNA and
priming DNA synthesis through it. (D) Amplification using primers P∆0/1-d and P2075-u
to detect CP mRNA that retains the 2/3 intron, i.e., that is not specific to the germ line.
The expected product is 1592 bp long.
3
Table S1. Primers used in PCR analysis of RT products.
Name
Sequence
Aub-d
5’-(1937)TACGGGAATGACGGACGCTATG(1958)-3’
Aub-u
5’-(2967)GTGTTCGTTTACGGTGGAGAAGTAG(2943)-3’
P318-d
5’-(318)GCACCTGCAAAAGGTCAG(335)-3’
P∆0/1-d
5’-(426)GGGAGTACACAAACAGA(442)/(501)GTCCT(505)-3’
TP5-d
5’-(424)GTGGGAGTACACAA(437)/TG/(1524)TCATT(1528)-3’
P1828-d
5’-(1828)CAAGCCGTCTCAACCAAG(1845)-3’
P∆2/3-u
5’-(2168)CCCGAATTTCTTAACATTTCTGTATTCCTGG(2138)/CTATTAT(1941)-3’
P2075-u
5’-(2075)CACAATAGACAGCACATAACTACC(2052)-3’
P2575-u
5’-(2575)CAACATCGACGTTTCGCGCTG(2555)’-3’
Nucleotide positions within the aub gene and P element are given in parentheses.
Intron and deletion breakpoints are indicated by a slash and nucleotides inserted at
deletion breakpoints are given between slashes. Sequences that prime DNA synthesis
downstream in the gene or element are denoted with the letter d; sequences that prime
DNA synthesis upstream are denoted with the letter u. The concentration of all primer
stock solutions was 20 ng/l except for P∆2/3-u, which was 40 ng/l; 1.9 l each primer
was added to each PCR. The temperature profile for all the PCRs involving the P∆2/3-u
primer was 3 min at 92o C, 2 min at 63o C, 3 min at 72o C for the first cycle and 1 min at
4
92oC, 2 min at 63o C, 3 min at 72o C for the subsequent 22 or 29 cycles. For all other
PCRs, the annealing temperature was lowered from 63o C to 60o C.
5
Table S2. Gonadal dysgenesis in the offspring of reciprocal F1 hybrids between TP or
H(hsp/P) stocks and the M5B#1 strain (experiment 1).
Cross I
TP or H(hsp/P)
Stocka present in F2
Cross II
 F1
Stock x M5B#1
No. of F2
%GD + SEb
Stock
x M5B#1  F1
No. of F2
%GD + SEb
yw
-
500
100.0 + 0.0
467
99.8 + 0.2
TP5-N
+
265
34.7 + 4.1
203c
82.3 + 3.9
-
241
46.7 + 5.5
186c
83.9 + 3.8
+
424
1.9 + 0.8
317
65.1 + 4.9
-
418
1.9 + 0.7
315
67.0 + 5.6
+
339
98.7 + 0.9
109
100.0 + 0.0
-
345
100.0 + 0.0
89d
100.0 + 0.0
+
410
99.0 + 0.5
179d
99.6 + 0.4
-
410
98.6 + 0.7
202
99.4 + 0.4
+
380
99.0 + 0.6
200
100.0 + 0.0
-
364
99.8 + 0.2
190
99.2 + 0.8
+
360
99.7 + 0.3
130
98.8 + 0.9
-
326
100.0 + 0.0
120
99.7 + 0.3
+
328
99.5 + 0.4
360
100.0 + 0.0
-
350
99.8 + 0.2
335
100.0 + 0.0
+
351
99.2 + 0.4
236
100.0 + 0.0
TP6-N
SP-B
KP-3
TP5-C
TP6-A
TP6-B
TP6-C
6
-
357
100.0 + 0.0
270
100.0 + 0.0
Except where indicated, the data in each group came from 25 replicate vials.
a
y w is an M strain. TP5-N and TP6-N are strains with the native telomeric P elements on
the X chromosome; other entries are strains with H(hsp/P) transgenes that contain the
indicated P element.
b
Unweighted mean percentage of GD + SE
c
These flies came from 16 replicate vials.
d
These flies came from 15 replicate vials.
7
Table S3. Gonadal dysgenesis in the offspring of reciprocal F1 hybrids between TP or
H(hsp/P) stocks and the M5B#1 strain (experiment 2).
Cross I
TP or H(hsp/P)
Stocka present in F2
Cross II
 F1
Stock x M5B#1
No. of F2
%GD + SEb
Stock
x M5B#1  F1
No. of F2
%GD + SEb
yw
-
457
100.0 + 0.0
362
99.7 + 0.3
TP5-N
+
327
46.5 + 5.2
321
83.1 + 4.2
-
283
58.9 + 5.1
279
87.4 + 3.5
+
310
3.8 + 1.5
252
64.6 + 5.3
-
267
1.2 + 0.8
221
68.1 + 6.0
+
299
99.5 + 0.5
121
100.0 + 0.0
-
304
100.0 + 0.0
122
100.0 + 0.0
+
379
99.4 + 0.4
262
100.0 + 0.0
-
257
100.0 + 0.0
159
99.5 + 0.5
+
173
99.5 + 0.5
211
99.6 + 0.4
-
160
100.0 + 0.0
179
100.0 + 0.0
+
204c
98.4 + 1.3
214
95.9 + 1.9
-
210c
99.7 + 0.3
184
93.3 + 2.1
+
276
98.5 + 1.1
48d
100.0 + 0.0
-
279
99.7 + 0.3
59d
96.9 + 3.1
+
298
99.2 + 0.5
118
100.0 + 0.0
TP6-N
SP-A
KP-7
KP-14
TP5-A
TP5-B
TP5-D
8
-
257
99.6 + 0.4
108
99.5 + 0.5
Except where indicated, the data in each group came from 24-25 replicate vials.
a
y w is an M strain. TP5-N and TP6-N are strains with the native telomeric P elements on
the X chromosome; other entries are strains with H(hsp/P) transgenes that contain the
indicated P element.
b
Unweighted mean percentage of GD + SE
c
These flies came from 23 replicate vials.
d
These flies came from 13 replicate vials.
9
Table S4. Gonadal dysgenesis in the offspring of reciprocal F1 hybrids between strains
with the native telomeric P elements (TP5-N and TP6-N) and the M strain Samarkand
(experiment 2).
Cross I
TP present
Stock
TP5-N
TP6-N
Stock x M
Cross II
 F1
Stock
in F2
No. of F2
%GD + SEa
No. of F2
%GD + SEa
+
414
97.8 + 0.9
247
94.6 + 2.9
-
434
98.6 + 0.5
219
100.0 + 0.0
+
398
89.2 + 2.5
294
99.1 + 0.5
-
391
90.3 + 2.2
291
98.5 + 0.8
The data in each group came from 24-25 replicate vials.
a
x M  F1
Unweighted mean percentage of GD + SE
10
Table S5. Gonadal dysgenesis in the offspring of the stocks used to analyze the effects of
the native telomeric P elements (TP5-N and TP6-N) on germ-line transposase mRNA
levels.
Stock
No. of vials
No. of
%GD + SEa
M cytotype
25
471
99.8 + 0.4
TP5-N
25
472
6.2 + 1.9
TP6-N
24
459
64.3 + 6.9
a
Unweighted mean percentage of GD + SE
11