P Transposons Controlled by the Heat Shock Promoter
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MOLECULAR AND CELLULAR BIOLOGY, May 1986, P. 1640-1649 Vol. 6, No. 5
0270-7306/86/051640-10$02.00/0
Copyright © 1986, American Society for Microbiology
P Transposons Controlled by the Heat Shock Promoter
HERMANN STELLERt AND VINCENZO PIRROTTAt*
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany
Received 25 November 1985/Accepted 11 February 1986
We have transformed Drosophila melanogaster with modified P-element transposons, which express the
transposase function from the heat-inducible hsp70 heat shock promoter. The Icarus transposon, which
contains a direct hsp7O-P fusion gene, behaved like a very active autonomous P element even before heat shock
induction. Although heat shock led to abundant somatic transcription, transposition of the Icarus element was
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confined to germ line cells. To reduce the constitutive transposase activity observed for the Icarus element, we
attenuated the translational efficiency of the transposase RNA by inserting the transposon 5 neomycin
resistance gene between the hsp70 promoter and the P-element sequences. The resulting construct, called
Icarus-neo, conferred resistance to G418, and its transposition was significantly stimulated by heat shock. Heat
shocks applied during the embryonic or third instar larval stage had similar effects, indicating that
transposition of P elements is not restricted to a certain developmental stage. Both Icarus and Icarus-neo
destabilized snW in a P-cytotype background and thus at least partially overcome the repression of
transposition. Our results suggest that the regulation of P-element transposition occurs at both the transcrip-
tional and posttranscriptional levels.
Transposable elements are a major cause of genome elements can no longer transpose autonomously, they can be
instability, responsible for a large fraction of spontaneous mobilized when the missing transposase function is provided
mutations in Drosophila melanogaster (1, 3, 7, 20, 25, 26, 31, in trans by an intact helper element.
43). The functional analysis of metazoan transposons has A functional analysis of P elements was started by Karess
been complicated by the fact that transposition events are and Rubin (19), who established lines transformed with
usually rare and the physiological conditions they require are single genetically marked P-element derivatives and investi-
not well defined. The P elements responsible for the phe- gated the effects of frameshift mutations on transposase
nomenon of P-M hybrid dysgenesis (21) in D. melanogaster activity. Their results suggested that all four major open
are of particular interest because their transpositional activ- reading frames (ORFs) in the P-element sequence contribute
ity, which can be readily detected by genetic tests (14, 19, to encode a single transposase polypeptide. P-element tran-
36), is potentially high but under strict genetic control. scripts are very rare, 0.001% of the polyadenylated RNA.
Transposition is induced at a high frequency only if males Neither quantitative nor qualitative differences could be
carrying functional P elements (P strains) are crossed with detected in the RNA prepared from dysgenic and
females lacking P factors (M strains), but not in the recipro- nondysgenic flies (19), raising the question of how transpo-
cal or in a P x P cross (for a review, see references 6 and 13). sition and production of the negative regulator are con-
Because the repressed state (P cytotype) is determined by P trolled.
factors themselves, it has been suggested that P elements To determine the role of transcriptional regulation in the
code for a negative regulator in addition to transposase control and tissue specificity of P-element transposition, we
function (10, 29). have constructed transposons in which P-element functions
Transposition of P elements is specific for germ line cells are expressed from the heat-inducible hsp70 heat shock
since no appreciable activity can be detected in the soma (11, promoter. This allows us to control externally both the
42). It is not known whether this is the result of tissue- amount and developmental stage of P-element transcription.
specific transcription of P elements or a requirement for We introduced these transposons, named Icarus and Icarus-
germ line-specific host functions acting posttranscription- neo, into the genome of M-cytotype flies and analyzed the
ally. effect of the promoter exchange on the rate and control of
P elements have been cloned (4, 32) and sequenced (29) transposition. Our results suggest that both transcriptional
and shown to transpose from injected plasmid DNA into the and posttranscriptional controls are important for the control
chromosomes of germ line cells (19, 33, 36). A conserved of P-element transposition. We also discuss how strains
element of 2.9 kilobases (kb) which is present in P but absent transformed with Icarus elements can be used for efficient
from M strains is autonomous in transposition (36). Smaller insertional mutagenesis and immediate cloning of the tagged
elements of heterogeneous size are found in both P and some gene by plasmid rescue.
M strains and are presumably derived from autonomous
elements by internal deletions. Although these defective MATERIALS AND METHODS
*
Corresponding author. D. melanogaster strains. Strains Canton S Heidelberg,
t Present address: Department of Biochemistry, University of C(1)DX, W cv; cn bwfs (2)PC42ICyO DTS513; al dp b pr cn
California, Berkeley, CA 94720. vg c a px bw 1 I spISMI and TM3, SerSb were from our stock
t Present address: Department of Cell Biology, Baylor College of collection. The P strain C(J)DX, y f wr2 and the snw, bw, st M
Medicine, Houston, TX 77030. strain were obtained from W. Engels. The y snwIC(J)DX, yf
1640VOL. 6, 1986 HEAT SHOCK PROMOTER-CONTROLLED P TRANSPOSONS 1641
H lH R XBSH H R S BH the DNA was repaired with Klenow polymerase and par-
I.-Y
NI. I I 11
or= Icarus tially digested with HindIII. The 2.9-kb P-element fragment
S-P pUC9 ha-p P-ORF 3'P was gel purified and ligated to pUC9 which had been
digested with HindIII and HindII. The resulting clones were
- Icmrus-Nso analyzed for the exact extent of the Bal 31 digestion.
pUCPT, which had lost 27 (±2) bp of the 31-bp 3' P-element
phs-W repeat was used for all further experiments.
For the construction of the Icarus plasmid, we first made
1 kb
an inverted P element. To do so we isolated the SalI insert
from p6.1 (29), circularized it by ligation under dilute condi-
FIG. 1. Map of heat shock promoter-controlled P elements. The tions, and gel purified the circular molecules. These were
Icarus transposon has an hsp7O heat shock promoter fragment
(black arrow) fused at nt 39 (Hindlll site), upstream of the P-element digested with XhoI, which has a unique site in the middle of
ORFs (gray boxes) (29; for details of the construction, see Materials the defective P element, and subcloned into the SailI site of
and Methods). The heat shock promoter-P-ORF fusion and the pUC9. Recombinant clones with both possible orientations
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entire pUC9 plasmid part (44) are flanked by P-element ends (p6.1inv-1 and p6.1inv-2) of the insert were obtained. For
(hatched boxes) derived from p6.1 (29). Icarus-neo was derived subsequent constructions, we used only p6.1inv-1, which is
from Icarus by inserting a 1,002-bp fragment containing the Tn5 neo oriented so that the 5' P-element sequences are adjacent to
gene (stippled box) (2, 38) between the hsp7O promoter and the 5' the HindIII site and the 3' P sequences are adjacent to the
end of the P element. The neo gene fragment contains 17 ATGs, EcoRI site of the pUC9 polylinker. This construction pro-
which precede the P-ORF initiation codon and attenuate translation vided the two extremities of the P element that frame the
of the transposase protein. The phs-irf helper plasmid lacks the 3' P
element end and is therefore defective in transposition. Restriction Icarus transposon. The final Icarus element (Fig. 1) was
sites in Icarus are: H, HindIII; R, EcoRI; B, BamHI; S, Sall; and X, obtained by cloning the 2.9-kb HindIII (partial)-BamHI
XhoI. P-element fragment from pUCPT together with a 460-bp
EcoRI-HindlII fragment containing the hsp7O promoter and
transcription start, from the XbaI to the XmnI sites (37)
'rr2 strain was made by crossing males from the M-cytotype y between the EcoRI and BamHI site of p6.1inv-1 in a three-
snW, bw st strain to fresh C(J)DX, y f 7r2 females and factor ligation. As all components used in these construc-
repeatedly crossing the male y sn' progeny to fresh C(J)DX, tions have been sequenced, the entire nucleotide sequence
y f Tr2 females for three more generations. All crosses were of the Icarus element can be predicted and is available on
carried out at 25C. request. Below we list the origin of the various constituents
Nucleic acid purification and analysis. For rapid, small- of Icarus, starting with the 5' inverted repeat of the P
scale DNA isolation, 1 to 30 flies were homogenized in 100 to element: Icarus nucleotides (nt) 1 through 732 correspond to
500 ,l of 0.1 M Tris (pH 9)-0.1 M EDTA-1% sodium nt 1 through 732 from p6.1 (29); nt 733 through 3383 are from
dodecyl sulfate-0.5% diethylpyrocarbonate (DEP) in an Ep- pUC9 (44); nt 3384 through 3837 contain the hsp7O promoter
pendorf tube and incubated for 30 min at 65°C. Thereafter, (17, 37); nt 3838 through 3858 are pUC9 polylinker se-
14 Ru of 8 M potassium acetate solution was added per 100 quences; nt 3859 through 6700 correspond to nucleotides 39
,1, and the tubes were transferred to ice for 30 min. The through 2880 from pur25.1 (29); nt 6701 through 6710 come
precipitate was sedimented by centrifugation at 4°C for 10 from the pUC9 polylinker (44); nt 6711 through 7099 corre-
min, and DNA in the supernatant was precipitated by the spond to nt 729 through 893 and 2686 through 2907 of the P
addition of 0.5 volume of isopropanol at room temperature. element (29); and nt 7100 through 8622 come from the white
For plasmid rescue experiments in which higher- gene (28).
molecular-weight DNA was required, we extracted DNA For the construction of Icarus-neo, we first removed the
from the nuclei of 1 to 20 flies as described by Steller and BamHI site close to the 3' P-element sequences to leave a
Pirrotta (40). unique BamHI site between the hsp7O promoter and the
Total RNA was prepared by a slight modification of our P-ORF P-element sequences (Fig. 1) by partial digestion
earlier protocol (37). Briefly, 1 to 50 flies were homogenized with BamHI, nuclease S1 digestion, and religation of the
in 100 to 500 pl of GHC1 buffer (7.5 M guanidine hydrochlo- blunt ends. Into the resulting plasmid, IcarusABam(+), we
ride, 0.025 M sodium citrate, pH 7.0, 5 mM dithiothreitol) inserted the transposon TnS neomycin resistance gene (neo)
containing 0.5% lauryl sarcosine and 0.2 to 0.5% DEP. The (2, 18) as a Bgl-BamHI fragment (38). In the final step, we
homogenate was extracted twice with an equal volume of deleted nonessential Tn5 sequences by redigestion with
phenol-chloroform-isoamyl alcohol (25:24:1), and nucleic BamHI and SmaI, S1 digestion, and religation. The entire
acids were precipitated by the addition of 0.025 volume of 1 nucleotide sequence of Icarus-neo can be predicted and is
M acetic acid and 0.5 volume of ethanol at -20°C for 5 to 24 available on request.
h. The pellet was dissolved in half the original volume of The phs-ir helper plasmid was made by digesting plasmid
GHC1 buffer and precipitated two more times to remove IcarusABam(-), which lacks the BamHI site between the
DNA (8). Northern blot hybridizations were performed as hsp7O promoter and P-ORF, with BamHI and EcoRV and
described by Steller and Pirrotta (37). religating the plasmid after repairing the BamHI site with
Southern blot hybridizations, plasmid preparations, and Klenow polymerase.
nick translations were done as described by Maniatis et al. Germ line transformation and establishment of transformed
(27). lines. Microinjection and preparation of selective G418 food
Plasmid constructions. Plasmid p'r25.1 (29), containing an was done as described by Steller and Pirrotta (38). Canton S
active full-length P element, was digested with XbaI, which wild-type embryos were injected with Icarus plasmid DNA
has a unique site in the flanking D. melanogaster DNA close at 200 ,ug/ml without addition of helper plasmid and Icarus-
to the 3' end of the P element. The linear DNA was neo at 300 ,ug/ml with (100 pug/ml) or without phs-' helper
incubated with Bal 31 nuclease under conditions which plasmid. Injected embryos were aged to 8 to 12 h and then
bidirectionally removed ca. 300 base pairs (bp). Afterwards, given a 20- to 30-min heat shock by putting the injection1642 STELLER AND PIRROTTA MOL. CELL. BIOL.
slides into the 37°C incubator. Potential Icarus transform-
ants were preselected by the snW assay (Fig. 2) and subse-
quently analyzed by Southern blot and in situ hybridization.
Flies transformed with Icarus-neo were obtained by select-
ing the F1 progeny on 1 mg of G418 per ml as described L..
I I
previously (38). Resistant transformants were crossed to
balancer chromosomes and reselected on G418 to genetically I .,
I
map the transposon and establish a balanced line. Lines -1
were further characterized by Southern blot and in situ
hybridization.
snW assay. Single male transformants or Xr2 control males (
were crossed to several y sn", bw, st virgins at 25°C. The
resulting y SnW F1 males were crossed individually to C(J)DX
w cv virgins, and the F2 males were scored for sn+ or sne
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bristle phenotype. FIG. 3. Southern analysis of transformants. DNA from individ-
For the reciprocal cross, single female transformants with ual F2 males of sne or sn+ phenotype obtained from the snH' assay
a C(J)DX w cv X chromosome or C(J)DX y f Wr2 control was restricted with EcoRI, separated on a 1% agarose gel, and
blotted. The blot was probed with nick-translated pUC9 DNA.
virgins were crossed to several y sn"', bw, st males. Single F1 Under these conditions, a single band is expected for each inte-
males were crossed to C(1)DX w cv virgins, and F2 males grated transposon. The size of that fragment depends on the
were scored for sn+ or sne bristle phenotype. distance from the integration site to the nearest EcoRI site in the
To test the sn' destabilization within the P cytotype, flanking D. melanogaster DNA. Lines 8b (sn+) and 131 (sne)
transformed males were first mated to y sn4', bw, st virgins. contained a single and line 13 c (sne) contained two Icarus
The F1 males, which carried the y sn"' X chromosomes and transposons.
were heterozygous for the transposon, were individually
mated to C(J)DX, y f T2 females. The sn"' males were
selected and individually crossed to fresh C(J)DX, y f Tr2 resistant females was verified by crossing them to (G418-
females (sne and sn+ males in the F2 generation were derived sensitive) Canton S males and reselecting on G418-
from the initial M x Icarus cross and were discarded). Males containing food.
in the F3 generation were scored for their sn+ or sne bristle Excision and reintegration of the Icarus-neo element was
phenotype. Some of the F3 sn"' males which carried a copy of measured with line 2-2 (see Fig. 6). Heat shock conditions
the transposon were tested for subsequent singed-weak were the same as above. To select against the CyO DTS513
destabilization by crossing them again to fresh C(J)DX, y f chromosome, flies which had received heat shocks during
females and scoring F4 male progeny for their bristle pheno- their development were mated at 25°C and allowed to lay
type. eggs for 12 to 24 h. After the flies had been removed, the
Transposition and excision assays. Transposition of the culture bottles were transferred to 29°C, and the eggs were
X-linked Icarus-neo transposon in line T-11 was tested by allowed to continue developing at that temperature. The
crossing male transformants individually to C(J)DX, w cv CyO+ survivors were tested for an Icarus-neo transposon at
virgins. The offspring were given three heat shock treat- a new chromosomal position as outlined in Fig. 6.
ments (1 h at 37°C, 2 h at 25°C, 1 h at 37°C), at the embryonic Plasmid rescue of integrated transposons. After an amount
or larval stage. The F1 flies were mated in pools inter se, of DNA equivalent to 1 to 10 flies was completely digested
their offspring were selected on food containing G418, and with the appropriate restriction endonuclease, the enzyme
the number of G418-resistant females among the total prog- was inactivated by heat (15 min at 70°C) or by 0.1% DEP
eny was determined. The presence of the transposon in the followed by 15 min at 70°C. The DNA was circularized by
ligation at a concentration of ,ug/ml (the DNA equivalent
-
of one fly in 100 to 200 RI), extracted with phenol-
chloroform, ethanol precipitated, and redissolved in 10 ,ul
30-60 mn. eabryos ------------- injection with plcarus per fly DNA equivalent. The DNA was used to transform
at 200 ug/ml competent Escherichia coli DH1 (16) or MC1061 cells.
Competent E. coli MC1061 cells were prepared by an
10-12 hrs. embryos ------------- heat shock for 30 min at unpublished protocol of Jill Gough (Ludwig Institute for
37 *C Cancer Research, Melbourne, kindly communicated by Nick
Gough). An overnight culture of strain MC1061 was diluted
FO adults: males x y sno females females x y snw males 1:100 into L broth containing 5 mM glucose, 10 mM MgSO4,
and 10 mM MgCl2. The culture was grown at 37°C with
Ft larvae: heat ShoCking lst to 2nd moderate shaking to an OD650 of 1.4 to 1.8 (depending on the
mnstar larvae for 30-60 amn.
at 37 t spectrophotometer). The cells were placed in ice for 5 to 10
min and then harvested at 3,000 x g at 4°C. The pellet was
Ft adults: y snu males x XX females brothers x sisters suspended in approximately half the culture volume of
ice-cold 0.1 M MgCl2 and immediately centrifuged again.
The supernatant was carefully removed, and the cells were
F2 adults: select y sne or y sn males select y sne males gently suspended in 1/20 volume of ice-cold 100 mM CaCI2,
FIG. 2. Isolation of flies transformed with Icarus. The protocol
50 mM MgCl2, 50 mM MnCl2, and 50 mM RbCl2. After the
for the isolation of Icarus transformants is outlined. Males with the bacteria had been left on ice for between 30 min and 3 h, cold
sne or sn+ phenotype obtained in the F2 generation were transfor- glycerol was added to a final concentration of ca. 10%, and
mation candidates and were analyzed by Southern blotting (see Fig. the cells were frozen in 500-RI portions in sterile Eppendorf
3) after being mated to different balancer chromosomes. tubes. For a transformation, the competent cells wereVOL. 6, 1986 HEAT SHOCK PROMOTER-CONTROLLED P TRANSPOSONS 1643
element promoter is inactivated by insertions at this HindIII
M 1 2 3 4 5 6 7 M site; hence, in our construction transcription should be
entirely dependent on the hsp7O promoter. To obtain the
final construct, named Icarus, P-element ends were added
20 back to the hsp-P fusion gene so that the transposon would
include a bacterial plasmid replicon and the ampicillin resist-
5.0 ance gene. This design should allow the convenient recovery
of the transposon together with flanking sequences from the
-a2.0
DNA of transformed flies by the plasmid rescue method (30).
To test the Icarus element for its ability to transpose from
1.5 injected DNA to germ line chromosomes, we used the
1.0
scheme outlined in Fig. 2. As Icarus does not itself contain
a genetic marker, we used the snw test (12, 36) to detect
transformation events. In this test, males presumably carry-
ing an active P element are crossed to females carrying the
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-w 0.5
hypermutable snW allele. If the male genome can provide
transposase function, it will destabilize the snW mutation and
-0. 2 result in the appearance of sn+ or sne alleles in the germ line
of the progeny. Flies destabilizing the snw allele were ob-
tained at a frequency of at least 20%, indicating a highly
FIG. 4. Analysis of the Icarus element after plasmid rescue. The successful transformation. Due to the premeiotic nature of
Icarus transposon from the SIca line was cloned by plasmid rescue the snW destabilization, the transposon can segregate inde-
using BgIIH (for details, see Materials and Methods), and the plasmid pendently from the sn allele. To establish a line carrying
DNA of pS'1 was analyzed on a 1% agarose gel after digestion with Icarus, it was therefore necessary to verify the presence of
various rstriction enzymes. Lanes: M, lambda marker DNA, EcoRI the element by Southern blot analysis of individual sn+ and
and HindIll digest; 1, pSICa, BglII; 2, pSIca, HindIll; 3, Icarus, sne flies after they had been allowed to mate. An example of
HindIII; 4, pS1cl, EcoRI; 5, Icarus, EcoRI; 6, pSIca, AvaIl; 7, this type of analysis is shown in Fig. 3. Altogether, six
Icarus, AvaIl. White arrows point to fragments derived from independent transformation events could be detected among
flanking white DNA sequences present in Icarus, which are lost 14 flies analyzed in that way.
during transposition. Fragment sizes (in kilobases) are shown to the Icarus transposon can be isolated from single transformed
right.
flies by plasmid rescue. To analyze integrated transposons
and the DNA flanking the insertion site, Icarus elements
thawed on ice, and 150 ,ul of cells was added to 10 to 20 ,ul of were reisolated from transformed flies by the plasmid rescue
ligated D. melanogaster DNA. The mixture was incubated method (30). The entire element was rescued by digesting
on ice for 5 to 10 min, heat shocked for 45 to 60 s at 37°C, and the DNA from transformants with enzymes that do not cut
immediately diluted with 500 ,ul of L broth. After incubation within the transposon, e.g., BglII, circularizing the frag-
at 37°C for 30 min, the transformation mixture was plated on ments with ligase, and using the entire ligation mixture for
four to six ampicillin plates. With this method, there were
routinely between 5 x 107 and 108 competent colonies per ,ug
of pUC8 DNA. Using the Hanahan procedure (16), we
obtained from 1 x 108 to 3 x 108 colonies per p,g of DNA. P 1I:i 10 181 1I IP 1;,. l)it 131 P1
_._- -t t,
RESULTS .--&-
dl.w- -
qpqp iL..r
Construction of the heat shock promoter-controlled Icarus
Mr! -.- .4
-.-
transposon. Since natural P elements are very weakly tran-
scribed, we replaced the P promoter by the inducible hsp7O ..4.
heat shock promoter to see whether enhanced transcription
of the P element would lead to increased mobility of the _ _
transposon. By varying the duration, frequency, and time of 4ft
o
heat treatment, we expected to control the amount and
developmental stage of expression and thereby avoid detri- _b I
mental effects such as dysgenic sterility or high instability
that might be caused by high levels of constitutive expres-
sion. The heat shock promoter can be activated in a wide
variety of cell types, particularly in germ line cells (5, 37), a FIG. 5. Heat shock-induced transcription from Icarus. Total
prerequisite to obtain transformed lines. RNA was extracted from Icarus transformants (lines 13c, 10a, and
To construct the transposons shown in Fig. 1, we replaced 131) which had been heat shocked for 1 h at 37°C (+) or not shocked
the P-element sequences upstream of the HindIlI site at (-). An amount of RNA equivalent to two flies was loaded on a 1%
position 40 of the P-element sequence (29; for details of the agarose gel and blotted to nitrocellulose after electrophoresis. The
construction, see Materials and Methods section) by a same amount of RNA from heat-shocked Xr2 P strain flies (P) was
456-bp fragment containing the functional hsp7O promoter included as a control. The filter was probed with radioactively
and 206 nt of the untranslated leader sequence (37). Since the labeled phs-'n DNA (left side) or P-specific pUCPT DNA (right
45-bp interval from this HindIll site to the normal transcrip- side). The phs-rr probe hybridized both to the hsp-P fusion RNA,
present only in heat-shocked transformants, and to the hsp7O RNA
tion start site at position 85 (19) contains no AUGs (29), we of 2.1 kb from the endogenous heat shock genes. The pUCPT probe
expected to obtain the normal polypeptide products. Fur- hybridized specifically to P transcripts. No transcripts from natural
thermore, Rubin and Spradling (37) showed that the P- P elements could be detected in the 1T2 strain under these conditions.1644 STELLER AND PIRROTTA MOL. CELL. BIOL.
TABLE 1. sn" destabilization by Icarusa
F, flies F2 flies
Cross (female x male) and
treatment No. tested No. with s Total no.
No. with
phenotype:
Ratio, sne +
flies/snw
snlI
+ sne + sn+
sne sn+ flies (%)
sn' (M) x Ica, no heat shock 10 10 (100) 841 104 93 23.4
sn' (M) x Ica, heat shock 10 10 (100) 926 144 126 29.2
sn' (M) X IT2, no heat shock 6 6 (100) 649 39 45 13
Ica x snw (M), no heat shock 8 6 (75) 607 33 29 10
I2 X snw (M), no heat shock 6 1 (17) 576 1 1 0.35
a
Flies were crossed in single-pair matings as indicated. The male progeny, in whose germ line destabilization events occurred, were then crossed to XX w cv
females, and the resulting F2 males were scored for sn+, sne, and snw phenotypes. The Icarus-carrying flies were from line 13c-1, but entirely analogous results
were obtained with line Sica.
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bacterial transformation (for details, see Materials and Meth- sequences. The nature of two minor transcripts (1.7 and 1.2
ods). For plasmid sizes between 15 and 20 kb, we routinely kb) is not clear.
obtained 1 to 10 ampicillin-resistant colonies per fly. Figure Icarus destabilizes the snW allele at a maximal rate even
4 shows the analysis of the Icarus transposon rescued from without heat shock. The previous experiments demonstrated
one of the transformed lines, designed Slca, after BglII that P transcription was strongly heat inducible in the
digestion. It is evident that all fragments from the mobile transformed lines. We next investigated the effect of heat
part of Icarus were present, whereas the flanking DNA shock-mediated overproduction of P RNA on transposase
sequences from the white gene were lost. activity. From Southern analyses done periodically after
It was also possible to reclone only the left part of the establishment of the transformed lines, it was already evi-
transposon together with flanking sequences by using en- dent that Icarus elements continued to transpose to new
zymes such as EcoRI, BamHI, and XhoI. This usually gave sites even in the absence of heat shock (data not shown). To
smaller plasmids and consequently slightly higher transfor- compare the amount of transposase produced in heat-
mation efficiencies. Analysis of clones obtained that way shocked and uninduced transformants and natural P strains,
also demonstrated that flanking vector sequences had been we used the snW test (14, 19, 36). The Icarus element
lost while the transposon remained intact (data not shown). destabilized the sn"' allele in an M x Ica cross at a very high
In situ hybridization with the recovered plasmid as a probe rate even without heat shock induction (Table 1). Transform-
showed that the Icarus insertion in the Sica line was at ants containing a single Icarus transposon were significantly
position 28D on the left arm of the second chromosome (data more active than flies from the 7r2 strain, which carry
not shown). Most of the subsequent experiments were multiple natural P elements. To our surprise, heat shock
carried out with this line or with another line, 13C-1, in induction apparently did not lead to a further increase in snw
which a single Icarus transposon is inserted at position 50C destabilization. We attributed this to a significant basal
on the right arm of the second chromosome. activity of the heat shock promoter. As we have shown in
Heat shock induces massive transcription of the Icarus other experiments, the uninduced level of this promoter,
element. To analyze the heat inducibility of P-element tran- although orders of magnitude lower than after heat induc-
scription, we heat-shocked transformed flies for 1 h at 37°C, tion, can be readily detected with sufficiently sensitive
extracted total RNA, and compared it with total RNA from assays (38, 39).
unshocked transformants and heat-shocked flies from the 7r2 The activity of P elements in natural P strains such as Tr2
strain by Northern blotting (Fig. 5). The same filter was depends on the orientation of the cross, i.e., transposition
hybridized twice, first with a P-specific probe and then with occurs only if females of the M cytotype are crossed to
phs-rr DNA to show the endogenous hsp7O RNA (Fig. 5). A P-cytotype males (M x P) but not in the reciprocal cross (P
major transcript of about 2.8 kb was strongly expressed in x M). The asymmetry is presumably due to the regulator
heat-shocked but not detectable in uninduced flies. No P product present in P-cytotype eggs. To determine the
transcripts from the 7r2 strain were visible under these cytotype of Icarus transformants, we measured the degree of
conditions. The 2.8-kb Icarus RNA most likely corresponds snW destabilization in the Ica x M cross by crossing at-
to the 2.6-kb transcript identified by Karess and Rubin (19), tached-X females carrying a single Icarus element on the
considering that our construct contained an additional 200 bp second chromosome (in either 50C or 28D2) to y snW males.
from the hsp7O leader region and 50 bp of upstream P The results (Table 1) show that a single Icarus transposon
TABLE 2. sn"' destabilization by Icarus-neoa
F1 flies F2 flies
Heat shock No. (%) with sX pheNo. with
phenotype:
Ratio, sne + sn+
No. tested instability Total no. flies/snw + sne + sn+
sne sn flies (%)
None 10 2 (10) 531 2 1 0.56
Embryo stage 10 6 (60) 347 13 10 6.6
Larva stage 10 4 (40) 263 11 4 5.7
a
sna (M) x Ica crosses were performed with and without heat shock treatment during development of the F, generation.VOL. 6, 1986 HEAT SHOCK PROMOTER-CONTROLLED P TRANSPOSONS 1645
1(2) Ica-Neo sition occurred in somatic cells. The same result has been
FO: males x females obtained (D. C. Rio, F. Spencer, and G. M. Rubin, personal
CyO DTS513 communication) with an hsp7O-P fusion similar to ours. We
1 heat shock embryos/larvae therefore conclude that the tissue specificity of P-element
transposition is controlled at the posttranscriptional level.
Construction of the attenuated Icarus-neo transposon. The
F 1: mate brothers x sisters in pools (20-50 flies) previous experiments demonstrate a high activity of the
kill CyODTS flies 4 by incubating at 29 OC
Icarus transposon even in the absence of heat shock. To
investigate the effect of transposase expression at different
developmental stages, we therefore had to construct a trans-
F2: only (2)rey survive, cross to SMI partners poson with reduced constitutive activity. This could be
1(2)Ica-Neo achieved by altering the hsp7O promoter to reduce the basal
level or by decreasing the translational efficiency of the heat
shock-promoted RNA. We obtained the latter by inserting a
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1-kb DNA fragment containing the TnS neomycin resistance
gene (38) between the hsp7O promoter and the 5' P-element
F3: cross Cy flies individually to Canton S partners sequences. In the resulting construct, called Icarus-neo (Fig.
1G418 selection 1), a polycistronic message encoding the TnS phosphotrans-
ferase [APH(II)] and the P polypeptide(s) should be tran-
scribed from the hsp7O promoter. Translation of eucaryotic
F4: resistant flies carry the /cirvs-N#o transposon on a mRNAs is usually initiated at the AUG closest to the 5' end
.new' chromosome
(23). Since in Icarus-neo the putative P initiation codon is
FIG. 6. Excision and reintegration of the Icarus-neo transposon. now preceded by 17 AUGs present in the TnS neo gene, we
Excision events occurring in the germ line of F1 flies produce expect that translation of P polypeptides should be signifi-
reversions of the 1(2)ca"neo mutation. Because these events are cantly reduced from that in the original Icarus element. In
premeiotic, eventual reintegration sites of the transposon will seg- addition, expression of the phosphotransferase should ren-
regate independently from the revertant second chromosome and der transformants resistant to G418 and thereby provide a
only half will be recovered in the F2 generation. In the subsequent dominant selectable marker (37). Embryos injected with
two crosses, the original second chromosomes are replaced by the
SMI balancer chromosome (marked with Cy) and a wild-type Icrus-neo were given a 30-min heat shock during develop-
second chromosome. G418-resistant flies with the dominant Cy ment, and the resulting flies were mated inter se. G418-
marker can be scored as transpositions to a new chromosome. resistant transformants were detected in the F1 generation at
Because only half of the transposons will segregate with the Cy a frequency of approximately 20%. The fact that these were
marker, only half of the transpositions will be scored. Overall, obtained without coinjection of a helper element shows that
therefore, the detectable transposition events represent one-fourth Icarus-neo still behaved like an autonomous P element.
of the original transpositions to a chromosome other than the Lines were established by crossing the transformed flies
second. with partners carrying balancer chromosomes and reselect-
ing the progeny on G418. For most of the subsequent
efficiently mobilized the defective P elements of the snW experiments we used line 2-2, which has a single transposon
allele in this cross as well, although at a slightly reduced integrated at 27A in the left arm of the second chromosome,
frequency compared with the "normal" M x Ica orienta- causing an embryonic lethal mutation, and line T-11, which
tion. We therefore conclude that Icarus transformants are has the transposon integrated on the X chromosome.
essentially of the M cytotype. However, in this cross we Transposition of Icarus-neo is stimulated by heat shock. The
observed significantly greater heterogeneity in the activity of effect of heat shock on the amount of transposase produced
individual ffies than in the Mx Ica cross; whereas some flies by Icarus-neo was assayed by sn"' tests. Flies transformed
showed very high activity, others showed relatively low with Icarus-neo produced a very low though detectable level
levels of snW mutability and hence were responsible for of transposase without heat shock (Table 2). Compared with
lowering the average rate compared with the M x Ica cross. the original Icarus element (Table 1), the rate of snw desta-
It is possible that the latter flies had acquired an intermediate bilization was reduced approximately 50-fold.
cytotype. In contrast to the previous results for the Icarus trans-
Icarus does not destabilize the snW allele in somatic cells. The poson, heat shock stimulated the destabilization of the snw
transposition of natural P elements is restricted to germ line allele roughly 10-fold (Table 2). The extent of snw destabili-
cells (11, 41). This could be due to either transcriptional or zation observed for Icarus-neo after heat shock induction (7
posttranscriptional control by germ line-specific host func- to 8%) was comparable to the levels obtained with natural P
tions. If the control is at the transcriptional level, we would elements (Table 1) or with a single Pc[ry+] transposon (19).
expect to obtain somatic transposition by inducing P tran- To test whether transposition of P elements is dependent
scription from the hsp70 promoter. With posttranscriptional on the developmental stage, we compared the effect of heat
control, on the other hand, the activity of the Icarus trans- shocks applied during the embryonic and third instar larval
poson would still be limited to the germ line. Somatic stages on snw destabilization. There was no significant
activity should be readily detected by the production of difference in heat shock induction between the embryonic
snelsn+ mosaic flies in the F1 generation of an M x Ica cross and larval stages (Table 2). We therefore conclude that
(11). We crossed males carrying Icarus to y sn' females and transposition is not restricted to a certain developmental
heat shocked the offspring for 1 h at 37°C daily during their stage but can occur during most of the, if not the entire, life
development. After carefully inspecting the bristle pheno- cycle of D. melanogaster.
type of the resulting F1 flies, we could not detect any Although the snw test is a very sensitive assay for
convincing example of sn mosaicism, implying that in spite transposase production, it does not allow determination of
of massive expression of the hsp-P fusion gene, no transpo- the transposition frequency of the Icarus element itself. To1646 STELLER AND PIRROTTA MOL. CELL. BIOL.
TABLE 3. snw destabilization in the P cytotypea
F1 flies F2 flies
Cross (female x male) and treatment No. with Ratio, sne + snI
No. tested No. with snw Total no. phenotype: flies/snw + snt + snI
instability fleM
sn' sn flies (%)
ir2 X snw Icarus, no heat shock 10 7 530 23 38 11.5
ir2 X snw Icarus-neo, heat shock 10 4 468 15 7 4.7
7T2 X snw r2, no heat shock 10 0 596 0 0 0
a
Crosses entirely analogous to those in Tables 1 and 2 were done with an sn" line in a r2 background.
deternmine this directly, we again made use of the G418 mined X-*A transposition rate (data not shown). This result
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resistance marker. In our first approach we measured the confirmed our predictions and demonstrated again that the
rate of X chromosome to autosome (X--A) transposition of activity of the Icarus-neo transposon is at least partially heat
the element integrated in line T-11. In this line, transformed shock dependent.
males carrying a single transposon on the X chromosome are It is not known whether P-element transposition is replica-
balanced against attached-X w cv females. In an attached-X tive, like that of bacterial transposons which generate a new
line the free (male) X chromosome is specifically inherited copy of the transposon at a new site (22), or involves
by the male progeny (and by XXX diploid metafemales, excision and reintegration. P elements can excise precisely
most of which die before the adult stage). Consequently only and imprecisely (9). To determine how frequently excision is
males (and a few XXX metafemales, which were discounted) accompanied by reintegration at a new site, we used the
will survive G418 selection unless the Icarus-neo element crossing scheme shown in Fig. 6. Transposition of the
has transposed from the male X chromosome to the Icarus-neo element from its original position at 27A on the
autosomes or the Y chromosome. The appearance of G418- second to a new chromosome will become apparent by the
resistant females is therefore indicative of a transposition segregation pattern of the G418 resistance marker. To facil-
event, and the transposition rate can be deduced from the itate the isolation of revertants in this experiment, we used a
ratio of resistant female to male flies (this method actually CyO DTS513 chromosome to balance the l,ca-neo insertion.
slightly underestimates the transposition frequency, as As this balancer chromosome contains a dominant temper-
X--X events will remain undetected). ature-sensitive lethal mutation, only revertants with the
Flies of the T-11 line were allowed to lay eggs on normal genotype l(2)rev./1(2)1ca-neo or 1(2)rev./l(2)rev can survive at the
food for 2 days, and the developing F1 embryos and larvae restrictive temperature (29°C). 1(2)Ica-neo/CyO DTS513 flies
were heat shocked or not shocked after removal of the that had been heat shocked at the embryonic or larval stage
parental flies (for details, see Materials and Methods). The or not shocked were allowed to lay eggs for 2 days at the
F1 flies were crossed in pools inter se, and their offspring (F2) permissive temperature. After removal of the parental flies,
were selected on food containing 1 mg of G418 per ml to the progeny culture was raised at 29°C to select the rever-
determine the ratio of G418-resistant female to male ffies. tants. In each case a control culture with a similar number of
Without heat shock, transposition events of the Icarus- eggs was kept at the permissive temperature (25°C) to
neo transposon are rare (2 of 1,326 flies; 0.15%). After heat determine the total number of offspring and thereby estimate
shock, X--A transpositions occur at a frequency of 1.2% (9 the reversion frequency.
of 782 flies), i.e., approximately 10 times more often than in Revertants were obtained at a frequency of -1% for the
the uninduced state and comparable to that of the Pc[ry+] heat-shocked and 0.1 to 0.2% for the unshocked flies,
transposon of Karess and Rubin (19). Again, no significant confirming our previous data. To see whether an Icarus-neo
influence of the developmental stage on heat shock induction transposon had reintegrated in a new chromosomal location,
could be detected (data not shown). we used the scheme illustrated in Fig. 6. Of 40 independent
Next we analyzed the transpositional behavior of the revertants (isolated from different culture vials), 7 (-17%)
Icarus-neo transposon in line 2-2. This line contains a single had the G418 resistance segregating with a "new" chromo-
element at position 27A on the left arm of the second some. Due to the independent assortment of the chromo-
chromosome. The insertion obviously disrupted an essential somes in the two selection steps, our crossing scheme would
gene, as the G418 resistance segregated together with a have detected only one-fourth of all transpositions. This
recessive, embryo-lethal mutation. Consequently, we pre- means that the reintegration frequency in our experiment
dicted that excision of the Icarus-neo element should lead to was 4 x 17%, i.e., approximately 60 to 80%. The actual rate
reversion of the lethality and, as the previous experiments is higher because reintegration events into the second chro-
suggested, should be stimulated by heat shock induction. As mosome would not have been detected in this experiment.
the chromosome carrying the lethal mutation (l(2)Ica-neo) was The observed high frequency of reinsertions among rever-
balanced with an SM1 chromosome marked with the domi- tants suggests that almost all precise excisions are associated
nant Cy mutation (curly wings), revertants with the genotype with reintegrations and argues against a replicative mecha-
1(2)rev./l(2)Ica-neo or 1(2)rev`/1(2)rev should be easily detectable nism for P transposition. These results suggest a method for
by their Cy' phenotype. efficient insertional mutagenesis and rapid cloning of the
To test our prediction we compared the offspring of tagged gene by plasmid rescue (see Discussion).
heat-shocked 1(2)Ica-neo/SMI flies with those of unshocked Icarus and Icarus-neo are not repressible in the P cytotype.
flies. Without heat shock induction we found revertants at a Transposition of natural P elements is repressed in the P
low frequency of -0.1 to 0.2%. After heat shock induction cytotype. The molecular nature and mode of action of the
revertants were found at an approximately 10-fold-higher postulated negative regulator are unknown. To investigate
frequency, in good agreement with the previously deter- the role of transcriptional control, we assayed the activity ofVOL. 6, 1986 HEAT SHOCK PROMOTER-CONTROLLED P TRANSPOSONS 1647
the Icarus and Icarus-neo transposons in the P cytotype by encode the transposase function. This leads us to propose
snw assays (for details, see Materials and Methods). Both that above a certain level of hsp-P expression the transpo-
Icarus and Icarus-neo destabilized snw in the P cytotype, sitional capacity is saturated because of limiting amounts of
while natural P elements were essentially inactive (Table 3). a host factor required for transposition.
Although the Icarus element largely overcame repression in Role of transcriptional and posttranscriptional control for
the P cytotype, there appeared to be a slight reduction of its P-element transposition. Transposition of natural P elements
activity compared with that in the M cytotype (Table 1). A is restricted to the germ line (11, 41). It is not known whether
similar effect had been noticed above when comparing the M this is due to germ line-specific transcription or to posttran-
x Ica (normal) and Ica x M (reverse) crosses (Table 1). scriptional regulation of P-element transposition. Our failure
The fact that the heat shock promoter-controlled to detect any appreciable transposase activity with the heat
transposons showed little sensitivity to repression in the P shock promoter-driven P elements in somatic cells argues
cytotype indicates dominance of the transposase over the strongly for the involvement of a germ line-specific host
negative regulator. We therefore propose that the negative factor at the posttranscriptional level. Laski et al. (24) have
regulator normally interferes with transposase production at recently shown that germ line-specific RNA splicing is
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the transcriptional level. required for transposase activity. If the specific factors are
present only in limiting amounts, that would explain why
DISCUSSION induction of the Icarus element did not further increase the
Icarus and Icarus-neo are heat shock promoter-controlled P transposase activity (see Discussion, above). In this case
transposons. In the present work we replaced the P promoter enhanced activity could be only achieved by overproducing
with the hsp70 heat shock promoter and studied the behavior the host function or eliminating the need for it. A plausible
of the modified transposon in transformed lines carrying alternative explanation is that the limiting transposition level
single inserts. We have shown that these constructs behaved is determined by competition between overproduced
like autonomous P elements; they did not require a helper to transposase and overproduced regulator.
transpose from the injected plasmid DNA to the DNA of How does the promoter switch affect the ability of P
germ line chromosomes. Both elements could undergo sub- elements to be repressed in the P cytotype? It has been
sequent transposition events and were capable of destabiliz- proposed that the P cytotype is determined by episomal
ing the snW allele. DNA (11, 12), by the three-dimensional structure of the
To our surprise, we f6und that even without heat shock a nucleus (9), or by a bifunctional transposase protein (29). If
single Icarus transposon showed higher activity in the snW the negative regulator is a specific transcriptional repressor,
test than the natural P elements present in the 1T2 stock. We we would expect its control to be overridden by the heat
have several reasons to believe that this is due to the shock-promoted P elements. However, if the regulator acts
leakiness of the hsp7O promoter rather than to residual at the posttranscriptional level, it should still operate on the
activity of the P promoter. We observed significant basal modified transposons.
activity when we used the heat shock promoter to express We found that Icarus and Icarus-neo transposons effi-
the APH(II) phosphotransferase of TnS (38) or the D. ciently mobilized the natural P elements from the snW locus
melanogaster white gene (39). According to unpublished in a P-cytotype background. This means that the trans-acting
experiments by Rubin and Spradling (cited in reference 29), transposase function is dominant over the negative regulator
the upstream P-element sequences present in Icarus should present in the Xrr2 stock. Since the changes introduced by the
have little or no promoter activity. This was confirmed by promoter switch affected only the promoter and 5' end of the
the greatly reduced constitutive activity of the Icarus-neo P RNA but not the coding region, we suggest that the
transposon, which differed from Icarus only by an insertion negative regulator normally prevents the production of
of the TnS neo gene. As this construct has otherwise transposase by interaction with the P promoter or with the 5'
identical (5') P sequences, any residual activity of the P end of the transcript. The weak effect of cytotype that we
promoter would have to be equal to or lower than the low detected could be explained by supposing that the target
activity of the uninduced Icarus-neo transposon. sequence is only partly deleted in our constructs, allowing
Heat shock induction leads to massive transcription of a residual interaction with the negative regulator. Unfortu-
2.8-kb-long RNA containing the 5' untranslated leader re- nately, since cytotype has little effect on Icarus activity,
gion from the hsp7O gene and P-element sequences. This these experiments cannot tell us whether Icarus is itself able
transcript most likely corresponds to the 2.6-kb RNA iden- to produce the negative regulator.
tified by Karess and Rubin (19). While they found a second Mechanism of transposition. Transposition of the Icarus-
transcript with a size of 2.4 kb, we could not detect any neo element from one chromosome to another can be easily
corresponding RNA for the Icarus element. It is very likely detected by following the G418 resistance marker. In the
that the 2.4-kb RNA is an artifact of the Pc[ry+] line, as it l(2)Ica-neo line, in which the insertion resulted in a recessive
could not be detected in flies carrying exclusively natural, lethal mutation, we could easily select flies in which the
autonomous P elements (24). element had been excised precisely from the lethal site by
Given the strong inducibility of the Icarus hsp-P fusion using the DTS balancer chromosome. Transposition events
gene, it remains to be explained why we did not detect a that are normally relatively rare even after heat shock (about
corresponding stimulation of snw destabilization. The fact 1%) rose to 17% among the flies selected for excision. When
that the activity of the attenuated Icarus-neo transposon we allowed for the fact that the chromosome carrying the
could be significantly stimulated by heat shock induction reinsertion would segregate independently from the chromo-
clearly demonstrates that the transpositional activity is, at some in which the excision occurred, this frequency became
least over a certain range, dependent on the amount of 60 to 80%. Furthermore, since reinsertions in the second
P-element expression. As the uninduced Icarus element is chromosome would not have been detected in our crossing
already more active than the induced Icarus-neo or natural P scheme, the actual figure becomes close to 100%, implying a
elements, it is also very unlikely that the 2.8-kb hsp-P RNA strong correlation between excision and reintegration
transcribed from Icarus is biologically inactive or does not events. Note, however, that by screening for revertants of1648 STELLER AND PIRROTTA MOL. CELL. BIOL.
the 1(2)Ica-neo mutation we most likely selected for precise Spierer, E. B. Lewis, and D. S. Hogness. 1983. Molecular
excisions and eliminated imprecise excisions. The latter genetics of the bithorax complex in Drosophila melanogaster.
would probably lead to loss of the transposon. Furthermore, Science 221:23-29.
this type of experiment can never exclude the possibility that 4. Bingham, P. M., M. G. Kidweli, and G. M. Rubin. 1982. The
the same individual could have high activity of both excision molecular basis of P-M hybrid dysgenesis: the role of the P
and transposition without a necessarily mechanistic relation- element, a P-strain specific transposon family. Cell 29:995-1004.
5. Bonner, J. J., C. Parks, J. Parker-Thornburg, M. A. Mortin,
ship. With these reservations, our results argue against a and H. R. B. Pelham. 1984. Use of promoter fusions in
replicative mechanism for transposition and suggest that, Drosophila genetics: isolation of mutations affecting the heat
while some excision events may be abortive and lead to loss shock response. Cell 37:979-991.
of the transposon, integration events are generally accom- 6. Bregliano, J. C., and M. G. Kidwell. 1983. Hybrid dysgenesis
panied by precise excision of the transposon from a previous determinants, p. 363-410. In J. A. Shipiro (ed.), Mobile genetic
site. elements. Academic Press, Inc., New York.
Use of Icarus elements for in vitro mutagenesis and gene 7. Carramolino, L., M. Ruiz-Gomez, M. Guerrero, S. Campuzano,
cloning. Gene tagging by insertional mutagenesis with P and J. Modolell. 1982. DNA map of mutations at the scute locus
of Drosophila melanogaster. EMBO J. 1:1185-1191.
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elements allows cloning of genes without knowledge of their 8. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. S.
products (4, 35). However, the relatively low rate of muta- Rutter. 1979. Isolation of biologically active ribonucleic acid
tion induction and the large number of P elements present in from sources enriched in ribonuclease. Biochemistry 18:
natural P strains mean that extensive analysis is generally 5294-5299.
required to identify the clone of interest. Mutator stocks 9. Daniels, S. B., M. McCarron, C. Love, and A. Chovnick. 1985.
carrying a single copy of Icarus or Icarus-neo should cir- Dysgenesis-induced instability of rosy locus transformation in
cumvent this problem. Drosophila melanogaster: analysis of excision events and the
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10. Engels, W. R. 1979. Hybrid dysgenesis in Drosophila
neo transposon into new chromosomal sites, using a bal- melanogaster: rules of inheritance of female sterility. Genet.
ancer chromosome carrying a dominant temperature- REs. (Cambridge) 33:219-236.
sensitive lethal (DTS) mutation, it is possible to preselect for 11. Engels, W. R. 1979. Extrachromosomal control of mutability in
transposition events and thereby improve the frequency of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 76:
mutation induction. From our preliminary data we estimate 4011-4015.
that lethal mutations are induced in approximately 1 of every 12. Engels, W. R. 1981. Hybrid dysgenesis in Drosophila and the
5 to 10 reintegration events (H. Steller and V. Pirrotta, stochastic loss hypothesis. Cold Spring Harbor Symp. Quant.
unpublished). Once a mutant has been isolated, DNA Biol. 45:561-565.
flanking the site of integration can be cloned by plasmid 13. Engels, W. R. 1983. The P family of transposable elements in
rescue of the transposon in less than a day's work. With Drosophila. Annu. Rev. Genet. 17:315-344.
14. Engels, W. R. 1984. A trans-acting product needed for P factor
high-efficiency bacterial transformation procedures (16; J. transposition in Drosophila. Science 226:1194-11%.
Gough, unpublished data, described in Materials and Meth- 15. Engels, W. R., and C. R. Preston. 1984. Formation of chromo-
ods), clones can be recovered from the DNA of a single fly, some rearrangements by P factors in Drosophila. Genetics
and hence even sterile mutants or flies that die before mating 107:657-678.
can be used. 16. Hanahan, D. 1983. Studies on transformation of Escherichia coli
The presence of single copy of the transposon should also with plasmids. J. Mol. Biol. 166:557-580.
help to minimize chromosomal rearrangements (deletions 17. Ingolia, T. D., E. A. Craig, and B. J. McCarthy. 1980. Sequence
and inversions), which are of little use for gene tagging and of three copies of the gene for the major Drosophila heat shock
induced protein and their flanking regions. Cell 21:669-679.
appear to result from the presence of multiple P elements on 18. Jorgensen, R. A., S. J. Rothstein, and W. S. Reznikoff. 1979. A
a chromosome (15). restriction enzyme cleavage map of TnS and location of a region
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ACKNOWLEDGMENTS 19. Karess, R., and G. M. Rubin. 1984. Analysis of P transposable
element functions in Drosophila. Cell 38:135-146.
We are grateful to C. Garber and H. Cambier for excellent technical 20. Kidd, S., T. J. Lockett, and M. W. Young. 1983. The Notch
assistance and to C. Christensen, A. Weischer, and A. Summerfield locus of Drosophila melanogaster. Cell 34:421-433.
for their help in photographic reproduction. We thank Frank Laski, 21. Kidwell, M. G., J. F. Kidweli, and J. A. Sved. 1977. Hybrid
Don Rio, Forrest Spencer, and Gerry Rubin for communicating dysgenesis in Drosophila melanogaster: a syndrome of aberrant
unpublished results, Jill and Nick Gough for their transformation traits including mutation, sterility and male recombination.
protocol, and Steve Mount and Frank Laski for comments on the Genetics 86:813-833.
manuscript. 22. Kleckner, N. 1981. Transposable elements in prokaryotes.
H.S. was the recipient of a European Molecular Biology Labora- Annu. Rev. Genet. 15:341-404.
tory predoctoral fellowship. Part of this work was supported by 23. Kozak, M. 1982. How do eukaryotic ribosomes recognize the
Public Health Service grant GM 34630 from the National Institutes unique AUG initiator codon in messenger RNA? Biochem. Soc.
of Health. Symp. 47:113-128.
24. Laski, F. A., D. C. Rio, and G. M. Rubin. 1986. Tissue
LITERATURE CITED specificity of Drosophila P element transposition is regulated at
1. Artavanis-Tsakonas, S., M. A. T. Muskavitch, and B. Yed- the level of mRNA splicing. Cell 44:7-19.
vobnick. 1983. Molecular cloning of Notch, a locus affecting 25. Levis, R., M. Collins, and G. M. Rubin. 1982. FB elements are
neurogenesis in Drosophila melanogaster. Proc. Natl. Acad. the common basis for the instability of the wDZL and wc
Sci. USA 80:1977-1981. Drosophila mutations. Cell 30:551-565.
2. Beck, E., G. Ludwig, E. A. Auerswald, B. Reiss, and H. Schalier. 26. Levis, R., K. O'Hare, and G. M. Rubin. 1984. Effects of
1982. Nucleotide sequence and exact localization of the transposable element insertions on RNA encoded by the white
neomycin phosphotransferase gene from transposon Tn5. Gene gene of Drosophila. Cell 38:471-481.
19:327-336. 27. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular
3. Bender, W., M. Akam, F. Karch, P. A. Beachy, M. Pfeifer, P. cloning: a laboratory manual. Cold Spring Harbor Laboratory,You can also read
