Download embryos) (1). Smaller P elements are also present

1 1 Number 18 1983
Vofume 11Nme
Voum
Nucleic Acids Research
818ucecAisRsac
Vectors for P element-{nediated gene transfer in Drosophila
Gerald M.Rubin + and Allan C.Spradling
Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway,
Baltimore, MD 21210, USA
Received 23 June 1983; Revised and Accepted 30 August 1983
ABSTRACT
We have constructed and tested several new vectors for P elementmediated gene transfer. These vectors contain restriction sites for cloning
a wide variety of DNA fragments within a small, non-autonomous P element and
can be used to efficiently transduce microinjected DNA sequences into the
germ line chromosomes of D. melanogaster. The P element in one vector also
carries the rosy gene which serves as an easily scored marker to facilitate
the transfer of DNA fragments that do not themselves confer a recognizable
phenotype. The failure of certain P element constructs to function as
vectors suggests that P element sequences, in addition to the 31 bp inverse
terminal repeats, are required in cis for transposition. Moreover, removal
of the first 38 bp of the autonomous 2.9 kb P element appears to destroy its
ability to provide a trans-acting factor(s) required for the transposition of
non-autonomous P elements. Finally, we describe a genomic sequence
arrangement that apparently arose by the transposition of a 54 kb composite
P element from a tetramer plasmid.
INTRODUCTION
P element-mediated gene transfer permits the introduction of cloned DNA
sequences into the germ line chromosomes of a complex metazoan organism,
Drosophila melanogaster (1,2). Moreover, the transduced DNA sequences
appear to be stably inherited and appropriately expressed in future
generations (3,4,5). Thus, this genetic transformation method should allow
the assessment of the functional properties of aenes that have been isolated
and subjected to in vitro mutagenesis.
The P element-mediated gene transfer method exploits the properties of
the P family of transposable elements (6,7,8). A 2.9 kb P element has been
isolated from the genome of a P strain and its DNA sequence determined (8).
This transposable element appears to be autonomous in that it is able to
transpose from plasmid sequences into the chromosomes of germ line cells
after microinjection into Drosophila embryos lacking such elements (M
embryos) (1). Smaller P elements are also present in the genome of P strains
© I RL Press Limited, Oxford, England.
6341
Nucleic Acids Research
(6,8) and the DNA sequences of these elements suggest that they arose by
internal deletion from the 2.9 kb element (8). These smaller elements are
non-autonomous; they cannot transpose when injected into M embryos (2).
When such non-autonomous elements are coinjected with the 2.9 kb element
however, they are able to transpose suggesting that the 2.9 kb element
encodes a tras-acting factor required for transposition (2). Other DNA
sequences can be caused to transpose into germ line chromosomes as well, by
constructing a P transposon carrying the DNA segment of interest within a
non-autonomous P element (2-5).
We describe here several new non-autonomous P element vectors. These
vectors are small plasmids which have many unique restriction sites for
cloning DNA fragments within a P element but lack sites in the plasmid backbone outside the element. The results of testing several intermediates made
in the course of constructing these plasmids reveal certain sequence requirements for P element transposition. Our results also suggest that P elements
as large as 54 kb can transpose.
MATERIALS AND METHODS
Restriction enzymes and BAL-31 nuclease were purchased from New England
Biolabs. T4 DNA ligase as well as Hpa I and Kpn I DNA linkers were
purchased from Collaborative Research. M13mplO DNA was purchased from P-L
Biochemicals.
Microinjection of Drosophila embryos and other aspects of the transformation procedure are described in detail in references 1 and 2. DNA
blotting and hybridization were as previously described (9). BAL-31
digestions were carried out at room temperature in 600 mM NaCl, 12 mM
CaC12, 12 mM MgC92, 1 mM EDTA and 20 mM Tris-HCl pH 8 for 5 or 10 min using
6 units of BAL-31 and 20 ag of plasmid DNA. Growth of recombinant M13 phage,
preparation of DNA templates and DNA sequencing using di-deoxynucleotides
were as described by Sanger et al. (10) except that the sequence reactions
were not in glass capillaries, but in 1.5 ml tubes as described by Heidecker
et al. (11).
RESULTS AND DISCUSSION
Vector Construction
Plasmid vectors were constructed according to the protocol diagrammed in
Figure 1. First, the Sal I fragment of p6.1 (2), which contains a nonautonomous P element inserted into white locus DNA (6), was ligated into the
6342
Nucleic Acids Research
HindMi
Sol
P
EcoRI
Smol
5.3 kb
u dM
HindMU Xhol Po.ub
Digest with BamHl
Digest with exonuclease Bal 31
Ligate with T4 DNA ligase
Tronsform E. co//
Figure 1.
Construction of
vectors. The indicated steps
were performed. DNA sequences
derived from the pUC8 plasmid
are shown stippled, those from
the P element are fully shaded
and those from the D. melcmogaster white locus are unshaded.
HindUm
Ps Il
((pCIW2~
\\i4.6 kb,
indm
HFlnd
Digest with
Digest with
Ligate with
Transform
Sall
exonuclease Bal 31
T4 DNA igose
E co/i
3.9kbg
HOndM
Hindm
Insert EcoRI linker into Pvu0
site to create pCIW4
Insert polylinker into pCIW4
between EcoRl and left HindIm site
Caonegie
ind M
Hind-1 Pstl Sol1 BamHl*Smal EcoRI
Carnegie 2
HindM Pstl Sal -BamHl Hpa EcoRI
Carnegie 3
HindlMPstl-Sall-BomHl.Kpnl EcoRI'
Carnegie 4
'HindE-Pstl Soll-Xbol-BomHt.Smol-Sstl-EcoRl
Sal I site of the plasmid vector pUC8 (12) to create the plasmid pCIW1.
Then two successive treatments with BAL-31 nuclease were used to eliminate
undesirable restriction sites and reduce the size of the plasmid. Finally,
the indicated linkers were inserted to create the plasmid vector Carnegie 1.
The Carnegie 1 vector is 3616 bp long and its nucleotide sequence is shown in
Figure 2. This vector carries the Hind III - Pst I - Sal I - Bam HI - Sma
I - Eco RI polylinker derived from pUC8. The Carnegie 2 and Carnegie 3
vectors were constructed by inserting a Hpa I (GTTMC) or Kpn I (CGGTACCG)
linker into the Sma I site of Carnegie 1, respectively. The Carnegie 4
vector was made by substituting the polylinker of M13mplO for the Hind IIIEco RI polylinker contained in Carnegie 1.
The vector Carnegie 20, which is diagrammned in Figure 3, was made by
inserting a 7.2 kb Hind III fragment containing the rosy gene (2) into
the Hind III site of the Carnegie 2 polylinker. This rosy DNA fragment
6343
Nucleic Acids Research
HindM
120
90
110
70
100
s0
00
s0
30
20
TAACA?AAGG ?GYCGCGAThOCCAA OCT?vACCGAA GUTAACAC? hhATTAG?G CAC0W"GCY 9T000-AOGW OAAMG7TCY?GTCGGACGAA ?7YP?
240
230
220
210
190
200
130
170
1S0
160
140
130
0AA~AACTAA rC~C0AATAAAAAA AAA?GAAAYA ?90MAATrY 90CYGAAAO CTO9GAC900 AOYAAAMTPA A?TCAC0T0C CGAAQ00TC TAIT7AAG AA TG?CGGG
3600
340
330
350
310
320
290
300
200
270
200
290
TG&O0c?0G70CAOC CTT?GTGAAA &CTCCCAAT ?TTGTAT0TCC CACTnTAA20. 0?YC AGCA" 0400aOCTA CCT=AAAAG GC0AaCAT TAAIAGG00GG CGACTCAACG
470
400
450
430
400
040
010
020
000
390
300
370
C0AG00CCGT ACC?AM?AAA GYGAThGAO CYGAACCAG.A AAAGAThAAAA G&AAGCTATA CCA0?GGGA0 TACACAAACA GAGTAAGTT GAATAGTAAA AAAAA?CATT TA?GTAAACA
10
MUMA0AA
340
590
330
910
520
S00
9000MGTCCYGTYVA T?GrYAAYG AAAATAAGAG CTTGAGOGAA AAAA?PGYA
HindM
000
070
090
640
630
610
TO0CGC9CAOCCAAG CTIr00CGTC TCGCA&ATPAL VYAAAAATAA AAC?T0AAAA A?AA?TTCGY
790
700
770
730
700
740
730
jCA~ACAA-f ATATCOCY COCACTCAGA CTCAATACGA CAC?CAGAT? ACTATTCC?T TCAC?CGCA
910
900
330
390
39
070
600
YGZAr.r:r A?-GrA.&T TZ:G..TG7 ZG':AThTZrG 3CTCTAA7AC
rZ
AYGPATTvYc AYA?t7
1030
1020
1010
1000
990
930
970
TTTrT'r'rTT TACcArTAIT ACCATCGr;T T-rACGTr!TA TTGCCZCCT1 k9AAAAGCThA rGrkArTATA
490
A?AAC0T
970
500
ClPrGAGWA CGATOA0COT
6000
EcoRi
So.
AGA7CC 00AGcCCGAA ?MCCG0GGA
720
710
700
090
000
CTAA?FAATA TTAICOA ATYCAAJk(C CAC0G-ACA C?AAGGGYA
340
030
020
300
I10
TTATTOCAAG CAThCGITAA GTGGAYOTCT CrtGCCGACG GGACCACCrY
9600
940
930
920
990
TTrCGGGCCCG AGSCAGGAG TAGC--GA-ZAT ArAr1-:C7AAA T9.Acr:"Z-rG
1060
1070
1000
1040
1090
TT~rGTGCCAA T&AAAAAZ9A7 ATATGACCTA TNSAATA~ZAA~;rA rTTZ.CZ:
1200
1190
1170
1110
1140
1130
1100
1130
1100
1120
1110
1090
TT-GA,ACATC CCCACAAGTA SACT1TGGAT TrGTZTtrCT kCC99AAGAC TTA7cACAcr G'-AThccrTA :ArC1AA%A9A T0GTTTATZG CTACATAAAA ZACCGGGATA rA rtrrrTAT
1310
1326
1290
1270
1300
1200
1240
1200
1230
1230
1220
1210
C rr :ATkATTCAZ CCACCA:A wA7JCGrrTCG TAkGTTGC'rCT rTCGCTG rCT :CCACCZGCT CTCCGCAAZA CA?1CACCTr ?TrGrTTGACG
~:TC
ATACATACTr T?CA.AA?CGC
1440
1430
1420
1410
1400
1390
1330
1370
1300
1330
1300
1330
ACCTTrGGAGC GACTGTCGTT AkGT'tCCGCGC GA?TT7GTGC OGYTIYTTCAC ACCGCATA'r G?GCACICT ATACAATCT GCYCYGATC CGCA?A09T AGCCAGCCO GACACCCOCC
A 39bp
19600
1940
1920
1930
1910
1300
A728bp 14940
1470
1490
1460
YCCOOGAOCT 0CA%GTCYGA GhaG?rrCA CCIACAC CGAAACGCGC
o&ocotGACCOCCCCY GACOGOCTYG TCTGCYCCCG OCAYCCOCY ACAGACAAGC T&ccGACG
1990
1000
1010
1970
1300
1930
1940
1990
1900
1970
2170
2130
2190
2200
2210
1090
1000
1070
1600
1630
1040
1900
1990
2000
2010
2020
2030
2040
2220
2230
2240
2290
2200
2270
2230
1020
G0AGaGo CCwGw
rACGccYATYr m"ATAGGT AATGTAAGA YAATAA0GOT TVCTPAGMM TCA0TOCA CTTlTCGGOG AA?CG=GO GGAACCCCTA TrrG?rrAFT
A 2bp
1730
1700 5[tort P-lactamnose gene
1740
1720
1730
18000
1710
1700
rTC-TAA&TA C&AIAAATrA 90TAICGC?C CATGOAC-AA TAACCCYGAT MAAACTMA ATAAT&ATA AAAAGGAAGA OTAYGAGTAT 1AACAWAV CGTGTCG=C ?FA~TFCC
1920
1910
1900
1300
1090
1090
1370
1000
1310
1040
1330
1320
?WYOGCOOCA ?PrCC-M CTGOT?TfMCACCCAGAA ACGCTOGYGA AAGTAAAAGA TGCYGOAGAT CAGTT0G¶ CACCAGT0G TTA?CrA CGGAFCICA ACAGCOGYAA
GAcTvCGwA harYTYcGcc ccGAAGAAcG. TrecAATG ATG.AGCAc?r TYAAAG?rT OCTAYGYGoC OCOGYTTAT ccTGTYA CGCCOGocAA GAGAACTM GTCOCcOAT
2140
2190
21600
2130
2110
2120
2100
2090
2000
2070
2090
2000
ACACTATYCYT cAcAAYGACT YGTPG?cAGTrA CYcAccwG ACAGAAAAGC ATCTTACGGA 9OGCAYGAA GTOAGAGAAT ravwCAGvC 0cccArAAC ATGOAaGYGA AcAcTc00C
CAACrrACTrr C90ACAACGA YCGGAGG1M GAAGGAGC?CA ACCGCYOTr"T OCACAACAT oooooatva
oTAAToCC ?rGATCOr OGGoACCG CT'AAC-M CCATXCAAA
24000
2390
2380
2360
2370
2390
C0AhAC0TQ GACACCACGA TOCCTGTAGC AAYOGCAACA ACGTYGCGCA AAC?AYTrAC TO0CGAACTA CTrACrCTAG CTTCOCG0CA ACAATrrAAYA GACrgGAY A00MGA?AA
2920
2900
2910
2490
2470
2430
2400
2490
2440
2430
2420
2410
GOaIOCOOGA ~cACrtcvCY acrCGcCY= Ycc0GcT0 TooYrYrArrG Cw?GAAArYc !oGGaGcG GA0cOa0T C1CGGaYwa cAivcAGA CYGGOCCAG AYGYAAGcC
2290
2300
OAOT!AY
C-2933
GTAYC
2940
2310
2320
2330
2340
end a-IoCtomoSe gene]
2600
2603
2990
2930
2900
2370
2990
WACGhCOAGO GAGYCAGOCA ACTAYGGAYG AACG.AAAAG ACAGAicYc GawATAGOYG cCYCcAcmrA YAACAYFOS YAACYGYCAG AcAAGT"A
2720
2720
2300
2320
2330
2300
2010
2790
2700
2773
gWCAGh0= G?AGAAAAGA ICAAAOOAYc TYCrTArGAY CCTrrYYYYC YOGcGGAAY CTGCTGCTYG CAAACAAAAA
2990
2960
2940
2930
2920
2910
2000
2390
OCWA cY CrWrcCGA AGOtOACYG CTIcAGcAGA GcGCArGAAc cAhATYAci* cc?TmCaraTG AOCCGww
3330
3000
3370
3090
3040
3030
3013
3020
CLICGCiCvc CTMICCIt TACCOTOW YGcYGCCAOn OOGAAAGY COYGYCTYAC COOGYFOGAC YCAAGACGAY
3200
3130
3190
3170
3130
3100
3140
3133
fCACA CAGCCCAGCY GOGAQGAGAC CACCTACACC CGOALCGAGAY ACCY.ACAOCO TGAGCA?OG GAAAGCOCCA
2390
2090
2000
2070
2030
2090
2700
2710
2700
2790
2700
CYCrAvATAT CYTwAGATY AtTrAhAAcYr YCArrrrAa YTrAA&AAGA YcTAaGOYAA GAYCCYTYTr GAYAAYTCWA YGACCAAAAYr CCCYTrAACOY GAa rfGTY TCACYGAGC
AACCA~CCC
2970
YhAOCCACC
3090
23300
2373
23060
GYGAYCAAGA
ACCAGGYGY G?TTrr
2930
2990
3000
3100
3110
3120
c?CAAGAhC YCYGYAGCAC cOCCTAATA
GAAC00GOG
3230
32400
3223
3210
COCTrTCCCGA AGOGAGAAAG OCOGACAGGY ATCGYAAG
AGYYACCG
YAOAOCOC.AGC00C
3300
3393
3300
3333
3310
3320
3300
3230
3290
3270
3290
3260
CG0CAG0GT WMACA0GAG AGCGCACGAG. 0GAGCTICCA 000GGAAA CCYGOYAY?CY YTAYAGICC @YCO0GM OCCACCYCYG ACTcGAaGGOY =&OA Y GIaYcYCm
34000
ORI CZ~7 3390
3470
3400
3490
3030
3000
3010
3020
34403
3370
AGGOOGOCOG AGOTAYGG A AAACC9Tr CT?Crt'GAAC TCGGGCrCGG TOGCCAGYATA CCTCIAAATGG rTGTCGTACC ?CTCATGGTT CCGTTACGCC AACGAkGGG?IC
TGCTGAkTTAA
3993
36000
3090
3903
A695bp 3920
3930
3S40
3993
3900
CCAATGGGCG GACG?GGAGC CGGGCGAAAT TAGCTGCACA TCGTCG%ACA CCACG?GCCC CAGTTCGGGC AAGGTCATCC
3S70
3900
rGGAGAC-GCT TAACT]1CTCC GC-CGCCGATC TGCCGCTGGA
3610
CTACGTGGGT CTGGCC
Figure 2. Predicted DNA sequence of Carnegie 1. The sequence is numbered
starting with the first nucleotide of the P element and proceeding clockwise
around the map as shown in Figure 1. P element DNA sequences (nucleotides
1-856) are shown both boldface and underlined. These are the sequences that
are transferred to Drosophila chromosomes during transformation. Sequences
derived from the Drosophila white locus (nucleotides 857-1358 and 3389-3616)
are shown in normal typeface and those derived from pUC8 (1359-3388) are
shown in boldface. The 31 bp inverse terminal repeats of the P element are
indicated by the arrows. The positions of cleavage sites for Hind III and
Eco RI, the start and end of the ~-Iactamase gene, and the plasmid origin of
6344
Nucleic Acids Research
replication are shown. The positions of the two BAL-31 deletions (728 and
695 bp) made during the construction of Carnegie 1 (see Figure 1) as well as
two smaller deletions of 39 bp and 12 bp are also indicated. The 12 bp
deletion apparently resulted from the Si nuclease treatment used by Vieira
and Messing (12) during the construction of pUC8 to remove an Eco RI site.
The origin of the 39 bp deletion is unclear; these nucleotides were deleted
from Carnegie 1 as compared to the corresponding region of pBR322 (13).
The Carnegie 1 DNA sequence shown was assembled as follows: The DNA
sequence of pCIW1 was predicted from the known sequences of pUC8 (12) and of
the Sal I fragment of p6.1 (8; K. O'Hare and G. Rubin, unpublished results).
The extent of the BAL-31 deletions made during construction of Carnegie 1
were determined by direct DNA sequence analysis of the Taq I - Sau 3A
fragment (nucleotides 1315 - 1871) and the Taq I-Taq I fragment (nucleotides
3340-3527) which span the deletion endpoints. The sequence of Carnegie 1
was then derived by modifying this sequence to account for the replacement
of the Eco RI-Hind III fragment of the P element in pCIW4 by polylinker
fragments.
does not contain cleavage sites for Hpa I or Sal I thereby permitting the
convenient cloning of additional Sal I, Xho I or blunt-end fragments into
this vector. In such constructs Carnegie 20 has been used successfully to
transfer DNA fragments that do not themselves confer a recognizable
phenotype by selecting for rosy+ transformants (see below).
All of the vectors grow well in standard bacterial hosts and confer
ampicillin resistance. A number of useful restriction sites found in
Carnegie 1 are listed in Table 1.
Tests of vectors and other constructs
Figure 3 summarizes the results of testing various vector constructs
by the Drosophila transformation assay. For these assays (Figure 3,
experiments 1-8), a 7.2 kb Hind III or 8.1 kb Sal I fragment containing the
rosy gene was inserted into the P element of each vector construct. The
resultant plasmids were microinjected into rosy- Drosophila embryos. DNA of
a plasmid carrying the 2.9 kb P element (either pff25.1 or pnr25.7) was coinjected to provide tras-acting functions required for transposition of the
non-autonomous rosy transposons (2). Adults developing from injected embryos
(GO adults) were mated to rosy- flies. The presence of rosy+ progeny in the
next generation indicated that the vector being tested was competent for germ
line transformation. Some of the GO adults themselves displayed a rosy+ eye
color which we believe to be due to expression of the rosy gene on the
injected plasmid DNA (2). While not directly correlated with germ line
transformation (see below) this transient expression does provide a convenient
indication of the ability of the rosy gene to function in a given construct.
We first had to verify that the plasmid created by the BAL-31 treatments,
6345
Nucleic Acids Research
TABLE I
Restriction Sites in Carnegie 1
Location
Enzyme
Recognition Sequence
39
588
593
593
598
604
610
618
860
914
1219
1375
2178
2325
3194
3400
Hind III
Eco RI
Ava I
Sma I
Bam HI
Sal I
Pst I
Hind III
Bal I
Apa I
BssH II
Nde I
Pvu I
Mst I
Hae II
Ava I
AAGCTT
GAATTC
CCCGGG
CCCGGG
GGATCC
GTCGAC
CTGCAG
AAGCTT
TGGCCA
GGGCCC
GCGCGC
CATATG
CGATCG
TGCGCA
AGCGCC
CTCGGG
No cleavage sites for
Bcl
Bgl
Cla
Eco
Hpa
Kpn
I
II
I
RV
I
I
Mlu I
Nae
Nar
Nco
Pvu
Sac
I
I
I
II
I
Sac
Sph
Stu
Xba
Xho
Xma
II
I
I
I
I
I
The location numbers refer to the sequence shown in Figure 2 and
correspond to the first nucleotide of the recognition site. All of the
enzymes shown cleave Carnegie 1 either 0 or 1 time except for Hind III and
Ava I which cleave twice. All of the enzymes shown in this table are
commercially available.
and from which all the vectors were derived, was still capable of acting as
an efficient transformation vector. We inserted the 7.2 kb rosy Hind III
fragment into the left Hind III site of plasmid pCIW4 (see Figure 1) to
create the plasmids pVlO and pVll. These plasmids differ only in the
relative orientations of rosy and P element sequences and both were able to
transfer the rosy gene at frequencies in excess of 50% (Figure 3, experiments
1 and 2). We also inserted the same rosy fragment into the right Hind III
site of pCIW4 to create the plasmid pV9. We did not observe transfer of
the rosy gene into the germ line when this plasmid vector was used, however,
suggesting that insertion of foreign DNA sequences at this site in the P
6346
Nucleic Acids Research
Experiment
Injected
(No)
DNAs injected
HR~Xtl
R.R X
Fertile
adults
expression
GO
r, +
progeny
4 (67%)
H
+pfI25.1
123
6( 5%
4 (67%)
i+pn25.1
121
9(7%)
6(67%)
6(67%)
133
5 (4%)
4 (80%)
0
pV1O:
HI~~~~X~~~I
RH
2
pVII:
3
pV9:
4
5
-..CarnegelI,
X ,
HR X5_H
HRF/s4
pVI.2:
f
""I)iitl,71
>-.'.vx'._:-.7l
6
Cornegie20:
,,,X,)+R
X R
......... .
H
If
R
HRSH
7
HRXH
.
111.Z
I
'.1
lineor ros S H
X R
HRoS
'i1 R
fragment:
8
frogment: t
9
pII25.7:
II
X UR
R S
BS
H XH R S
BS
HXH
k_
_
u
pn25.7A1:
H
777777777M,")il
156
10(6%)
4(40%/)
5(50%)
171
21 (12%)
7(33°%)
0
+prl 25.7
116
20117%/)
11(55%)
[
RHB
_
+prl25.7
83
+p[125.7
188
14 (70%)
9 5)
0
0
(14%)
0
0
S
+pry
26
+pry 3
121
17 (14%)
6 (35%)
5 (29%)
+pry3
208
21
(Y10)
3 (114%)
0
+pry 3
114
6 ( 5)
2 (33%/6)
0
RH
_
+_'.
+pry)
H XH R S
RHB
k -.rD,
m -l
plI25.7A2:
+pry
r
+pfl25.7
+pl125.7
H
/
*.5
linear pryl S HF X,/Sp
10
X
+pI125.1
Figure 3. Testing of vector constructs. The structures of the microinjected DNAs are diagrammed. Rosy sequences are shown cross-hatched and
P element, white and pUC8 sequences are indicated as in Figure 1. Cleavage
sites for Hind III, Eco RI, Xho I, Sal I, and Bam HI are indicated by H, R,
X, S, and B, respectively. X/S indicates the product of ligation of Xho I
and Sal I ends. All DNAs were microinjected as supercoiled plasmids (except
for experiments 7 and 8) at the following concentrations: p7r25.1, pir25.7 and
their deletion derivatives, 50 pg/ml; rosy-containing DNAs in experiments
1-8, 300 pg/ml; pryl and pry3 in experiments 9-11, 150 pjg/ml each. The
number of embryos microinjected and the number and percentage surviving to
become fertile adults are given. Also shown are the number and percent of
fertile adults showing GO expression of the rosy gene, and the number and
percentage of fertile adults giving transformed rosy+ progeny.
element destroyed its ability to transpose (Figure 3, experiment 3). We
cannot rule out the formal possibility that it is not the transposition of
this rosy transposon which is affected, but rather the expression of the
rosy gene it carries. This seems unlikely, however, as the percentage of
embryos showing GO expression was similar to that obtained with pVl0 and
pVll.
We inserted the pUC8 polylinker into pCIW4 in two different positions between the Eco RI site and the Hind III site located to its left to create
the Carnegie 1 vector, and between the Eco RI site and the Hind III site to
its right to create the plasmid pVl. The Carnegie 1 vector was shown to be
capable of promoting efficient germ line transformation when tested with an
inserted 8.1 kb rosy Sal I fragment (plasmid Carnegie 1.1, Figure 3,
experiment 4). In contrast, no rosy+ transformants were recovered when pVl
carrying the rosy gene (plasmid pV1.2) was injected, although normal GO
6347
Nucleic Acids Research
expression was observed (Figure 3, experiment 5). In the pV1.2 construct,
sequences from position 45 to 588 of the P element have been replaced by the
polyl inker.
The apparent failure of the rosy transposons of pV1.2 and pV9 to
transpose suggests that sequences, in addition to the 31 bp inverse
terminal repeats of the P element, are required in cis for transposition.
Previous studies of the structures of non-autonomous, but transposition
competent, P elements indicate that no more than the first 139 or last 216
nucleotides of the 2.9 kb P element are required in cis for transposition
(8). Taken together with those findings, the results presented here suggest
that, in addition to the 31 bp inverse terminal repeats, a cis-acting
sequence essential for transposition is present in the first 140 bp of the
P element.
Carnegie 20 was shown to be effective in transferring the rosy gene
(Figure 3, experiment 6) and has subsequently been used in our laboratories
(unpublished results) to transfer additional DNA fragments inserted into its
Hpa I site. In these latter experiments, transformants were identified by
their acquisition of a rosy+ phenotype. In this way, DNA segments that do
not themselves confer a recognizable phenotype can be routinely transferred
into the germ line.
Attempted improvements in P element-mediated gene transfer
P element insertions into host chromosomes occur at a wide variety of
locations (2,3); no evidence for homologous recombination has been obtained.
In yeast, the frequency of recombination between introduced plasmids and
homologous host sequences can sometimes be substantially increased by using
linear rather than circular DNA molecules (14). Consequently we attempted
to transfer rosy genes carried on linear DNA fragments. However, our
attempts were unsuccessful regardless of whether the rosy gene was within a
P element or not (Figure 3, experiments 7 and 8). Moreover, we did not
observe any GO expression in these experiments suggesting either that the
microinjected DNA was degraded or that linear fragments are poor templates
for RNA transcription.
Although the relative amounts of autonomous and nonautonomous P elements
injected into embryos are designed to favor introduction of the non-autonomous element, occasionally transformed strains are obtained which have
acquired an autonomous element as well. This problem could potentially be
eliminated by the use of a transposition-defective P element which was still
able to provide transposition functions in trans to non-autonomous elements.
6348
Nucleic Acids Research
..
..
.s.
...
........
....
. ................
.... .... ......
Figure 4, Analysis of the structure of transferred rosy genes in line
R310.1. The line R310.1 was generated by transformation of the ry42 host
strain with pryl, a rosy transposon plasmid constructed by inserting the 8.1
kb rosy Sal I fragment into the Xho I site of p6.1 (2). DNA was isolated
from the host strain ry42, which carries an apparent point mutation in the
rosy gene (W. Bender and A. Chovnick, personal communication), and from two
individual sublines of line R310.1. Sal I and Hiind III digests of these
DNAs were electrophoresed on 0.5% agarose gels and transferred to nitro-
cellulose filters to make thiree sets of identical filters. Each set of
filters was hybridized with a different 32p-labeled plasmid probe as
indicated: pDm2844S8.5 contains th .1k roy SlIfamn lndi
pBR322. pm12.8 is a pBR322 clone of the 1.5 kb white locus Sal I fragment
into which the P element is inserted in p6.1. The third probe was pBR322
alone. Autoradiograms of the resultant hybridizations are shown. The sizes
of labelled bands are indicated in kb. A diagram of the genomic arrangement
of pryl sequences deduced from the data in the autoradiograms is also shown.
P element sequences are shown fully shaded, pBR322 sequences stippled, rosy
sequences cross-hatched and white sequences unshaded.
However, we were unsuccessful in our initial attempts to construct such a
mutated 2.9 kb P element. Removal of either the first 39 (pTr25.7A1) or
last 492 bp (pir25.7A&2) of the 2.9 kb P element carried on the plasmid pw25.7,
destroyed its ability to provide the required trans-acting functions (Figure
3, experiments 9-11).
Evidence for transposition of a 54 kb P element
For many purposes it would be desirable to introduce large segments of
DNA into Drosophila chromosomes. However, we have observed that 15 kb
6349
Nucleic Acids Research
transposons containing the rosy gene are transferred at only about onethird the frequency of 9 kb rosy transposons, suggesting that transformation
frequency may be highly size-dependent. We have carried out a series of
experiments, reported elsewhere (3), to assay the level and tissue
specificity of rosy gene expression in transformant lines. In the course of
those experiments, we recovered one transformant line which produced
approximately four times the expected level of the product of the rosy gene,
xanthine dehydrogenase. DNA blotting experiments indicate that this line,
R310.1, contains a tandem array of rosy transposons (Figure 4). The relative
intensities of the hybridizations to various bands indicate that there are
four copies of the ryl transposon in this tandem array. This can be most
clearly seen in the hybridization of pDM2844S8.5 to Sal I digests (Figure 4,
leftmost panel). The 8.1 kb fragment derives from the two mutant genomic
copies of the rosy gene (one on each homologue) present in the ry42 host
strain. R310.1 DNA was prepared from flies heterozygous for the chromosome
carrying the transferred ryl transposon DNA. Thus, the equivalent
intensities of hybridization to the 8.1 and 10.8 kb fragments indicate
that the 10.8 kb fragment is present twice in the tandem array. The 18
and 22 kb Sal I fragments both contain a single rosy transposon as well
as genomic sequences adjacent to the tandem array (see diagram in Figure 4).
The structure of this array suggests that it arose by the transposition of
a large composite P element from a tetramer plasmid. The tetramer plasmid
could have been generated by homologous recombination of monomer plasmids
either in E. coli, during growth of the plasmid-containing strain, or in
the Drosophila embryos following microinjection. Such recombination between plasmids to produce multimers is known to occur both in recA- E.coli
(15) and in mammalian cells (16). Alternatively, such a tandem array
could be generated by a normal P transposon integration followed by successive events of homologous recombination between the integrated chromosomal sequences and free circular plasmid DNA as has been observed in
yeast (17). Given the failure to observe homologous recombination between
injected DNA sequences and their chromosomal homologues in Drosophila (2),
we feel that this latter mechanism is highly unlikely in this case. Thus,
although the frequency of transfer may be greatly reduced, these results
suggest that P elements as large as 54 kb can transpose from plasmids into
chromosomal sequences.
6350
Nucleic Acids Research
ACKNOWLEDGEMENTS
This work was supported by grant from the National Institutes of Health
to G.M.R. and A.C.S. We are grateful to Kevin O'Hare and Christine Murphy
for invaluable help with the DNA sequence determination of the Carnegie I
vector.
+Present address: Department of Biochemistry, University of California, Berkeley, CA 94720, USA
REFERENCES
1. Spradling, A.C. and Rubin, G.M. (1982) Science 218, 341-347.
2. Rubin, G.M. and Spradling, A.C. (1982) Science 218, 348-353.
3. Spradling, A.C. and Rubin, G.M. (1983) Cell, in press.
4. Scholnick, S.B., Morgan, G.A. and Hirsh, J. (1983) Cell, in press.
5. Goldberg, D.A., Posakony, J.M. and M1aniatis, T. (1983) Cell, in press.
6. Rubin, G.M., Kidwell, M.G. and Bingham, P.M. (1982) Cell 29, 987-994.
7. Bingham, P.M., Kidwell, M.G. and Rubin, G.M. (1982) Cell 29, 995-1004.
8. O'Hare, K. and Rubin, G.14. (1983) Cell, in press.
9. Levis, R., Bingham, P.M. and Rubin, G.M. (1982) Proc. Natl. Acad. Sci.
USA 79, 564-568.
10. Sanger, F., Coulson, A.R., Barrell, B.G., Smith, A.J.H. and Roe, B.A.
(1980) J. Mol. Biol. 143, 161-178.
11. Heidecker, G., Messing, J. and Gronenborn, B. (1980) Gene 10, 69-73.
12. Vieira, J. and Miessing, J. (1982) Gene 19, 259-268.
13. Sutcliffe, J.G. (1978) Cold Spring Harb. Symp. Quant. Biol. 43, 77-90.
14. Orr-Weaver, T.L., Szostak, J.W. and Rothstein, R.J. (1981) Proc. Natl.
Acad. Sci. USA 78, 6354-6358.
15. Doherty, M.J., Morrison, P.T. and Kolodner, R. (1983) J. Mol. Biol.
166, in press.
16. Foler, K., Wong, E., Wahl, G. and Capecchi, M. (1982) Mol. Cell Biol.
2, 1372-1387.
17. Orr-Weaver, T.L. and Szostak, J.W. (1983) Mol. Cell Biol. 3, 747-749.
6351