Proc. Natl. Acad. Sci. USAVol. 89, pp. 10753-10757, November 1992Genetics

Genomic sequences with homology to the P element of Drosophilamelanogaster occur in the blowfly Lucilia cuprina

(trnpoblk elements/evolutln/Lu-Pl)

HARVEY D. PERKINS AND ANTONY J. HOWELLSDivision of Biochemistry and Molecular Biology, School of Life Sciences, Faculty of Science, Australian National University, P.O. Box 4, Canberra,Australian Capital Territory, 2601, Australia

Communicated by M. M. Green, July 21, 1992

ABSTRACT We have cloned two DNA elements (Lu-P1and Lu-P2) from the Australian sheep blowfly Lucioia cupruaathat are similar to the tsble P element of Drosophilameknogaster in both structure and sequence but have divgfrom it and from each other c ably. Hybrizatistudies indicate that a third reated element probably exists Inanother, as yet unsequenced, done. Neither Lu-P1 nor Lu-P2appears to be active In terms of mobilt, and It Is not knownwhether any t tn-competent pies of other relatedelements occur in the genome of the blowfly. However, theisolation of any P-like seq s from a species outside of thefamily Drosophilidae ailows comparisobs to be made of morewidely divergent P-related elements than has been possiblepreviously. We are unaware of any report of the presencefomultiple P-like family members within a single species. Thediscovery ofLu-P1 and Lu-P2 in the blowfly fuels the posibitythat similar elements may be widespread in insects, andperhaps in other orders of animals.

The transposable P element ofDrosophila melanogaster hasbecome an invaluable tool for genetic manipulation, experi-mental mutagenesis, and germ-line transformation in thisspecies (1, 2); however, it is essentially nonfunctional inheterologous species. As a consequence, the basic and ap-plied opportunities afforded by such germ-line transforma-tion systems (3) have stimulated efforts toward their devel-opment in other organisms based on endogenous elementswith similar properties.The P element belongs to a class of transposable elements

called transposons, which transpose at theDNA level and arecharacterized by the presence of a gene encoding a "trans-posase" enzyme and by inverted terminal repeat sequences.Other elements of this type are common in prokaryotes (4)and plants (5-7) and have been found in some invertebrates(8), but they appear to be relatively uncommon in insects orthe higher orders of the animal kingdom, where most mobileDNA belongs to a class of elements that transpose by way ofa reverse-transcribed intermediate.However, a number of recent observations suggest that

elements of the transposon type may be more widespreadthan has previously been thought. First, P-like elements havenow been characterized from a range of Drosophila species,and a P-like element has also been described from Scapto-myza pallida (9), a more distantly related member of thefamily Drosophilidae. Second, elements of the transposontype have now been isolated from a range of insects andinclude mariner (10) and hobo (11) elements from variousDrosophila species and TECthl from Chironomus thummi(12); mariner-like elements have also been found in thesilkmoth Hyalophora cecropia (13). Finally, it has beenreported that there are regions of significant homology be-

PL72 PL81 PL82 PL83 PL91

X E H xE H X EH EH X E H

FIG. 1. Southern blot of five genomic clones probed with P-el-ement DNA. Multiple hybridizing bands in HindIl digests of PL81and PL83 are due to incomplete digestion; however, genuine hy-bridization occurs to several bands in both digests ofPL82. The filterwas hybridized and washed at 54TC and then exposed to x-ray filmfor 24 hr. Lanes: A, HindIIl-digested A phage DNA; E, EcoRIdigests; H, HindIll digests.

tween the transposase genes of hobo and two plant trans-posons, the Ac element of maize, and Tam3 from Antirrhi-num majus (14), suggesting a distant but common evolution-ary origin.

In this paper we report the cloning of two P-like elementsfrom the blowfly Lucilia cuprina, providing further evidencethat transposons may indeed be widely distributed in in-sects. *

MATERIALS AND METHODSFly Stocks. All fly strains used in this study were obtained

from the Blowfly Genetics Group, Commonwealth Scientificand Industrial Research Organisation Division of Entomol-ogy (Canberra, Australia). They were originally collectedfrom various geographical locations in Eastern Australia,from Queensland in the north to Flinders Island in Bass Straitin the south.

Library Construction. Genomic DNA, prepared from stan-dard wild-type (SWT) embryos as described (15), was par-tially digested with Sau3A1 and, after partial end filling,ligated into commercially prepared half-filled Xho I-cutAGEM-il vector (Promega).Su log and PLmid DNA. DNA was subcloned into

pBluescript II KS vectors (Stratagene) by standard ligationand transformation techniques, using Escherichia coli hoststrain JPA101. Plasmid DNA was prepared by the alkalinelysis method (16).

*The sequences reported in this paper have been deposited in theGenBank data base (accession nos. M89990 and M89991).

10753

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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10754 Genetics: Perkins and Howells Proc. Natl. Acad. Sci. USA 89 (1992)

Southern Analysis. DNA was transferred to nitrocellulose (United States Biochemical). Manipulation and analysis ofin 20x standard saline citrate (SSC) (17) and hybridized in 50 sequence data were performed with MACVECTOR (IBI) soft-mM Hepes/lOx Denhardt's solution/3x SSC/0.1% SDS ware.containing salmon sperm DNA at 50 ,g/ml. Washing was in2x SSC/0.1% SDS. DNA probes were labeled with RESULTS[a-32P]dCTP (1700 Ci/mmol, Amersham; 1 Ci = 37 GBq) bynick translation. We have used an internal sequence of the P element [2.4-DNA Sequencing and Analysis. Exonuclease III was used to kilobase (kb) HindIII-Sal I fragment of pir25.1; see ref. 21]

generate progressively deleted sequencing templates (18). from D. melanogaster as a hybridization probe to search forWhere a protective 3' overhang could not be generated, similar sequences in the genome of the Australian sheepexonuclease digestion was blocked with a-phosphorothioate blowfly L. cuprina. Genomic Southern blots were initiallydeoxynucleotides (19) according to the Promega protocol. hybridized at normal and reduced stringency. Even under theSequencing of single-stranded templates was by the chain- least stringent conditions (52°C) no specific bands of hybrid-termination method (20) as modified for use with Sequenase ization were obtained, although some faint diffuse hybrid-

a 1 GAATTCCAACGCTATTTAGTATATTTAGCCTCTTTTATATGGAGAAAACATATAGTACTGATGTTTCCACCAAAATGAAATTTTATTCATTCAAATTTTATTTGATTTTTACGAAACCAA12 1 CGTTTATCTCAAAACATGGCTTAATATAGTAAAATACTTATAATGTTATTTACAM XNGW GCCTACAACAGTGGTATAG;AAATAAACAAGTGAAGAAGTATAGTCGGGCGTGG241 CCGACCATATGTAATATTATTGTGCTGAAATTCATCACGATATTAGTTTTATATGTGAATTTTGGACATTCAAGTCATATTCTGAAGGWACTTGGTATGGGAGCAGGGTCAAATGAGG3 61 TCCGATCTTTATGAAATTTGGCAAGGTCATC :AAGGCTTGTATAAA ACTTATTTGTGCCGATTTTTTCAA AATACAGGTAATTA ATAAT ATAGCTAAGCCTTTTTCGGGGGG4 81 TTCTGTTGTATGWGGCCTAGGTGAAbAATTGGGCCGATTTTAACCATTTTCGTCCTTGCAAAACAGCGCTTGTACCAAATTTCATCCAATTATATTGAAAATTGCGACCTGTATCT601 TGCGCACAAGGTTTAAGCACATGGACAGTCAGCCAGACGGACGGACAGTCGGACATAGCTCAATCGACTCAGAMAxTGTTTCAAAGGCGATTGGTATACTATAAGGTGGTTATAAGACGT

721 ATATTTGTGTGTGTTACAAACATCAGCACAAACCCAATATACCCTTCCCACTAAAGTGGTGTAGGGTAAAAGGAATAATTCTGTAACTTCTAAACGAGAGAkTTCTGTATATGT

841 AAGTTTCATGAGATCGCAACACAAACGAAAAAGTAGGATCGTTATAAAAsATTTTATATATCCGGTCTCCTAAGCCCTGGTGGGCCCATGAAGTCCAC TTCAGTCGACAAAAM R S Q H X R X S R I V I X I L Y I R S P X P N K A H E V H I Q S G T I K T

961 AAATTTTCTTAGAATTTTATTTTAATTTAGAATGCCAAGTGACATCAATATGGAAACAGATGAATGTTTATTTTTAAGTCGCCCTAGCCCTTCTTGGACATCGAATGTTGATCCGTCTGTN F. L R I L F * F R M P S D I N Mt E T D E C L F L S R P S P S W T S N V D P S V

1081 TCCGAGTGCTGTATTAACGGAAAGTTTTCCCACAGAATATACCAAAACTTATGTAAATCAGACCAWGAZ&&WTAATTAAAATAACATCAGTTATGTTTTTACTAATGTTAP S A V L T E S F P T E Y T X T Y V N Q E T Q T E

1201 TGTT,= =TCCCGATATTTTTGCAGCGGAATCAGCAAAATATGTTGACAAAATCCGCGAACTCGAAAGGGAAAACAAAGAGCTAAATTTTAGACTTAAGATGCAGGTCCACAATTCAAAP D I F A A E S A K Y V D K I R E L F R E N R F L N F R L R M Q V H N S N

1321 TATGGCTCTTCACGGAGTCAATAAAATATTTACTGAkGGGCCAAATTCGAAAGTTGGAAGGATGCAAAAATTGTATG&AAATCCCCCGATATTTCAAGTGCTATTTGCTTACACGCCGCM A L H G V N X I F T E G Q I R K L E G C K X I V N X S P D I S S A I C L H A A

14 41 TGGACCAAGGGCTTACAATCACTTGTAAGAAAGGATTTCCGCTACCTCACGTCAGCACTTTACAGCGCTGGTGTCAGAAAGTTGATGTGCATGAGGCCTTCTTAAAACGTCACTAGGG P R A Y N H L * X X G F P L P H V S T L Q R W C Q X V D V H E G L L K T S L G

15 61 ATTTATGCAACAAGCAACATATTTGTCACAAGATGAGAAAATTTGTGTGCTAGCCTTTGACGAAATGAAGGTAGCGGAAACTTTCGAGTACGATTGTACTAATGATGTCGTAAGACAGCCF M Q Q A T Y L S Q D E X I C V L A F D F M X V A E T F E Y D C T N D V V R Q P

1681 AGCAAAATATGTGCAGGTGGTAATGGCACGTGGTCTAAAGAAATCCTGGAAGCAGCCAGTCTTTTATGATTTCGACTGTAGAATGTCGAAACAAATTTTAGACAGCATAATTTCAGAATTA K Y V Q V V M A R G L R K S N R Q P V F Y D F D C R H S K Q I L D S I I S E L

1801 GTCTAAAGCTGGTTTTCCGGTGCTGGCAATTGTCTGTGATATGGGGCCAACGAATCGTAAGCTGTGGAGTGATTTAGGTGCAACGACAGrIAAZTTATTAAACACATGCCACATGTAAS K A G F P V L A I V C D M G P T N R X L K 5 D L G A T T

192 1 GATACATCACACAGCACATTTAACACATGGCTTACGATTTTTAATTCGGhAGAAgTTAAATTTTCTCTACTCGATCGGTTCCTGTTTTTGCCTAGCAGAAGGGATTTTTACATTCGTTGT2041 AATAATCGGTAATTAAAAATATGAAATTTTTAACAATAQATAATTGCTTTCGTTTTTAAAGATCAAATAAQATTTGATACCAATAAGAACAGCATATTTCATATTTCGGCATTAAGGCTT21 61 TAATTTACTTTATTAAAAATGTCACCZ=WAAAGCCTTGGTTCCCTCATCCAGTGAATACAGAGG^AAATATATACTTTTGTTGATGCTCCACATTTGTTGAAGTTAATCCGAAAC

E K P V F P H P V N T E E X V Y T F V D A P H L L X L I R NH{nd III

2281 CATTATATCGACACCGGCTTGATTTATAACGGCGAACACTTAACGTCACGTACTATCGCGGATGTTTTACAACATACMAAAAAATGTGATACATCTATAACATTTAAGCTTAGTGATGAAH Y I D T G L I Y N G E H L T S R T I A D V L Q H T N K C D T S I T F K L S D E

24 01 CACCTCTTAGTCAAAGGCGCCGMA=3GAATATATTGTATACATATGTATTTCAAAACTTTATTTCAAAATATTIWGAGCzAAAAGGTAAAATTGGCTGCACAGCTATTTTCAAAH L L V R G A G R Q X V X L A A Q L F S N

25S21 TACCACAGCTAGTGCAATCCGACGTTGCTATACTCACGGAAAGGACATCTACAAACCACTCGAGACTGCCGAACTTATTCAAACAGTAAACAACTGGTTTGATGTTGTTAACTCTTCAATT T A S A I R R C Y T H G X D I Y X P L E T A E L I Q T V N N W F D V V N S S M

2641 GAATACGTTCGGTCTCCCTGGCAAGGTIAAGTATGTTATTTTTATTTATAAGTAATTTTAAAGCGTAGTTTTATATCZTAMGAACCATATGGAGTTGATCTGGAAAGCCAGCAAAATAAN T F G L P G K E P Y G V D L E S Q Q N K

2761 GTTGGAACAGATGAACCAACTAATGGCTGTACCAATTATACCAGGTAGGAAAAGTCTGGAACCTTTCCAAAAGGCATTCTAATGACAAACCGGGCCTTGGTAATGCTTTATGACGATGTL E Q M N Q L M A V P I I P G R X S L E P F Q X G I L M T N R A L V M L Y D D V

2881 TAAAAAGTATGACATGACATACATTTTAACAAATCGTCTAAATCAAGACGTCTTGGAGCATTTTTTTGGTGCGATTCGCAGCAAGGGTGGACTGAACGACCATCCCTCTCCACAAGACTTX X Y D M T Y I L T N R L N Q D V L E H F F G A I R S X G G L N D H P S P Q D F

3001 CAAGTTTAGATTAAGAAAATACATATTGGGTAAGTTAATTATATAAGCACACTGGGTAATGCTAGTATAAATAAAAATTTCTACAATAAATTTTTTTTTATTTAATAZIQGCGAAAX F R L R X Y I L A R N

312 1 TACCGAATACTTAAATGGCACTGGAAATGTCGAAAACGATAACACTGAATGGCTCAATTCCACAAATTTTACAATAACAAGTTTGGCAAAAGTTGACGAAGTATCGGAAGAAATCCATCTT E Y L N G T G N V E N D N T E N L N S T N F T I T S L A X V D E V S E E I H L

32 41 TGATTGCCAAGCTCCGATATCGATACATGCCCCGACGGAAGAACAGTTTTTAGACCTAGACGAAACGGATTTAGACGACATTGGTATTCTTCAAGAGGATGCTCTAGAATATATATCCGGD C Q A P I 5 I H A e T E E Q F L D L D E T D L D D I G I L Q E D A L E Y I S G

a1~~~~~~~~~~~~~~~~~S1

33 61 ATATATCATTCGAAAGCATAACCTTGAAGAATACCAATGTCGTGAAAATACGTTTACGTGGGTCGACGAAGTATCGAAAGGTTCATTAAAAAAACCATCAAACTTTTTCCTCCAAAAGATY I I R X H N L E E Y Q C R E N T F T W V D E V S X G S L X X P S N F F L Q X I

34 81 AAAAAGTTTAGAAGTAGTGTTCTACAATG;TCAATGGAAGAGAAATATCGCACAG.AACAAACCTCCGTCAAkCATTTACTTAATGAAAGTTCATATGTAG;ACCTACCTGAkACGCATTAAGCAkK 5 L E V V F Y N V N G R E I S H R T N L R Q H L L N E S S Y V D L P E R I K Q

3601 GTTCTTTTTTAGATGCCGAATATTTTTTCGGATACGGAATTTAAATAAGCACATTAAGGTCCAAA.AACAAGTGCTAATGGGTAAAAAAAGAT.ATAAAACTATATTATAAATGTAAGT

3721

3841

3961

4081

4201

4321

4441

456146814801

F F F R C R I F F R I R N L N K H I K V Q K Q V L M G K K K M I K T I L

ATGTACTATTCACGGATTATTCTATTTTTTGATTTTTTTCTGTCGTATACTTATAGGCTCAGTATTTTTGTAAATTACTACACATCGTATCAACAGCCTATGTCCGATTTTGATAATAGC

TCTAAGGATTACG

TAATTTTGGTCTCCTACCA AAATTTTTAGGACAATTTATCCCCTTTGGTTTTCTAATTCATTACTAACTATTAATACATATTATTCACAAGTATTAATTTTAGTCTTGTCTCTTATT

ATTTAATACTGATCTAAAATTTGACTTCGCTATTTGTTTGTCCAGTAATAAAGATTGAGGTGTACAGAATCTCTAATTGGTGCAGAAAt agCCtACGGCATCAGGATTTACAAATAATTA

TATGAACTTAATGCTTAAAATATATTAACTTTTATTTTATGACAAAATCTTTTAAATTTAAAATTGTTACAAGATGTGACTTATAAGTGTACGTAATAAAGGCGAAGTGTAACGAAAAATCGTATTTTGGACATTTATTTCCTAGGGAAAAAACTTTCGGTTGATACAGCTGTCGCTGzGGTTGAGAATCTTTGACCAGAGCGAATATCTAATTGCCGTGGGAACGATAAGAGTTTGTTTTCATTGGTTGTCATACAAAACAAAATTACATTAGAGAAACAAGACCTATACACAATTAAATAACTCCACTTACTCCAGGTA

FIG. 2. (Figure continues on the opposite page.)

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Proc. Natl. Acad. Sci. USA 89 (1992) 10755

h 1 GAATTCGATTTTCCCAAAATTAAGCATTAATAATOCTTTGAGCTTCATAGATATAAGTTTCATACCAAATTTAAGTTCCTTTAAAATTGTTTTTTTTTTTGGGTATTTGGTTTCGCTATTHind III

12 1 AAGCTTTAAAATAATTATTCGTAATTAAACTTAATTTTAAATAATTTTAATGCCAATTTTATATGTGAAATTAACCTATTGTTTC=LQLCAQGACCATGGTTTCCTAATCCCACAAATG2 R P 3 F P N P T N

241 CGGCTGATAAAGTTTTCGCTTTTGCMGATGCCCCCCACTTGTTAAAGCTAATAAGGAATCACTTGTTAGATAGTGGATTAGTGGTTAATGAACAGCTTTTGACGCCGCGGACAATAGCCGA A D K V F AF A D A P H L L K L I R N H L L D S G L V V N E Q L L T P R T I A

361 ATGTTATTAATCACACATCTGCCTCCGATGTTTCTATAACATTTAAGCTGAATGATGACCATCTGACTGTTAAAGGCGCA ZTAAATACGAACATTTATTATTAATTCGTATCCD V I N H T S A S D V S I T F K L N D D H L T V K G A

481

601

721

ATATTGTTAATTAACATTAjAAAWGGCAAAAGGTAAAACTGGCGGCTCAGTTATTTTCGAACACAACTGCTAACGCAATCCGCCGATGCTTCTCCCTTGGTCTGGATGTCTACCAAGE R QK V K L A A Q L F S N T T A N A I R R C F S L G L D V Y Q

CCACTGAGACAGCGGATTTTATACAGCTTGTAAACGATTGGTTCGACATTCTTAATTCAACGTTAAGTACTTTCGTTTATCCGGGCAAGGAACCATTTGGACATAATTTGGCAACACAAAA T E T A D F I Q L V N D W F D I L N S T L S T F V Y P G K E P F G H N L A T Q

T TTGGACGAAATTACCAAAATCATGGCTTCTCCAATAATACCAAATAA TGCTTGAACCATTCCAAAGGGGCGTCGTCATCACCAACAAAGCATTGATAATGCTTCATGI R X L ,D E I T R I M A S P I I P N R R V L 13 P F Q R G V V I T N R A L I M L H

841 AATATGTTGGCGAAAGATTTAATATGCAATACATATGCACCAGCAGGCTCAATCAAGACGTTTTGGAACACTTCTTTGGTGCCCTTCGTAGCAAAGGGGGCTTAAACGATCATCCGTCGCE Y V G E R F N M Q Y I C T S R L N Q D V L E H F F G A L R S K G G L N D H P S

961P K D F K Y R M R K Y I L A R

1081 ACACCGAACACCTTAGTGGTACTGGTAACGTTGGCTACGACACCACCGAATGGCTCAGCACTTCAGACACCAATGTTTCTTTCATTGAAAGTGAAACAAAACTTTCCAATGAGCCGCTTGN T E H L S G T G N V G Y D T T E W L S T S D T N V S F I E S E T K L S N E P L

1201 AACTGGAAGAAATTTCAGAAGAACCCCTAACAAATGTACTTGACGACCTAGATGGATCGAATTTTGATGATATGGGTACTCTCCAAGTTGATGCTTTGGAATACATTGCTGGCTACACTAE L E E I S E E P L T N V L D D L D G S N F D D M G T L Q V D A L E Y I A G Y T

Hind III1321 TTCGAAAGCTTAAACTTAGAGAATATGAATCTC ATTCGTATACTTGGGTTGATCAAGTATCAAAGGGTATGCTGATTAAGCCATCTTCAGAGTTTCTCGAACAATTGAAGAAAC

I R K L K L R E Y E S Q R N S Y T N V D Q V S K G M L I K P S S E F L E Q L K K

1441 TGGAAGCTGTGTTTTACACTTTAAATGGCTTGGAAATATCGCACTGCGACAACATCCATCAACGTTTAGTGGAACGAAGTAACAACATTGACCTTCCAGACGTTGTTAAATGTTTTTTCTL E A V F Y T L N G L E I S H C D N I H Q R L V E R S N N I D L P D V V X C F -F

1561 TTAAATGTCGCATATACTTCAGAATCCGCCATTTGAATCAAAGACTCAAAAAACAAAAATGGAACAAGGAAGTAAGCAAAAAGATGCTTAAAATAAAGCTTTAATAACTGCAAGTAAGTTF K C R I Y F R I R H L N Q R L X K Q X W N X E V S K K M L K I K L

168118011921204121612281

ATTACCTTTACTTAAAAACATTTCTTAACTTAATACATATTCTTTCCTTTAATTCATAGGCTTATGCAGAAAAC CTTGCCCTTCTACACTTTACAAATTGCTTTACGTTATCAAAACCCATTGTCCCTGAGTTGTGTTTGGAIACATGTTTGGACCGAAA&ATACCTATGGCAAGTATGTAATTAACAGTTAAAAATAAAATCTTTTACATATTAATGTTTTTCTTTGAAATATTCTGATTTCAGAAACAACTTTTGTTTGGGTCTACGTGATCCTGCCGTTAATCGTCTTACTTTATAGTTCGGGGAIATGTTAAGCATTTGTTTAAGTCTCTTGCCTTCAGAACAAAACCGGGAGALCACTACATTTGAAAATGAGTTTCATTTGTTATCACCAGTACACCCTCTGTAGCCACAGTTTTCCACCCTCCACCTGGATTGTTTACAATMAGCCAAAAGGALCCAGCCGAGAATAAGTGMATAAGTCCAGTTTCCATATTTCTTGGAAAAACACTATTTTTTTTACTAAATATATGTCAAACTTAAATAAATTGCTAACACATTTTTTATTTATTTGCATTTTTATTTATTCTTTACTCAAATATTATTTTT

FIG. 2. Nucleotide and derived amino acid sequence for Lu-P1 (a) and Lu-P2 (b). Splice consensus sequences are indicated by singleunderlines; inverted repeats mentioned in the text are shown by double underlines. Restriction sites used to generate internal probes (see textand Fig. 4) are shown above the sequence. Also indicated by underlining is the 495-base-pair (bp) region of repeated sequence mentioned inthe text. A short stretch of ambiguous sequence from PL83 (positions 4529-4534) is indicated by lowercase letters. The sequence from PL83(Lu-P1) was obtained by cloning a 5.6-kb EcoRI fragment as overlapping 3.6-kb EcoRI-Sal I and 2.8-kb Xho I-EcoRi subclones, which weresequenced from both strands. The sequence ends about 550 bp upstream ofthe second EcoRI site. The 4.0-kb EcoRI fragment from PL91 (Lu-P2)was sequenced from one strand only for the first 2.3 kb.

ization was observed that appeared to correspond to satellitebands visible in the gel (results not shown). However, workin our laboratory aimed at the cloning of eye color genes (22)and retrotransposons from L. cuprina by cross-species hy-bridization has shown that, due to the amplification of theparticular sequences, appropriate clones can often be iso-lated from genomic libraries despite a lack of clear signals ongenomic Southern blots. Consequently a L. cuprina genomicDNA library was screened with the P-element probe underconditions of low stringency (54°C). From a screen of 35,000clones (approximately one genome equivalent of L. cuprinaDNA), about 30 positively hybridizing clones were detected.Five of these were plaque-purified and analyzed further.Preliminary restriction mapping indicated similarities be-tween only two of the five clones (PL72 and PL81), and Fig.1 shows the results obtained when DNA from all five wasSouthern blotted and probed with P-element DNA. Distinctbands of hybridization occur in all cases and the intensity ofthe signal varies amongst the five clones, with clone PL83having the strongest.DNA sequence was obtained from the P-hybridizing region

of PL83 (Fig. 2a) and later, for comparison, from a smallerregion of PL91 (Fig. 2b). These obviously related but differ-ent elements have been named Lu-P1 and Lu-P2, respec-tively. Analysis of the sequences produced a number ofopenreading frames and amino acid sequences with striking sim-ilarity to those of the P element of D. melanogaster. Align-ments based on this similarity have enabled some structuralfeatures of Lu-P1 and Lu-P2 to be deduced (Fig. 3a) and abasis on which to assess the similarities and differencesbetween these elements. While sequence from Lu-P1 appearsto cover the corresponding full length of the Drosophila Pelement, amino acid homology does not begin until part wayinto the putative exon 1, then continues to the end of exon 3.No homology is detected between exon 0 of the P element

and the putative exon 0 of Lu-P1. The incomplete sequencedata obtained for Lu-P2 begin in the second intron, where-after homology can be traced throughout exons 2 and 3. Theupstream part of Lu-P2 is presumably present in the adjacent6-kb fragment, which in EcoPJ digests remains attached tothe right arm of the vector and hybridizes to the P-elementprobe (see Fig. 1). Interestingly, in Lu-P1, "exon 2" isinterrupted by two small introns not present in theP element,while Lu-P2 possesses only one of these additional introns.Otherwise the insertional position of intron sequences for thethree elements is identical. Comparisons of the exon 2sequences from p7r25.1, PS18 from S. pallida (9), Lu-P1, andLu-P2 (summarized in Table 1) show that pir25.1 and PS18are more closely related than Lu-P1 is to Lu-P2, and thatdespite the -34% difference between them the Lu-P elementsare about equally divergent from both pir25.1 and PS18.

Despite the availability of 850 bp of sequence upstream ofthe proposed exon 0, and nearly 1200 bp downstream of theend of exon 3 of Lu-P1, no good evidence for the presence ofinverted repeat termini could be found. (The 31-bp perfectinverted terminal repeats of the P element lie within 200 bpof the start and end of the transcription unit.) The bestcandidates are a 10-bp sequence (TCGGACATAG) and a12-bp sequence (AGAGATTCTGTA) indicated in Fig. 2a;interestingly the 12-bp repeat shows some similarity to the12-bp inverted repeat (CAGAGAACTGCA) of the hobotransposable element of D. melanogaster (11). However,neither of these inverted repeats is flanked by a direct repeat(transposable elements characteristically produce a "targetsite" duplication as a result of insertion) and neither ispresent in the 3' sequences of Lu-P2. In the case of the 12-bprepeat, the situation is further obscured by the presence of astretch of DNA between the end of exon 3 and the down-stream 12-bp repeat which appears to encode a series of 24tandemly repeated 7-amino acid motifs similar to those

CAAAGGATTTCAAATATCGAATGAGAAAGTACATCTTAQQTA=TTCAAACTGATTTTATTTCATTGTTATATTACATTAAATTGTTTTTTTTTTTTGTTATAACAATZATWCTAGAA

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a pit25.1 2

* *

Lu-PI =

Lu-P2

b pc25.1 SK WFSHPADDHHLI VFSDTPHLIKLVRNHYVDSGTINGKKLTKK[K QPS18 SK WFSHPTDDNSKIJVFSDTPHLI KLVRNHYVDSGFT NGKK LTjKTTyQLu-P1 EKPWFPHPVNTEEKVYTFVDAPHLLKLIRNHYIDE3G IYNGEHLTSRTIALu-P2 RPFWF PNUPTNAADKVAF A DAPH LLK LI RNHLLDSG VVEQ LLTP R A

V (Lu-P 1 and Lu-P 2)

pW25.i EALHLCNKSDLSI LFKINEN INVRSLAK|QKVKLTLFSNTTASSIRRCPS18 QTLNHCAKSDVSI LFKISENIH NIVIRSLEKIQKVKLAITQLFSNTTASSIIRRCILu-P1 DV LQHTNK DTS ITFKLSDEH LLVKGAGRQKVK LAAQLFSNTTASAIIRRCILu-P2 DVINHTSA D SI FKLNDD[DjTMKGAERQKVK LA|AQLFSNTTA|NAII RjRC

V (Lu-P 1 ody)pc25.1 YSLGYDIENJT|T ADFFK MNDWFDIFJD| F TF1SNCIECSQPGK QEDIOPS18 YSLGYDVEN[CETS LFKNLINDWFD|VF|NSIKLTIANF IOST Q|PYG|K Q|L|DF|Q|Lu-P f7RGIKIDII YKPLETA LIQTVINE8 WFDIVVINSISITI-FGLPGKEPYGVDIL ESIQ|Lu-P 2 L JV Y BT AF I Q[gVIN D W F DII L"T -FV Y P G KEFH NjA T

P25.1 NDI LNRMSEIMRTGILDK4P[RL-PFQKGG IVNNASLDGLYKILQENF SMQPS18 RDVLEKMTQLMCSNILGKSQKL-PFQKG IIVNNASLDGLFIYLKDKYNIM|ELu-P 1 QNKLEQMNQL|AVPIIPG SLEPFQKG ILMTNR| ALVM|L|YDDVK-KY DMTLu-P 2 IK KULDEITKI ASPUIIPNNKVLE VTVVITKIATIMtjHEJVGERF F JQ

p251 [IYILTSRLNQDI E SMfRRGQF[DHP|TLQFKYRLRKYIIPS18 YjLTSRLNQ DI E8FFG MRSRGGQFDHPTPLQFKYRLRKYLILu-P' IYILTE8RLNQDIV LEHFFGAI|RS|K|GG|LN|DHP|S|P|QD|FKE]RLRKYILLu-P 2 NVLEHFFG ALRSKGGLNDSUPKD L

present at the 3' end of the largest subunit of RNA polymer- between thenase II (23, 24); although possible, it seems unlikely that these apart from Liare part of the Lu-P1 structure. Hence the ends of these third elementelements remain undefined. ently phylogeThe presence of single stop codons in both exon 0 and exon but, given the

1 of Lu-P1 indicates that it cannot produce an active trans- Notably thereposase. While there are no obvious defects in Lu-P2, the lack under these bof the entire sequence prevents us from speculating on its trating the dihpotential activity. To gain some indication of whether thereare transposition-competent copies of either of these ele-ments in the genome of the blowfly, we probed genomicSoutherns of five different wild-type strains of L. cuprina The discoverwith internal sequences from each element. The results (Fig. considerable4a) indicate that there is only a single copy of Lu-Pl, which isolated fromappears unchanged in all five strains, whereas Lu-P2 is and because,apparently present in 5-10 copies, again with little difference related but sin the pattern of hybridizing bands between strains. This species has nindicates that neither of these elements is transpositionally One of theactive; similar findings were reported by Capy et al. (26) for given their dithe mariner element in Drosophila sechellia. The same degree of inteprobes from both Lu-P1 and Lu-P2 were also used to probe and preservalDNA from the five PL clones to determine the relationships helix-turn-he

of the transpsTable 1. Matrix of the proportion of nucleotide and amino acid would argueasdifferences between the various P-like elements

pw25.1 PS18 Lu-P1 Lu-P2pi25.1 0.219 0.496 0.475PS18 0.219 0.533 0.492Lu-P1 0.410 0.426 - 0.354Lu-P2 0.429 0.436 0.327

Values have been determined from the exon 2 region of eachelement only. The P-element sequence used was pir25.1 (21), andPS18 is the Scaptomyza pallida P-like element (9). Nucleotidedifferences are shown in roman type, and amino acid differences initalics.

cBecome immatained in theperhaps morrepeat terminniscent of th4Drosophila siof transpositielements alsoa mechanismthe productioP-element rel

FIG. 3. Structural and amino acid sequencesimilarities between the P element (pir25.1), Lu-P1,and Lu-P2. (a) Schematic representation of thestructures ofthe three elements. The 31-bp invertedrepeat termini of the P element are represented byarrowheads; stars mark the positions ofstop codonsin putative exons 0 and 1 ofLu-P1. Minor variationsin exon size occur among the three elements; how-ever, the main structural differences are the smallintrons interrupting exons 2 of the Lu-P elements.(b) Amino acid alignments for the exon 2 regions ofthe P element, PS18 (a P-like element from Scap-tomyza; see ref. 12), Lu-P1, and Lu-P2. Dashesindicate gaps introduced to optimize the alignment,and the positions of introns in Lu-P1 and Lu-P2 aremarked by arrowheads. Identical amino acidsshared by at least three of the four sequences areboxed. Similar levels ofhomology are also found forexons 1 and 3.

n. The results shown in Fig. 4b indicate that,Lu-Pl and Lu-P2 (present in PL82 and PL91), at also exists (in PL72 and PL81) that is appar-enetically intermediate between the other two: stronger signal, more closely related to Lu-P2.e is no hybridization between Lu-P1 and Lu-P2ugh-stringency conditions (630C), further illus-Lstinctness of these two elements.

DISCUSSIONry of the Lu-P sequences in L. cuprina is ofinterest because they represent P-like elementsa species outside of the family Drosophilidae,as far as we are aware, the isolation of threestrongly divergent P-like elements from onelot been reported before.most interesting aspects of the Lu-P elements,ivergence and apparent immobility, is their high-grity (e.g., absence of deletions or duplicationstion of splice junction sequences). In Lu-P1 theelix motif and two of three leucine zipper motifsosase also appear to have been preserved. Thiseither that the Lu-P elements have only recentlyobiie or that they have been selectively main-genome as non-mobile elements. The latter is-e likely given the apparent lack of invertedii (at least for Lu-P1), a situation that is remi-e P-like elements of Drosophila guanche andubobscura, for which a role in the suppressionion has been suggested (27, 28). If the Lu-P)have some regulatory function, they may act bydifferent from that of the P element. Certainly,n of a protein product equivalent to the 66-kDapressor (29) would require some read-through

Proc. Natl. Acad Sci. USA 89 (1992)

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Proc. Natl. Acad. Sci. USA 89 (1992) 10757

aLu-PI probe

X 1 2 3 4 5

44

.......

am~ ~

Lu-P2 probeX 1 X2 3 4 5 A

Jn

bLu-P1 probe

X 72 81 82 83 91

O-

*V,f..._i

Lu-P2 probe

X 72 81 82 83 91

FIG. 4. Southern blots of genomic and cloned L. cuprina DNAprobed with the two Lu-P elements. (a) Genomic DNA from fivewild-type strains of L. cuprina digested with EcoRI and probed at63°C with internal fragments of either Lu-P1 or Lu-P2. The Lu-P1probe was a 1-kb HindIII-Sal I fragment, and the Lu-P2 probe a1.2-kb HindIII fragment, both from regions spanning exons 2 and 3(see Fig. 2). The presence of two bands in each digest probed withLu-P1 appears to be due to polymorphisms between individual flieswithin each strain [the DNA used for these blots was prepared by themethod of Lifton as described by Bender et al. (25), using five adultflies per preparation]. The slight variability in banding pattern withLu-P2 is also likely to be due to polymorphism; however, the lessdistinct bands may represent cross-hybridization to other relatedelements. Lanes: A, HindIII-digested A phage DNA; 1, LBB; 2, SWT(standard wild type); 3, FBWT; 4, Weller; 5, Llandilo. (b) DNA fromthe five PL clones digested with EcoRI and probed as for a.

mechanism to permit translation of the stop codons in exons0 and 1 of Lu-P1.The extent of the sequence differences between the Lu-P

elements, the Drosophila P element, and the P-like elementsof Scaptomyza suggests that these sequences have beenevolving independently for a substantial period of time, andcomparisons of the nucleotide sequences from the variouselements fit well with the phylogenetic relationships of therespective host species. It therefore seems likely that P-likeelements were present in the common ancestor ofDrosophilaand Lucilia and have been evolving independently since theirdivergence, estimated to be about 110 million years ago (30).This suggests that the apparent horizontal transmission oftheP element from D. willistoni to D. melanogaster (31) may bean isolated incidence of such transfer and that transmissionof P-like sequences occurs primarily by normal verticalinheritance. Certainly our data provide no supportive evi-dence to suggest that lateral transfer has occurred betweenthe Drosophila and Lucilia genera in the recent past.

In conjunction with the currently known distribution ofP-like elements, these results suggest that the P-elementfamily is much more diverse and widespread than previouslythought and indicate that further work on the distribution ofsuch elements is warranted. The sequences ofLu-P elements,when taken together with the sequences of the P element ofDrosophila and the P-like elements from Scaptomyza, pro-vide us with a better idea of the highly conserved regions ofthese elements, which enables the design of oligonucleotideprimers to use in searching for similar elements in otherspecies.

We thank P. East and P. Atkinson for comments on the manu-script. This work was supported by a postgraduate scholarship fromthe Australian Wool Corporation.

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