Proc. Nati. Acad. Sci. USAVol. 87, pp. 1686-1690, March 1990Genetics

"Site-selected" transposon mutagenesis of Drosophila(P element/polymerase chain reaction/sib selection/singed/transposon mutagenesis)

KIM KAISERt AND STEPHEN F. GOODWINDepartment of Genetics, University of Glasgow, Church Street, Glasgow G11 5JS, Scotland

Communicated by Seymour Benzer, December 4, 1989 (received for review September 11, 1989)

ABSTRACT Despite the wide range of techniques that canbe brought to bear on the study of basic processes in Dro-sophila, there are still deficiencies in our armory. One of theseis an ability to select mutants in cases where the gene is knownand has been cloned, but where we are ignorant of theassociated phenotype. We describe here a solution to thisproblem as applied to a model system, the singed (sn) locus.Our method is a combination of classical genetics and molec-ular biology: sib selection plus the polymerase chain reaction.We have used the method to isolate rare individuals withP-element-induced alleles of sn merely by recognition of theDNA structures induced at the locus by transposon insertion.Phenotypic criteria were used only retrospectively to verify ourdiagnoses. There are obvious implications of this technique forthe mutagenesis of other organisms.

There is a rapidly growing number of loci in Drosophila forwhich cloned genes are available but for which we neitherhave phenotypic information nor can be sure what kind ofscreening procedure would be likely to provide it. ManyDrosophila genes, for example, have been cloned by homol-ogy with those of other organisms (1); other genes have beencloned on the basis that they exhibit an interesting pattern ofdifferential gene expression when two tissues or develop-mental stages are compared (e.g., ref. 2). We can sequencesuch genes, study their patterns of expression in detail, andgenerally adduce a wealth of circumstantial evidence insupport of a belief that they play an interesting role in thebiology of the fly. Without a phenotype, however, we areseverely handicapped in the scope of our investigations,being unable either to demonstrate function by a study ofindividuals in which gene expression has been perturbed orto make use of the most important characteristic of the fly,the wealth of accumulated genetic knowledge. We describehere a simple, fast, and general solution to this problem asapplied to a model system, the singed (sn) locus.The logic of our method is as follows: The polymerase

chain reaction (PCR) allows the detection, even within smallamounts of DNA, of specific DNA sequences and configu-rations of sequences (3). Thus, should a transposon liewithin, or close to, any genetic locus we can detect thischromosomal state by PCR between one primer specific forthe transposon and one primer specific for the genetic locus.Moreover, due to the extreme sensitivity of the PCR, we candetect one individual with such a chromosomal configurationwithin a large excess of flies that do not carry a transposonat the locus. We have exploited these findings to detect andisolate rare individuals (within a population of mutagenizedflies) that carry a transposable P element inserted into a targetgene, in this case sn. Rather than sacrifice the flies them-selves we have carried out PCR on eggs laid by mutagenizedflies. Then, by subdivision of the population in successivestages, we have been able to "home-in" on a rare fly with the

desired characteristics merely by identifying at each stage thesub-population in which at least one individual is laying eggswith the appropriate chromosomal configuration.

MATERIALS AND METHODSFlies and DNAs. CFL3 and CFL5 (sn strains, ref. 4),

Birm-2; ry5 and w; SbP[ry'A2,3]/TM6 (5), ir2 and Harwich(strong P strains; ref. 6) and P-cytotype balancer strains wereobtained from J. Paterson and K. O'Hare (Imperial Collegeof Science, Technology and Medicine, London), as was thesn9 DNA clone. Consult ref. 7 for a description of the geneticmarkers and fly strains not described here.

Mutagenesis. Both strategies began with a cross involving20 males and 50 females. Batches of 40 F1 males were matedwith 100 females and transferred to new bottles regularly toavoid overcrowding. This generated more than sufficient F2females for our experiments.

Strategy A. F1 male progeny of ir2 males and Oregon R(M-cytotype) females were mated with P-cytotype FM7/Basc females. Batches of 100-200 F2 virgin females (sn?/FM7or sn?/Basc) were placed in small cages with 50 youngOregon R males, matured for at least 24 hr, and thereaftereggs were collected for 24 hr on yeasted grape juice agaroseplates (all at 200C).

Strategy B. Birm-2; ry5-' males, homozygous for a secondchromosome bearing 17 defective P elements, were mated at16'C with w; Sb P[ry+A2,3]/TM6 females (5). P[ry'A2,3]provides a nonmobilizable source of transposase. F1 maleprogeny (w/Y; Birm-2/+; Sb P[ry+A2,3]/ry5os) were matedwith Oregon R females at 18'C. Approximately 900 Sb+ F2virgin females were placed in a cage at 20'C with 200 OregonR males and matured, and their eggs were collected as above.DNA Isolation. Eggs were washed with tap water and

homogenized in 500 ttl or more of 10mM Tris HCl, pH 7.4/10mM EDTA/60 mM NaCl/0.15 mM spermine/0.15 mM sper-midine/0.5% Triton X-100. The homogenate was extractedonce with phenol/chloroform [1:1 (vol/vol)], ethanol-precipitated, and taken up in an aqueous solution of RNaseA (10 ,ug/ml). After a 30-min incubation at 37°C, two furtherphenol/chloroform extractions, one chloroform extraction,and a further precipitation, the DNA was taken up in 100-1000 ,u of 10 mM Tris'HCl, pH 7.8/1 mM EDTA. Approx-imately 1 ,ug of egg DNA was obtained per F2 female. AdultDNA was prepared essentially by the same method.PCR and Analysis of Amplified DNA. HPLC-purified oli-

gonucleotide primers were provided by the Oswell DNAService (Edinburgh University). Their approximate locationsare shown in Fig. 1. [PI is 5'-CGACGGGACCACCTTAT-GTTATTTCATCATG, derived from the 31-base-pair (bp)terminal inverted repeat sequence of the P element (8). [snA]is 5'-GTCTGTCGTCACACCCTTCACTTCGCC, derivedfrom the sequence beginning 150 bp upstream of the right-hand EcoRI site of Fig. 1 (within the sn gene). [snB] is

Abbreviation: PCR, polymerase chain reaction.tTo whom reprint requests should be addressed.

1686

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Proc. Nati. Acad. Sci. USA 87 (1990) 1687

[PI P-element [PI

snA].__

EcoRI

FIG. 1. Promoter-proximal and 5' flanking region of the sn locusin the Oregon R and w; SbP[A2,3]/TM6 stocks used in this study (4).Several independent sites of P-element insertion have been docu-mented within the hot-spot region. sn9 is a cloned EcoRI fragment.PCR primers are shown in square brackets.

5'-GCTGCCCTTCTAATCCTCGCGTCC, derived from thesequence beginning 11 bp downstream of the HindIII site ofFig. 1 (5' to the sn gene). Egg DNA (-0.2 ,ug per 10 F2 femalesor 2 pug per 100-900 F2 females) was amplified in 20 A.l of 50mM KCI/10 mM Tris-HCl, pH 8.3/1.5 mM MgCl2/0.1%gelatin/200 ,uM dATP/200 puM dCTP/200 ,M dGTP/200AMdTTP/the two primers, each at 1 ,uM/1 unit of Thermusaquaticus DNA polymerase (Cambio, U.K.). The samples,overlain with 20 ,.l of mineral oil (Merck) in 1.5-ml micro-centrifuge tubes, were placed in an Intelligent Heating Block(Cambio); heated to 940C for 3 min; taken through 30 cyclesof 1 min at 500C, 3 min at 700C, and 1 min at 940C; and finallyincubated for 5 min at 50'C and for 20 min at 700C. Afteramplification, the PCR mixtures were separated in 1.5%agarose gels and alkaline-blotted to Hybond-N membranes(Amersham) as instructed by the manufacturer. The mem-branes were prehybridized and hybridized at 650C in 6x SSC(lx SSC is 0.15 M NaCl/0.015 M sodium citrate, pH 7.0)/0.2% bovine serum albumin/0.2% Ficoll/0.2% polyvinylpy-rollidone/1% SDS/0.005% sodium pyrophosphate/dena-tured herring sperm DNA (0.1 mg/ml). Hybridization was for3-12 hr with nick-translated sn9 DNA (up to 106 dpm/ml) (seeFig. 1). The membranes were washed briefly in 2x SSC atroom temperature, twice for 15 min in lx SSC/0.1% SDS at650C, and twice for 10 min in 0.1x SSC/0.1% SDS at 650C.The membranes were blotted dry, covered in Saran Wrap,and exposed to Cronex film (DuPont) for at least 2 hr, wherenecessary with intensification screens.

RESULTSP-Element Mutagenesis. Transposable P elements can be

mobilized by mating males bearing P elements with femaleslacking them. This is the basis of strategy A. Recently,crosses between flies with many defective but mobilizableelements and flies that provide a nonmobilizable source oftransposase from an engineered P element have been used (9)to achieve more efficient mutagenesis (strategy B). In bothcases, since the transposition events of relevance occurindependently in different germ-line cells of the F1 progeny,one further generation is required to obtain flies with thecapacity to breed true with respect to a newly mutagenizedlocus. P elements are bounded by 31-bp inverted repeats(Fig. 1). Thus, regardless of the orientation of a P element ata given locus, a PCR primer derived from this 31-bp se-quence, together with a locus-specific primer can be used toamplify locus-specific DNA.

Detection of P Elements at the sn Locus. The sn gene islocated on the X chromosome. Mutant alleles of the gene,readily produced by P element mutagenesis, result in more orless severe defects in bristle morphology. sn has been cloned,characterized, and sequenced, and mutants are available in

which the precise location of the relevant P element is known(ref. 4; J. Paterson and K. O'Hare, personal communication).Fig. 2 shows that amplification products of the expected sizesand hybridization characteristics are produced when mutantDNA is amplified with [P] and [snA] primers. "Spurious"amplification also occurs in both sn and sn' DNAs. Furtherevidence of spurious products is shown in Fig. 3. They aregenerated in all strains tested, even when the primers areused singly (but not in their absence or in the absence ofDNA; data not shown). Moreover, their generation is inde-pendent of the number ofP elements in the genome (Fig. 3)and of the PCR annealing temperature (in the range 50-70'C;data not shown). We are ignorant of their origin, and theirintensity varies from experiment to experiment. Since theyare not detected by hybridization and do not confuse ouranalysis, we do not consider them further.Can a Rare sn Fly Be Detected Within a Population Of sn'

Flies? Serial dilution ofCFL5 DNA into Oregon R DNA, andamplification as above, shows that the equivalent of 1 fly in1000 can be detected easily by hybridization, if with somedifficulty in the stained gel (Fig. 4). Emboldened by thisresult, we proceeded to generate mutant flies by strategy Aand to screen for F2 females that were laying eggs with a Pelement insertion at the sn locus. Approximately 4500 fe-males were screened in batches of 100-200, from which wedetected and isolated three sn lines (snGl-3; see below).However, although we had naively assumed that the maleprogeny of a selected F2 female would be of only two types,those with a balancer X chromosome and those with an snphenotype, this was not the case. Among the males that hadinherited theirX chromosome from their F1 male grandparentwere both sn and sn' flies in various ratios. Probably this wasthe result of remobilization of P elements even when the F1males are mated with P-cytotype females (6). Consequently,we opted for an alternative approach, strategy B, that al-lowed us to select for F2 females that had not inherited asource of transposase.

Isolation of sn Mutants by Strategy B. One or more specificamplification products of the anticipated size can be detectedin the DNA of eggs laid en masse by a population of 900 F2

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FIG. 2. P-element-dependent amplification ofthe sn region. As inthe experiments described below, amplification was with [P] and[snA] primers and the probe was sn9 DNA. sn mutants CFL3 andCFL5 have P elements at different sites within the hot spot of Fig.1 (4). The predicted sizes of the amplification products are 400 bp and378 bp, respectively. A CFL3 product of inappropriate size, notinvariably found, also hybridizes. n(123 bp) indicates integral mul-tiples ofa 123-bp DNA size marker (BRL). (Left) Ethidium bromide-stained gel. (Right) Autoradiograph.

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Genetics: Kaiser and Goodwin

1688 Genetics: Kaiser and Goodwin

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FIG. 3. Specific and spurious amplification products from vari-ous Drosophila melanogaster strains. [P] and [snA] primers, eitheralone or in combination, were used to amplify DNA isolated from thesn mutant CFL3, from the sn' and M-cytotype laboratory strainsOregon R (OR), Canton S (CS), and M56i (ref. 10), and from strainsthat carry P elements, Birm-2 (Birm; 17 elements) and Harwich (Har;'40 elements). Although amplification products were seen in all

cases (Upper), only the bona fide product of CFL3 DNA wasdetected by hybridization (Lower). (Upper) Ethidium bromide-stained gel. (Lower) Autoradiograph.

females produced by strategy B (Fig. 5, lane 1). The femaleswere 4 days old when this result was obtained: 1 day tomature, 1 day to collect eggs, and 2 days to isolate, process,and detect the DNA. Immediately thereafter the 900 femaleswere divided into nine batches of 100, each batch was placedin a small cage (here as in further subdivisions with anappropriate quota of the accompanying males), and eggcollection was continued during day 5. Thus, on day 7 we had

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FIG. 5. Detection of P-element insertions at the sn locus afterP-element mutagenesis. Lanes: The first lane (labeled 1-9) representsDNA from eggs laid en masse by a population of 900 F2 femalesproduced by strategy B. The next nine lanes represent DNA fromeggs laid by batches of 100 F2 females after 9-fold subdivision of theoriginal population of 900. Females carrying P elements at the snlocus were inferred in batches 1, 2, and 6. (1:1000) and (1:10) arealiquots of the dilution mixtures shown in Fig. 4.

narrowed down the population to three batches of 100females, batches 1, 2, and 6 (Fig. 5). In turn, each of thesethree batches of 100 was further subdivided 10-fold, and eggcollection was continued during day 8. On day 10 we hadnarrowed down the population to three batches of 10, batches1.9, 2.7, and 6.5 (Fig. 6). At this stage single females, in thecompany of younger males, were placed in individual vialsand allowed to generate progeny. An example showing thata single vial (1.9.3) within batch 1.9 generated progeny withthe anticipated chromosomal configuration is shown in Fig.7. Vials 2.7.2 and 6.5.10 were similarly selected (data notshown). Consistent with our diagnoses, phenotypically snmales were indeed found in the three selected vials. Bycontrast with strategy A (see above), sn males comprisedapproximately half of the male progeny in each vial. Selectedfemales had, therefore, bred true in the absence of a sourceof transposase. Fig. 8 summarizes the results of both muta-genesis strategies: sn males inherited the capacity for specificDNA amplification (their sn' brothers were discarded with-out further testing). Fig. 9 is consistent with disruption of thesn locus in snGl-3 having occurred within the EcoRI-HindIIIfragment that spans the hot spot shown in Fig. 1. Similar data

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FIG. 4. A reconstruction experiment to model the detection of arare fly with a P element at the sn locus within a population of sn'flies. Oregon R (OR) and CFL5 egg DNAs were mixed in the ratiosshown and amplified. Bona fide amplification products are easilydetected by hybridization down to a dilution of 1:1000. (Left)Ethidium bromide-stained gel. (Right) Autoradiograph.

FIG. 6. Further subdivision of batches 1, 2, and 6. Femalescarrying P elements at the sn locus were inferred in batches 1.9, 2.7,and 6.5. We suspect the faint band in lane 6.9 to be due to crosscontamination. Beware! (Top) Subdivision of batch 1. (Middle)Subdivision of batch 2. (Bottom) Subdivision of batch 6. Each panelis an autoradiograph of a blot probed with sn9.

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Proc. Natl. Acad. Sci. USA 87 (1990)

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Proc. Natl. Acad. Sci. USA 87 (1990) 1689

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FIG. 7. An example of further subdivision of batch 1.9. DNA wasisolated from half of the female (F3) progeny produced by eachmember of the batch. An amplification product diagnostic of aP-element insertion at the sn locus was detected in the daughters ofthe female 1.9.3. (Upper) Ethidium bromide-stained gel. (Lower)Autoradiograph.

(not shown) have been obtained for snG4-6. During thesibling-selection procedure, unselected batches of femaleswere also investigated for their ability to generate sn progeny.

None were so able.Genetic tests of our new sn mutations show them all to be

allelic with that carried by CFL3. In addition the experimen-tally induced chromosome configurations segregate with theX chromosome since they are not transmitted by the femaleprogeny of a cross between sn males and compound Xfemales (data not shown). However, truly unequivocal dem-onstration that our mutations arose by insertion ofP elementswould require cloning and characterization of the sn locusfrom a mutant strain.

DISCUSSIONWe have described the application of "site-selected" muta-genesis to the isolation of sn mutations. The method issimple, fast, and would seem to be general. Very little needbe known about a gene other than a minimum of DNAsequence, and the only phenotype one has to score is a bandon a gel. Furthermore, insertion of the transposon is detectedin the heterozygote, at least one generation before themutation is uncovered. Frequencies of P-element mutagen-esis are somewhat locus-dependent (11), sn being a relativehot spot. Nonetheless, by using the more efficient of the twostrategies described here, only on the order of 104 mutagen-ized flies would be required for there to be a P element

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FIG. 8. Analysis of DNA prepared from sn males. snGl-3 wereproduced by strategy A; snG4-6 were produced by strategy B.Different sizes of amplification product are indicative of differentsites of insertion within the hot spot of Fig. 1. Hybridization (notshown) reveals only the predominant amplification products shownin the figure.

OR

sn

{;<fiW snO1

FIG. 9. Restriction fragment length polymorphisms at the snlocus in the mutants snGl-3. DNA from sn males was digested withEcoRI and HindIll, separated in a 1% agarose gel, blotted, andprobed with sn9. Two EcoRI-HindIII fragments are apparent in ourOregon R (OR) DNA (see Fig. 1). The mutagenized X chromosomein the three mutants was derived from their Oregon R great-grandmother. Consistent with P-element insertion indeed havingtaken place at the sn locus, the larger EcoRI-HindIII fragment hasbeen disrupted in the mutant individuals. As expected, the size of thesmaller fragment remains unchanged. Apparently, the event thatgenerated snG3 was independent of that which generated snG1 andsnG2.

inserted on average every 1 kilobase along the Drosophilagenome (11). Since it would be straightforward to screen 105or more flies by our method, one could be relatively confidentof success for many if not most genes. Moreover, othertransposon mutagenesis systems are available for Drosophila[e.g., inducer-reactive (IR); ref. 12], and these have differentinsertion preferences. Thus a gene that is rarely mutagenizedby one transposon may be more readily mutable by another.Our method also allows the detection of "silent" insertions

or those that confer phenotypes too weak or obscure to scoreby conventional methods. Silent insertions, near but not atthe gene of interest, may widen the spectrum of genes thatcan be mutagenized since P elements, once inserted, may beremobilized by a cross that provides a source of transposase.At a significant frequency, loss of a P element from a locusis accompanied by deletion of a segment of flanking DNA,often no more than a few kilobases (11). We hope to detectsuch events by inverse PCR (13) or by PCR with primers thatflank the integrated element.A natural extension of our strategy would be to the

mobilization and detection of marked P elements. Severalsuch engineered elements have been described (e.g., ref. 14),although they are rather large and usually low in copynumber, characteristics that would generally militate againsttheir use for saturation mutagenesis (11). However, giventhat a "silent" insertion can be used to generate a deletion,marked elements even relatively far from a gene could bemobilized, loss of the marker selected for, and markerlessstrains examined for loss of the gene in question.There are some general features of our method that are

worthy of consideration. (i) A mere 50 bp or so of DNAsequence will allow the design of two divergent primers. Inunpublished experiments we can detect amplification, suffi-cient for our method, between aP element at the sn locus and[snB], a primer t2.3 kilobases away (Fig. 1). Thus a regionas large as 5 kilobases, perhaps up to 10 kilobases or more(15), could be scanned at one time. (ii) If sufficient DNAsequence information is available, it may be possible to useseveral locus-specific primers in the same reaction mixture,each pointing in the same direction but separated by a fewkilobases. (iii) Far more egg DNA than is required for PCRis produced at each stage. Thus several genes can be exam-ined at the same time, perhaps by a consortium of users. (iv)DNA saved from previous experiments can be used toprovide a rough guide to the scale of experiment that will berequired to mutagenize a new gene. (v) Apart from the source

of transposase, all of the strains used in our experiments canbe virtually wild type. Hence the F2 females are healthy and

- ON (N

Genetics: Kaiser and Goodwin

1690 Genetics: Kaiser and Goodwin

fecund, helped by the fact that they carry their mutations inheterozygous form. (vi) In combination with inverse PCR(13), our method would be applicable to the detection of a

range ofDNA rearrangements, ofwhich transposon insertionis merely one. (vii) Lastly, given the apparent generality ofour method, there are obvious implications for its applicationto other organisms, even those not yet the subject of formalgenetic analysis.

Note. While this manuscript was in preparation we discovered thatsimilar, although not identical, experiments had been carried out byBallinger and Benzer (16).

It is a pleasure to acknowledge the patience and generosity ofJamie Paterson and Kevin O'Hare, who made available to us strains,DNAs, and unpublished sequence information. Their enthusiasmand that of our collegues in Glasgow, especially Steven Russell, isgreatly appreciated. We also thank Seymour Benzer for his open

discussion and communication of data prior to publication. S.F.G. issupported by a postgraduate studentship from the Department ofEducation of Northern Ireland.

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254-258.10. Schalet, A. & Lefevre, G. (1973) Chromosoma 44, 183.11. Engels, W. R. (1989) in Mobile DNA, eds. Berg, D. E. &

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13. Ochman, H., Ajioka, J. W., Garza, D. & Hartd, D. L. (1989) inPCR Technology: Principles and Applications for DNA Am-plification, ed. Erlich, H. (Stockton, New York), pp. 105-111.

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Proc. Natl. Acad. Sci. USA 87 (1990)