Chapter 4 From Clone to Mutant Gene

CHAPTER 4

From Clone to Mutant Gene Bruce A. Hamilton* and Kai Zinnt *Whitehead Institute for Biomedical Research Cambridge, Massachusetts 02412 ?Department of Biology California Institute of Technology Pasadena, California 91 125

I. Introduction A . Screens Based on Chemical or Radiation Mutagenesis B. Screens Based on Transposon Mutagenesis 11. Overview A . Steps in Targeted P-Element Screens Using Flanking Sequence Rescue Hybridization B. Planning a Targeted Mutagenesis Screen 111. Protocols A. Protocol I: Generating Local Transposition Lines B. Protocol 11: DNA Preparation C . Protocol 111: Plasmid Rescue of Flanking Sequences D. Protocol IV: Hybridization Screening IV. Conclusion References

I. Introduction Targeted mutagenesis has become a second essential paradigm in molecular genetics for correlating mutant phenotypes with molecular properties. The ability to produce null mutations in cloned genes has been invaluable in the yeast and mouse genetic systems. In Drosophila, phenotypic screening for mutations has remained the primary means of genetic analysis because methods for targeted mutagenesis have until recently been rather limited. However, many METHODS IN CELL BIOLOGY. V O L . 44 Copyright 0 1994 by Academic Press. liic All righrr nfrcproducr~onin any form reserved

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newly identified fly genes are isolated on the basis of sequence similarities (Michelson et al., 1990; Tian et al., 1991), bulk expression pattern (Levy et al., 1982; Palazzolo et al., 1989), or biochemical properties of their products (Goodrich et al., 1993). Even in the most genetically tractable organisms, large numbers of transcripts for which mutations are not known to exist have been identified (Oliver et al., 1992; Palazolo et al., 1989; Sulston et al., 1992).Isolating a null mutation in a molecularly defined gene can be immediately useful in at least three ways: for direct assessment of any nonredundant functions provided by the gene, as a host background in which to test transgene constructs for restoration or alteration of function, and as a starting stock for phenotypic suppressor and enhancer screens to identify interacting genes. In this chapter, we briefly review several methods for isolating mutations in cloned Drusuphila genes and provide detailed protocols for generating P-element-induced mutations at targeted loci. In these protocols, local transposition of P-elements is used to increase the probability of insertion into the site of interest. We also describe flanking sequence rescue screens to identify lines bearing insertions into the gene of interest from collections of P-element transposants.

A. Screens Based on Chemical or Radiation Mutagenesis

In those cases in which the phenotype of a desired mutation can be reasonably guessed, standard phenotypic screens of mutagenized chromosomes are often feasible (e.g., Saxton et al., 1991). In this approach, mutagenized chromosomes are crossed into a marked stock carrying a deficiency that spans the cytological interval of interest. The progeny of this cross are assayed for a predicted phenotype (usually lethality of individuals heterozygous for a mutagenized chromosome and a deficiency chromosome). In this way, genes outside of the region defined by the deficiency can be ignored and only genes within the defined interval that can be mutagenized to the predicted phenotype are isolated. This method is limited primarily by the ability to predict a mutant phenotype, since for many genes a null mutation will not confer lethality. In addition, for some regions of the genome suitable deficiency stocks may not be available. Another approach that has been used successfully is screening for loss of an epitope by antibody binding (Katz et al., 1988; Van Vactor et al., 1988). An antibody raised against the gene product is used to assay crude protein extracts from animals carrying the mutagenized chromosome, either as homozygotes or as heterozygotes over a deficiency. This approach does not require a prediction of phenotype, except that the mutation must not be lethal prior to the time when readily detectable levels of the gene product accumulate in unaffected animals. This tactic does require a good antibody for each gene targeted and a separate assay for each chromosome screened. Dolph et al. (1993) recently used this method to isolate three mutations in the arrestin-2 gene among 20,800

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third chromosomes screened and two arrestin-1 alleles among 15,481 second chromosomes screened. B. Screens Based on Transposon Mutagenesis

Several properties of P-elements (in addition to being nontoxic) make them attractive as mutagenic agents for targeted genetic screens. Recombinant Pelement derivatives whose mobility is controlled by mating to a source of transposase and whose transposition is selectable by phenotype (either a change in eye color or G418 resistance) are widely available, making the generation of single or multiple hit transposants straightforward. Since P-element transpositions tag the inserted site with a known DNA sequence, insertions into the target region can be detected at the DNA level without a prediction of phenotype. Pelement insertions isolated in or near a gene of interest can be used as substrates to generate new alleles at very high efficiencies. For example, remobilization of a P-element often generates flanking sequence deletions by imprecise excision (Daniels et al., 1985; Salz et al., 1987; Voelker et al., 1984). These can extend into an adjacent target gene to produce a null mutation. Remobilization also generates local reinsertions that can often be selected by scoring the dominant marker on the transposon. This local transposition property is especially useful in light of the large number of enhancer detector P-elements that have been carefully mapped and collected. Spradling and coworkers have reported insertion rate enhancements for linked sites up to 100kb away from a starting P-element (Tower et al., 1993)on a minichromosome derived from the X. We have likewise observed several instances of local transposition that cross an entire lettered division on ordinary autosomes (Hamilton, 1993). These findings suggest that saturation of P-mutable sites within a region might be obtained efficiently using a local transposition strategy. Several detection assays have been used for targeted mutagenesis experiments with P-elements. These include genomic Southern blot hybridization to detect RFLPs, PCR assays to detect the juxtaposition of P-element sequences to known sequences in a gene of interest. and flanking sequence rescue techniques in which genomic sequences adjacent to P-insertion sites are isolated and screened by hybridization to target DNA. Each of these designs has inherent strengths and weaknesses, which we outline below. Genomic Southern blot hybridization can be an effective screen for insertions generated by local transposition. The high targeting efficiencies achieved in some local transposition experiments and the definitiveness and relative simplicity of the assay argue that this is a reasonable approach for screening up to a few hundred lines. Furthermore, rehybridizing the same filter membranes with transposon-derived probes can provide useful information about the success of the transposition strategy, such as the true rate of transposition, the existence and dominance of insertion hot spots among recovered lines, and the fate of

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the initial insertion site in local transposition experiments. Our lab has isolated PlacW insertions in or near two receptor-linked protein tyrosine phosphatase genes using this type of screen (B. A. Hamilton, A. Ho, and K. Zinn, in preparation: C. Desai and K. Zinn, unpublished experiments). Two insertions adjacent to the 3' end of DPTP99A were isolated from 132 transposants from a PlacW element at 99B, and an insertion into the DPTP69D locus was identified from about 300 transposants from an element at 69D. PCR strategies have been designed to detect novel fragments amplified between a P-element terminal repeat primer and one or more gene-specific primers (Ballinger and Benzer, 1989; Kaiser and Goodwin, 1990). This scheme has the advantages of being rapid, assaying large numbers of mutagenized chromosomes in a single experiment, and allowing the use of naturally occurring defective P-elements such as are found in the Birmingham and Harwich strains. This kind of screen has also been adapted to local transposition experiments in the mutagenesis of the Drosophila synaptotagmin gene (Littleton et al., 1993). The limitations of PCR-based screens are the size of the target (effectively about 2 kb per gene-specific primer) and the potential for false positives if the primer sequences and PCR conditions are not carefully controlled. Flanking sequence rescue screens present a third alternative for P-element insert detection. The essence of this method is to isolate genomic DNA fragments that flank the transposon insertion sites and hybridize them against the targeted sequences. In the original version of this screen, a P-element that carries a miniplasmid in one end (PlacW) is used as the mutagen and sequences that flank insertion sites are rescued by digesting transposant genomic DNAs with EcoRI or SacII, cyclizing the fragments with DNA ligase and transforming the circles into Escherichia coli. Circles capable of transforming E. coli to drug resistance carry both the plasmid end of the transposon and the flanking genomic restriction fragment. The resulting plasmid library is then used as a hybridization probe against segments of the targeted genes. This method was used to isolate insertions near or within several genes represented by cDNA clones in an array of hybridization targets (Hamilton et al., 1991)and to isolate insertions adjacent to DPTP99A, using an array of genomic DNA clones (Hamilton, 1993; B . A. Hamilton, A. Ho, and K. Zinn, in preparation). The strengths of this approach are that little molecular characterization of the targeted gene is required before the screen is begun and that relatively large pools of transposants (up to 200 or more) can be represented in each probe. Furthermore, each probe can be hybridized against a large array of targets representing the entire genomic region spanning each targeted gene. The advantage of screening large targets is that they allow detection of insertions into introns and nonessential flanking regions, and these insertions can be used to generate null alleles at very high frequencies by local transposition or imprecise excision of the element. Recently, Dalby and Goldstein have used inverse PCR (Ochman et al., 1988) on genomic DNA to generate flanking sequence probes in a successful screen of similar design (personal communication, 1993). Because it does not require a transformation

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step, this adaptation streamlines probe preparation and broadens the variety of useful starting transposons.

11. Overview A. Steps in Targeted P-Element Screens Using Flanking Sequence

Rescue Hybridization

1. Design the experiment and select the materials, including one or more transposon stocks, a source of transposase, and target clones. Sources for these materials and some considerations in their use are summarized in Table 1. The version of flanking rescue described here requires the use of a transposon that contains a plasmid in one end, such as PlacW. 2. Set up matings to generate transpositions, select transposants, and establish lines (Fig. 1). 3. Prepare blots of clones representing the mutagenesis targets. 4. Prepare DNA from pools of mated transposants. 5. Rescue the transposon-flanking sequences by transformation into E. coli (Fig. 2). 6. Hybridize labeled rescue plasmids to blots. 7. Identify mutant lines from positive pools. B. Planning a Targeted Mutagenesis Screen

For local transposition experiments, it is wise to gather several P-element single insert lines as potential starting stocks. As a rule of thumb, one-half to one lettered division of the salivary gland chromosome map from the target gene is a reasonable proximity from which to start. Not all P-element derivatives will transpose with the same efficiency, and the distribution of second insertion sites may vary both with the type of element and the starting location. Furthermore, local hot spots may act as sinks for insertions in some regions. Beginning with transposons at distinct sites near the gene of interest may therefore offer an advantage in some cases and having several alternatives already passed through your quarantine procedures would be worthwhile. Transposons marked with partially complementing W + minigenes, such as PlacW (Bier et al., 1989), are the most useful for scoring increases in transposon copy number caused by local transpositions. Most PlacW single-insert lines have yellow or orange eyes, and increases in PlacW copy number produce easily visible eye-color changes. Many mapped single-insert lines of this type are available through stock centers and individual researchers. In designing the crossing scheme, it is also worth bearing in mind that the distribution of inserted sites is more random in female germlines than in male (Zhang and Spradling, 1993).

Table I Starting Materials Type of material

Transposons

Transposase source Balancer stocks

Target clones

Description

Availability

Advantages

Disadvantages

Attached X chromosome that canies eight PlacW elements

Bloomington Stock Center

Low targeting efficiency for any one locus

Single-insert enhancer trap lines P[v]A2-3(99B)

Bloomington Stock Center; individual labs Many sources

X-to-autosome transpositions, targeting several sites at once Local transpositions Stable

May recombine away from chromosomal markers

TMS, Sb P[v]A2-3(99B)

Bloomington Stock Center Many sources

Lambda and cosmid genomic clones

Listed in Drosophila Information Service (DIS), Vol. 72, pp. 180-183; updated in Drosophila Information Network (DZN) Listed in DIS, Vol. 72, pp. 180-183; updated in DIN

PI genomic clones

Genome Systems

cDNA

Stable, not recombinogenic Select stocks that are compatible with markers on the chosen P-elements Detects insertions into the gene

Span entire gene with a short walk, represent as discrete fragments on a gel blot May span an entire gene with a single clone

Starting site remains linked

May not detect insertions into introns or control sequences

4. From Clone to Mutant Gene

87 W; P l m W

x W;

I

w; PlacW PlacW TM3 or 6

SbPA2-3 TM2, Ubx PA2-3

'

x

w; TM3.SbSer

I T"" establish line

DNA preparation

Fig. 1 A typical mating scheme for generating local transpositions on the third chromosome.

A line bearing a single w + PlacW element (light eye color) is mated to a source of P transposase. Somatic transpositions in the progeny are evident as a mosaic eye. Selecting F2 animals for eye color darker than that of nonmobilized controls ( w : PlacW/ +) greatly enriches for lines carrying multiple transposons. The majority of these are likely to be local transposants that retain the original insertion site intact.

Several alternatives are also possible for target clones; we recommend a combination of cDNA and genomic clones to span the entire gene. Full-length cDNA clones are useful targets because any insertions detected by them are likely to disrupt the function of the gene. However, insertions into introns or flanking control regions may also be mutagenic. Insertions into these sites can be detected by genomic clones that span the length of the targeted gene. Insertions of this kind may also be useful for generating deletion alleles by imprecise excision in a subsequent mobilization. This approach may be especially useful for genes that are refractory to direct insertion by P-elements.

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R

RS

R

V

P

S

R

targeted gene

1

EcoRI or SacII digest Dilute ligation

4

Electrotransform E. coli Drug Selection

PlasmidDNA miniprep

/

Hybridization Fig. 2 Transposon insertions can be screened by plasmid rescue of flanking genomic DNA. Pools of DNA are digested with EcoRI (R)or SacII (S) to release the plasmid carried in the transposon (P) along with a flanking genomic fragment. DNA fragments are ligated in dilute solution to form circles and transformed into E. coli. Plasmid DNA from drug-resistant colonies is labeled by random priming and used as a hybridization probe against Southern blots of cloned fragments from the genes of interest. Modified from Hamilton et al. (1991).

111. Protocols A. Protocol I: Generating Local Transposition Lines

Most dysgenic P-element mating schemes use the stable transposase source P[ry+]A2-3(99B),also called PA2-3 (Robertson ef al., 1988). A typical mating scheme for local transposition on the third chromosome in a female germline

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is illustrated in Fig. 1. For dysgeneses in female germlines, the balancer chromosome TM2, Ubx PA2-3 has been used; however, it tends to be a weak source of transposase. Recombinations between PA2-3 and visible markers on noninverted chromosomes are only slightly problematic as they are usually evident by eye-color mosaicism and can be discarded. A recent alternative chromosome is TMS, Sb PA2-3. Mutagenesis on other chromosomes or in male germlines follows essentially the same design, except for the choice of balancers. The steps of a typical mating protocol are illustrated in Fig. 1 and enumcrated here. 1. Mate the stock carrying the starting insertion to a PA2-3 stock en masse. This can be done in either direction with respect to sexes. Ten to 15 virgin females to 3 or 4 males in a half-pint bottle, transferred every 3 days for a total of four bottles, provides a good density of offspring without overcrowding. 2. Collect dysgenic progeny. These should be evident by mosaic patches of eye color caused by the somatic activity of PA2-3. For stocks in which the transposition rate is low, mosaicism in the eyes of F1 progeny may be a good indicator of which animals will produce frequent transpositions in the germline.

3. Mate single dysgenic F1 animals to an appropriate balancer stock, such as w ; TM3/TM6, in a small vial. Selecting one F2 transposant from each vial guards against repeated isolations of the same insertion event. At the same time, set up several control vials, mating the original transposon strain to the balancer stock. Progeny from this vial will be controls for scoring transposants in the next generation. Use two or three flies from the balancer stock for each vial, as single-pair matings often have a high failure rate. If the transposition rate is low, including two to three dysgenic flies per vial may be more efficient. Although dysgenesis in female germlines is thought to produce a more dispersed pattern of insertion sites, it may be of use to set up matings from a limited number of males as well. 4. Select transposants by scoring for darker eye color. Taking only one transposant from each vial ensures that each line analyzed represents an independent transposition. These flies will be enriched for lines that carry multiple inserts. Most of these lines will carry the original plus one new insertion, although single, apparently new insertions are relatively common and lines carrying mutliple new insertions are occasionally seen as well. Comparing the F2 dysgenesis offspring with control offspring of the same age is important, as eye color deepens with age. Males are preferable at this point because they can be mated to several balancer females for a prodigious stock vial before being used in DNA preparation and because eye-color changes are often more difficult to score in younger (virgin) flies. 5 . Mate the selected transposants to the balancer stock, again in vials. Once larvae appear in the vial, remove the original transposants for DNA preparation.

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B. Protocol 11: D N A Preparation This protocol is based on the method described by Bender et al. (1983) and is intended to provide consistent quality and yield among many samples. 1. Collect 20 F2 flies per 1.7-ml microcentrifuge tube and freeze on dry ice. Each tube constitutes a subpool in the following screening protocol. It is essential to faithfully record which fly lines are collected in each tube, so that mutant lines can be identified after screening. 2. Remove a tube from dry ice and add 100 p1 grinding buffer (5% sucrose, 80 mM NaCl, 100 mM Tris, pH 8.5,0.5% SDS, 50 mM EDTA). Grind the flies quickly but carefully with a small baked glass rod. Rinse the end of the rod into the tube with 100 pl grinding buffer. Return the tube to dry ice until all samples have been processed. 3. Incubate the homogenate at 70°C for 30 min. 4. Add 35 p1 8 M KOAc. Incubate on ice for 30 min. Pellet the precipitate by spinning for 10 min in a microcentrifuge. Decant the supernatant into a clean, prelabeled tube. 5 . Add 150 p l isopropanol. Mix well by inverting the tube several times. Let stand at room temperature for 5 min. Centrifuge for 10 min to pellet the DNA. The pellet should be visible, but may be dispersed along the side of the tube. Rinse the pellet once with about 0.5 ml 70% EtOH and a brief spin in the centrifuge. Aspirate the supernatant. Dry the pellet briefly by leaving the tube inverted on a paper towel or in a finger rack. 6. Resuspend the pellet in 100 pl TE + RNase A (approximately 0.1 mg/ ml). Dissolution of the pellet is aided by incubation at 5540°C for 1-3 hr.

C. Protocol 111: Plasmid Rescue of Flanking Sequences 1. Restriction digests release the plasmid and flanking genomic DNA on the same fragment. Pool 50 pl from each of 5 to 10 DNA preparations (100 to 200 fly lines). Digest each of four 30-pl aliquots of DNA (approximately 3 pg each) with 20 units of the chosen restriction enzyme for 1 hr in a 40-p1 reaction. Heat inactivate the enzyme at 70°C for 15 min. 2. The fragments are then ligated to form closed circles. To each digest, add 120 p15 x ligase buffer (Sambrook et al., 1989)and 440 p1 sterile distilled water. Mix well. Add 1 p1 T4 DNA ligase (400 U/p1; New England Biolabs) and mix by inverting the tube several times. Incubate at room temperature for 4 to 16 hr to cyclize the fragments. 4. Precipitate the DNA circles by adding 12 p18 M LiCl and 600 pl isopropano1 to each tube. Incubate on ice for 20 min. Recover the DNA by centrifugation for 10 min. Aspirate the supernatant. Wash the pellet once with 0.5 ml 70% EtOH and centrifuge for 5 min. Aspirate the supernatant. Dry the pellet in

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uacuo. Resuspend the pellet in 8 p1 sterile distilled water. Centrifuge 10 min to pellet any remaining insoluble material. 4. Transformation is accomplished by electroporation. Add 4 pI resuspended DNA to 40 pI electroporation-competent LE392 cells (Dower et al., 1988) on ice. Incubate 5 to 10 rnin on ice. Electroporate (we use a Bio-Rad Gene Pulser, following the manufacturer’s recommended conditions for E. coli) and immediately take up the cells with 1 ml SOC medium into a 15-1111 culture tube. Shake the cells 200-220 rpm at 37°C for 45 min. Spread 250 pl of the culture onto each of four LB agar plates containing 100 pg/ml carbenicillin. Incubate overnight or until colonies are 1 to 2 mm in diameter. The yield should be at least two to three colonies per fly line used. 5. Plasmid DNA is prepared by the rapid boiling method (Holmes and Quigley, 1981). Resuspend the colonies from all four plates in 2-3 ml LB with a sterile pipet. Split the suspension into two 1.7-ml microcentrifuge tubes and pellet the cells with a brief centrifuge spin. Resuspend each pellet in 0.6 ml STET (8% sucrose, 5% Triton X-100,50 mM EDTA, 50 mMTris, pH 8.0) with a vortex mixer. Incubate with 35 p1 lysozyme (10 mg/ml) for 0.5 to 2 min. Boil for 2 to 2.5 min. Centrifuge for 10 min. Scoop out the viscous pellet with a toothpick and discard it. Precipitate DNA with 0.5 ml 75% isopropanol, 2.5 M NH,OAc. Spin down the precipitate and wash the pellet with 0.5 ml70% EtOH. Dry the pellet in uacuo and resuspend in 100 p1 TE, 50 pg/ml RNase A. 6. To estimate the DNA concentration and complexity of recovered plasmids, compare restriction digests of each sample to known standards by electrophoresis through an agarose gel containing ethidium bromide.

D. Protocol IV: Hybridization Screening

1. Preincubate the clone blot filters that represent the targeted loci with hybridization buffer and agitation at 42°C while preparing the probe. Our hybridization buffer is 50% formamide, 5x SSPE, 1% SDS, lx Denhardt’s solution, and 100 pg/ml denatured salmon testes DNA. The buffer should be heated above 80°C for 5 min before use to denature the DNA. 2. Label approximately 100 ng of restriction-digested plasmid DNA by random priming method (Feinberg and Vogelstein, 1983). High-quality kits are available from several commercial sources. Follow manufacturer recommendations in their use. Dilute 100 ng digested plasmid DNA with sterile distilled water so that the final reaction volume will be 75 p1. Boil for 2 rnin to denature the DNA and immediately chill it on dry ice. Most of the reaction components can be added while thawing the denatured DNA between fingers. Add 8 p1 [a3*P]dCTP(80 pCi at 3000 Ci/mmole) to a reaction mix that otherwise lacks dCTP before adding the polymerase. 3. Purify the probe through a Sephadex G-50 spin column (Sambrook et al.,

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1989). Add hybridization buffer to approximately 1 ml. Determine the specific activity of the probe by Cerenkov or scintillation counting. 4. Denature the probe by heating to 80-100°C for 4 min. 5 . Add the probe to the preincubated filters to a final concentration of 1-3 x lo6 cpm/ml and incubate at 42°C with agitation for 18-36 hr. 6. Wash the filters with three changes of 0.1 x SSPE, 0.3% SDS prewarmed to 50°C. 7. Wrap the filters between single layers of SaranWrap and expose to film between two intensifying screens (the order is screen, film, filter, screen) at -70°C or below. Signals are often evident after 24 hr, although signals from 200-line pool probes have required as long as 1 week with a single intensifying screen. 8. Identify lines that carry the relevant insertions. Once a pool is identified as containing a line that has a candidate mutation in a gene of interest, each DNA preparation from that pool should be digested, Southern blotted, and hybridized to a probe from the appropriate target clone. This identifies a subpool of 20 lines. Genomic DNA from each line in this subpool is prepared and used to make a Southern blot. Probing this blot with the identified target clone fragment indentifies the line. 9. Depending on the characteristics of the identified insertion lines, generating derivative deletion alleles by imprecise excision of the P-element may be desirable. To do this, the insertion is remobilized using essentially the same crossing scheme by which it was produced. The principle differences are that in excising the element, one scores for a reduction in eye color and molecular screening is done by genomic Southern blot hybridization of individual lines to probes flanking the insertion sites.

IV. Conclusion We have used the protocols presented here routinely and with success. We have isolated insertions at several target sites from relatively small numbers of examined transposant lines (Hamilton, 1993; Hamilton et ul., 1991). We have not yet observed any artifacts that obscure the results of this screening procedure. We note, however, that modifications to these protocols could streamline this approach. In particular, the inverse PCR probe adaptation of Dalby and Goldstein may save substantial effort when the target sites are represented by large, contiguous clones. Enhanced chemiluminescence detection may offer additional time savings over conventional autoradiography . The availability of large collections of mapped single-insert enhancer detector P-element strains makes targeted mutagenesis of almost any locus in Drosophifu feasible without the need of specialized reagents. Although targeting efficiencies will vary by target site and the availability of nearby elements, local transposi-

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tion strategies, combined with the ability to generate flanking deficiencies by imprecise excision, offer a powerful genetic approach for cell biologists.

Acknowledgments We thank M. J. Palazzolo, K. VijayRaghavan, and E. M. Meyerowitz for their encouragement and contributions to the development of these procedures. We thank C. Desai, 8. Dalby, and L. S. B. Goldstein for discussing their results prior to publication. Our work cited in this manuscript was supported by NIH Grant NS28182, Basil O’Connor Starter Scholar Research Award 5-816 from the March of Dimes Birth Defects Foundation, a Pew Scholars Award, and a McKnight Scholars Award to K.Z. B.A.H. was supported in part by National Research Service Award 5 T32 HG00021-02 from the National Center for Human Genome Research during the development of these protocols and is currently supported by afellowshipfrom the Helen Hay Whitney Foundation.

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