Actin Genes in the Mediterranean Fruit Fly, Ceratitis capitata David S. Haymer," Joan E. Anleitner," Mei He,* Sujinda Thanaphum,* Stephen H. Saul,+ John Ivy,* Kathy Houtchens* andLoretta Arcangeli" *Department of Genetics, University o f Hawaii, Honolulu, Hawaii 96822,?Department of Entomology, University o f Hawaii, Honolulu, Hawaii 96822, and*Hawaii Biotechnology Group Inc., Aiea, Hawaii 96701 Manuscript received December 1 , 1989 Accepted for publication February 3, 1990 ABSTRACT We have undertaken the study of actin gene organization and expression in the genome of the Mediterranean fruit fly (medfly), Ceratitis capitata. Actin genes have been extensively characterized previously in a wide range of eukaryotic organisms, and they have valuable properties forcomparative studies. These genes are typically highly conserved in coding regions, represented in multiple copies per genome and regulatedin expression during development. We have isolated a gene in the medfly using the clonedDrosophila melanogaster 5C actin gene as a probe. Thismedfly gene detects abundant messages present during late larval and late pupal development as well as in thoracic and leg tissue preparations from newly emerged adults. This pattern of expression is consistent with what has been seen for actin genes in other organisms. Using either the D. melanogaster 5C actin gene or the medfly gene as a probe identifies five common cross reacting EcoRI fragments in genomic DNA, but only under less than fully stringent hybridization conditions.

A

CTIN genes produce contractile proteins which are involved in a variety of fundamentally important cellular processes such as muscle contraction and motility (POLLARD and COOPER1986). Molecular studies of actin genes in a variety of eukaryotic organisms (listed in BURN,VIGOREAUXand TOBIN 1989) have helped shape contemporary ideas on the regulation of gene expression and the evolution of multigene families. During development, actin genes are typically expressed in very precise temporal and tissue specific patterns(FYRBERG et al. 1983; DAVIDSON 1986). Except for the single actin genes foundin yeast (GALLWITZ and SEIDEL1980) and Tetrahymena (CUPPLES and PEARLMAN 1986), these genes are also present in multiple copies pergenome in eukaryotes (BURN,VICOREAUX and TOBIN 1989). The coding sequences of actin genes often exhibit extensive homology at the nucleotide and amino acid sequence levels (SANCHEZ et al. 1983). There is, however, often considerable variation in 5' and 3' flankinganduntranslatedregions as well as forintron sequence and positioning. This has been shown to be true both across phylogeny and between the individual members of a multigene family (FYRBERG et al. 1983; SANCHEZ et al. 1983). As an example in Drosophila rnelanogaster, variation in the 3' untranslated regions of the six actin genes has made the construction of probes specific to each gene possible (FYRBERG et al. 1983). How actin genes are organized in agenome can (;tnetic\

125: 155-1liO (\1,1\. 11t:tO)

vary as well. The six actin genes of D. melanogaster are found at dispersed genomic locations (FYRBERG et al. 1983) whilein other organisms such as the sea urchin, as many as three actin genes have been found clustered within 28 kb of contiguous DNA (DAVIDSON 1986). Copy number is highly variable as well. The number of actin genes per genome varies from asingle gene in yeast (GALLWITZ and SEIDEL1980) and Tetrahymena (CUPPLES and PEARLMAN 1986)upward to of 15 to 17 found in sea urchins (DAVIDSON 1986) and the slime mold Dictyostelium (MCKEOWNet al. 1978). Patterns of actin gene expression have also been studied in detail in several of the organisms named above.Althoughactin genes are typically broadly categorized as cytoskeletal or muscle specific, regulation of their expression during development is seen as very precise in both temporal and tissue specific terms. In the sea urchin Strongylocentrotus pupuratus, for example, even the threeclosely linked cytoskeletal actin genes exhibit differences in their tissue specific pattern of expression (DAVIDSON1986).D. melanogaster appears to have four muscle specific actins, two of which are expressed predominantly in larval musculatureand two which arefound in adult tissue. Combinedgenetic and developmentalstudies have demonstrated the role that one of these adult muscle specific actin genes (88F) plays in the formation of myofibrils in indirect flight muscles (KARLICK,COUTU and FYRBERG 1984). We have undertaken the study of actin genes in the genome of the medfly Ceratitis capitata. The medfly

D. S. Haymer et al. E

s

P

RV

RV

I

I

1

1

I

B X B I

I

1

-

23.79.56.8-

-

43-

SCALE

,

n

E

I

I

PROBE

1

2

3

lkb

232.0-

FIGURE1 .-Southern blot of medfly genomic DNA probed with the 1 .X-kb Hindlll fragment of the D. melanogaster 5C actin clone I h A 2 (FYRRERG, KINDLE^^^ DAVIDSON1980). Each lane contains three tnicragrilms of genomic DNA. The blot was hybridized and washed under reduced stringency conditions (see MATERIALS AND METHODS).

is an agricultural pest species of considerable importance in several parts of the world (SAUL1986), however to date, relatively little in the way of contemporary molecular studies have been conducted on this species. We report here on the identification of sequences and the cloning of a gene from the medfly which shows homology to the actin genesof D. melanogaster. This medfly gene hybridizes to transcripts which are temporally regulated in expression during development, and in different tissues it detects transcripts in a pattern of expression typical for Dipteran actins. MATERIALS AND METHODS

Adult DNA isolation and genomic library construction: Highmolecularweightgenomic DNAwas isolated from adult C. capitata fliesusing a strain which hasbeen in continuous laboratory culture forseveral years.Adult DNA was isolated by homogenizing approximately 1 g of flies in a Dounce homogenizer, filtering through rough gauze and spinning homogenate at 2500 X g for 10 min at 4" to isolate nuclei. Lysis buffer (20 mM EDTA, 50 mM Tris pH8.0) was added to resuspend the pellet with the addition of 1% Sarkosyl after resuspension. Cesium chloride was added to achieve a final density of 1.7 and the genomic DNAwas isolated by equilibrium gradient centrifugation at 40,000 rpm for 40 hr at 18". High molecular weight DNAwas partially digested with EcoRI, size fractionated on a step gradient and ligated into Charon 4 arms to construct the library. Probes and hybridizations: Probe DNAs for the initial library screen [conducted as described by BENTONand DAVIS(1977)l were labeled by random priming using y2PdCTP of high specific activity. Full stringency hybridizations were conducted in 50% formamide, 42" with SSC,Denhardt's solution and salmon sperm DNA as per MANIATIS, FRITSCHand SAMBROOK (1982) with washes at 65" in de-

FIGURE2.-Restriction map of insert cloned in pmed 2 1. The vector is pUC 9. Probe 1 is the same as described in Figure 1. Probe 2 is total cDNA labeled by randomly primed reverse transcription of poly(/\)+ RNA. Probe 3 is a cDNA clone isolated from a late pupal cDNA library. The thickness of the bar beside each probe corresponds to the relative intensity of hybridization. See text for explanation. Restriction sites are: E = EcoRI, S = Sall. P = Pstl, R V = EcoRV, B = BglII, X = Xbal, H = Hindlll.

creasing concentrations of SSC down to 0.I X . Reduced stringency conditions were 43% formamide, 42" hybridization temperature with wash temperatures at 55" at afinal SSC concentration of 0.1X. Autoradiography was performed usingFujiX-rayfilm and DuPont lightning plus intensifying screens. For Southern blots, genomic DNA was digested with various enzymes from Boehringer Mannheim Biochemicals under conditions described by the manufacturer. Southern blotting of DNA to nitrocellulose membranes was conducted as described by SOUTHERN (1975). Hybridizations for Southernblots were conducted using the nonradioactive DNA labeling and detection kit from Boehringer Mannheim Biochemicals, again under either full or reduced stringency temperature and formamide conditions as described above. RNA extractions and Northern blots: R N A extraction was performed using slight modifications of the procedure et al. (1 981). Briefly, materialwas homogenized of LEMEUR in an extraction buffer consisting of 3 M LiCI, 5 M urea, 10 mM sodium acetate (pH 5.0), 0.2 mg/ml heparin and 0.1 % SDS. After an overnight incubation at 4", RNA was pelleted by centrifuging at 12,000 X g for 15 min at 4" in a microcentrifuge. The pellet was washed with 4 M LiCl and 8 M urea and centrifuged again. The pellet was then dissolved in 0.1 M sodium acetate and 0.1% SDS. After phenol/ chlorophorm extraction,the RNA was precipitated with sodium acetate and ethanol at -20". Northern analysis was performed using formaldehyde gels asdescribed (MANIATIS, FRITSCHand SAMBROOK 1982). Probes for northern blots were also labeled by random priming as described above. Isolation of poly(A)+RNA, cDNA and libraryconstruction: Poly(A)+RNA was isolatedusingoligo-dt-cellulose chromatography. Double-stranded cDNA used in the construction of a cDNA library was prepared from poly(A)+ RNA isolated from the late pupal stage (20 days after egg laying). Kit components for double stranded synthesis were from Promega Biotec.First strand synthesis was initiated using oligo-dtpriming. After second strand synthesis, EcoRI adaptors were added and inserts were ligated into lambda gtlO. Total cDNAused as a probe was generated using randomly primed reverse transcription of poly(A)+ RNA as described by SARCENT (1987).

157

Medfly Actin Genes

23.7-

23.7-

9.56.8-

9.56.8-

43-

43-

23-

232.0-

2.0-

FIGURE 4.-Southern blot of medfly genomic DNA probed with pmed 21 under full stringency hybridization conditions.

FIGUREJ.--Soutllern blot of medfly genomic DNA probed with pmed 2 1 under conditions itlenrical to those of the blot in Figure 1 .

RESULTS

The representation of actin sequences in the medfly genome was determined by probing a Southern blot of medfly genomic DNA with the D. melanogaster 5C actin probe at reduced stringency. Figure 1 presents such a blot. In the EcoRI lane, there are three bands of relatively intense hybridization seen with an additional four faint bandsvisible. In the HindIIIlane, six bands can be easily resolved. The isolation of the medfly gene was accomplished as follows. Approximately 60,000 phage plaques of the Charon 4 library were plated out and screened initially with a clone of a sea urchin actin gene (prounder reduced stringency vided by T. HUMPHREYS) (see MATERIALS AND METHODS). Six plaques which produced intense hybridization signals were picked for rescreening with both a Drosophilamelanogaster and a actin cDNA clone (provided by S. BERNSTEIN) genomic clone containing the 1.8-kb HindIII fragment from the 5C actin gene of D. melanogaster (proDNA was extracted from plate vided by E. FYRRERG). lysates of t w o of these plaque purified clones, and these two were found to contain apparently identical inserts. Insert fragments from one of these (X 2-1) were shotgun subcloned into pUC9 for further characterization. A Southern blot was made of several insertcontaining subclones. Using the 1.8-kb fragment from the 5C actin clone mentioned above as a probe, a subclone was identified containing a 4.7-kb fragment with homology tothe Drosophila actin probe. This probealso identified a 4.7-kb EcoRI fragment in the original phage isolate, and this fragment is apparently identical with the 4.7-kb fragment in genomic DNA (Figure 1). This plasmid, designatedpmed21, was mapped

with 6 cutter restriction enzymes and the results are shown in Figure 2. T h e homology to the D. melanogaster probe was initially determined to reside within the 2.8-kb internal HindIII-PstI fragment. A refined map of the transcript was developed by comparing the hybridization pattern of three different probes to restrictionfragmentsfrom this region (Figure 2). Probe1 was the 1.8-kb 5C actin probe described earlier.In this case, hybridization was seen tothe EcoRV and EcoRV-BglII fragments. Probe 2 was total cDNA madefromrandomlyprimedreversetranscribed poly(A)+ RNA. Here, hybridization was seen to the same two fragments as above, but in addition, weak hybridization was seen to the adjoining BglIIXbaI fragment. Probe 3 was a cDNA subclone isolated from apupal cDNA library using pmed 2 1as a probe. This library was constructed using oligo-dt primed first strand synthesis. In this case, hybridization was seen to theEcoRV-BglII and BglII-XbaI fragments. Figure 3 shows thepattern of hybridization observed when pmed 2 1 is used to probe genomic Southern blots of the same type as those shown in Figure 1, under identical conditions. Of the six bands visible in the EcoRI lane of this blot, five are in common with fragmentsidentified using the D. melanogaster 5C actin probe. T h e sizes of these common fragmentsare 9.6, 5.9, 4.7, 3.1 and 2.6 kb. In the HindIII digest several common fragments are identified as well, although at least two additional HindIII fragments not seen previously are also visible. Under more stringent conditions,areduced number of hybridizing fragments are visible (Figure 4). The 4.7-kb EcoRI fragment, which is essentially the probe hybridizing back to itself, is most easily visible while the 5.9- and 2.6kb fragments are also identified. Finally, when pmed 21 is used to probe a blot of D. melanogasterDNA digested with EcoRI, a pattern of hybridization con-

D. S. Havmer et al. I

PC 0 0

Y

23.7-

956.843-

7.5232.0-

4.4-

’ 0

2.4-

6

.

FIGURE.5.-Southern blot of D. melanogaster DNA (Oregon R) prohrtl w i t h p l e d 21 under conditions identical to those of the I d o t i n Figurr I .

sistent with that known for actin sequences of this species is seen. This can be seen in Figure 5. At the RNA level, the putative medfly actin gene hybridizes to transcripts present at specific stages during developmentand in specific tissues. Figure 6shows that pmed 21 hybridizes to messages present in total and poly(A)+ RNA but not in the poly(A)- fraction. T h e transcripts are approximately 1.6-kb in size for both the early (up to3 hr) and thelate (approximately 24 hr) embryonic stages. In Figure 7, abundant messages are detected in late third instar larvae corresponding to 9 days after egg laying (AEL) and in late pupae (20 days AEL). During the early (10 days AEL) and mid pupal (14 days AEL) stages the transcripts are much less abundant. Also during these pupal stages, weak hybridization to transcripts 2.2-kb in size can be seen. The tissue specificity of the medfly gene expression is shown in Figure 8. Here, equivalent amounts of RNA extracted from either whole bodies or various body parts from newly emerged adults (1 day post emergence) are probed with pmed 2 1. Abundant levels of transcripts are detected in whole body, thorax and leg preparations. Much weaker hybridization is seen in head material and virtually none in the abdominal preparation. DISCUSSION

The evidence presented here suggests that we have cloned a medfly actin gene. T h e 4.7-kb EcoRI fragment in pmed 21 contains regions thatcross hybridize with the 5C actin probe (DmA2) fromD.melanogaster. Thisprobe consists of a 1.8-kb HindIIIfragment containing most of the 3’ major exon of this gene as well as a portion of the intron (FYRBERC et al. 1980). Transcript mapping of the pmed 21 insert indicates

.3FIGURE6.-Northern blot of RNA from earlv (up to 3 hr old) embryos and late (up to 22 hr old) embrvos. One microgranl was loaded into the poly(/\)+ lanes, 2 ag were loaded into the polv(A)lanes and 3 pg were loaded into the total lanes.

that the region of homology is transcribed. In probings of medfly genomic DNA, both pmed 21 and the DmA2 fragment identify a series of common fragments in EcoRI and HindIII digests. Using the pmed 21 insert to probe D. melanogaster genomic DNA, a pattern of hybridization consistent with that known for actin genes of this species is produced. T h e pattern of transcripts detected during development by the gene in pmed 21 is also consistent with that of an actin gene (see discussion below). There is an apparent change of representation of actin homologous sequences in the medfly genome seen as a function of the stringency of hybridization. When using pmed 21 to probe medfly genomic DNA at reduced stringency, up to six cross reacting fragments, possibly representing six distinct actin genes, are identified in EcoRI digests. This is a typical number of actin genomic sequences which have been seen in a variety of different Drosophila species (FYRBERG et al. 1983; LOUKAS and KAFATOS 1986). However, a reduced number of cross reacting fragments are observed when the blot is done atfull stringency (Figure 4). This could possibly reflect the extentof homology between the genes contained in these different fragments. Although actin genes are typically thought of as being highly conserved within an organism, there are examples of sequence differences which can reliably distinguish the coding regions of the cytoplasmic us. the muscle specific actins (SANCHEZ et al. 1983). An extreme example of such distinction between cytoplasmic and muscle specific isoforms of a func-

L

7.5-

7.5-

4.4

4.4

*

-

2.41.4-

.3-

tionallv related gene is apparently the case in D. melanogaster. Here,the myosinheavy chain genes are referred to as a gene family although there is little or no sequence homology between the genes encoding the cytoplasmic and muscle specific isoforms (KIEH A R T et al. 1989). I t is also possible that only those sequences identified on the full stringency blot are true actins. Based on the BcoRI pattern alone, this would reduce the number of detectable medfly actin sequences to three.Again although Drosophila species KAFATOS1986), typically have six actins (LOUKAS and there is certainly precedence for wide copy number variation in different species for actin genes as well as for genes encoding other contractile proteins. Actin genes, forexample,range in numberfromone in yeast and Tetrahymena to 15 to 17 in sea urchins and Dictyostelium (listed in BURN,VIGOREAUXand TORIN 1989). T h e muscle specific myosin heavy chain gene, which is single copy in D. melanogaster, is found in multiple copies per genome in most eukaryotes (BERNSTEIN et al. 1983; ROZEKand DAVIDSON 1983). T h e tubulins, another set of genes whose products often act in concert with actins, also exhibit a range of copy number variation between species (CLEVELAND 1983). For the sequences identified here, distinguishing between these possibilities w i l l have to await isolation of additional genes and ultimately DNA sequence analySIS.

The pattern of expression exhibited by this medfly gene clearly demonstrates several points. First, it detects messages that are polyadenylated (Figure 6) with temporal peaks seen in the late larval and late pupal stages of development. Transcripts are also detectable, albeit at lower levels, during embryogenesis, the early and midpupal stages as well as in adults. The early embryonic message population could be in part

1.4-

.3-

maternal in origin, while the late embryonic stage corresponds to a time whenlarval musculature is known to be developing. The late larval and latepupal stages of expression (Figure 7) arealso times when a considerable amount of muscle differentiation is oc1978). In curring(FYRRERGet al. 1983, CROSSLEY terms of tissue specificity, in newly emerged adults there arealso abundant transcripts detectablein those tissues where muscle differentiation is taking place (Figure 8). T h e thorax is the primary example of this. Indirect flight musculature develops in the thorax with the buildup of myofibrils continuing on through the first few days after adult emergence. In addition, both leg and thoracic musculature develop de novo from imaginal discs as opposed to the cephalic and abdominal muscles which are reorganized from larval et al. 1983, CROSSLEY 1978). The tissue (FYRRERG weak hybridization seen in the abdominalpreparations is also consistent with the fact that in Diptera, abdominal muscles are apparently fully differentiated during the pupal stage (CROSSLEY 1978). Although thepattern of expression seen forthe medfly gene in pmed 21 is similar to some of the individual expression patterns reported for D. melanogaster actins (FYRRERG et al. 1983), at this point it is not possible to make direct comparisons. FYRRERG et al. (1983) used gene specific probes derived from 3' untranslated regions to study individual expression patternsforthe six actins of D. melanogaster. The medfly clone we have identified here clearly contains coding region. However, the results reported in Figure 2 suggest that we have in fact identified the 3' end of the medfly gene as well. In this figure, a short XbaI-BglII fragment is identified which does not cross react with the Drosophila probe.This Drosophila probe contains only internal regions, not either ex-

D. S. Haymer et al.

160

trenle end of the gene (FYRBERG et al. 1980). However, this same short fragment is identified as part of the transcribed region weakly by randomly primed reverse transcribed RNA and strongly by a cDNA clone (Figure 2). This cDNA clone was isolated from a library generated by oligo-dt primed first strand synthesis, and it will therefore tend to strongly represent the 3' end of the gene. Using this information, it may n o w be possible to study gene specific patterns of transcription in the medfly as well. Transcripts 1.6-kbin size are the predominant messages found in all stages and tissues. However, during the early and midpupal stages of development, weak hybridization toa second sizeclass (2.2-kb) can be consistently seen. This could be dueeithertothe presence of another message with sequence homology or alternatively processed transcripts from the same gene. In Drosophila actin, at least three distinct size classes of hybridizing messages have been seen (FYRBERG et al. 1983) and the existence of alternatively processed actin messages have now been documented (BURN,VIGOREAUX and TOBIN 1989; VICOREAUX and TOBIN 1987). In conclusion, the medfly gene cloned here detects abundanttranscriptsat times and in tissues where muscle differentiation is occurring. The transcripts are atpeaks during late larval and late pupal stages of development, although they are detectable at several other stages. The predominant message size class is 1.6 kb with additional slightly larger transcripts detectable during certain stages of development. Both the tissue specificand temporal patternsof expression of this gene are consistent with those known for other actin genes. This medfly gene appears tocross hybridize with all six known Drosophila actin genes. It also appears tobe one of at least three conserved sequences in the medfly genome identified under full stringency conditions, withpossibly three additional sequences observable under reduced stringency hybridizations. The authors would like to acknowledge the very constructive criticisms given by two anonymous reviewers of an earlier version of this manuscript. This work was supported in part by U.S. Department of .4griculture grant 88-34135-3583 awarded under a program administered by the Pacific Basin Advisory Group.

LITERATURECITED BENTON,W. D., and K. W. DAVIS, 1977 Screening Xgt recombinant clones by hybridization to single plaques in situ. Science 196: 180-1 8'2. BERNSTEIN, S. I . . I(. M A G A M I , J . J. DONADY andC. P. EMERSON, JR.,1983 Drosophila muscle myosin heavy chain gene in a cluster of muscle nlutations. Nature 302: 393-397. BURN,T . C . , J .0. VIGOREAUX and S. L. T O B I N , 1989 Alternative 5(: ;tctin tl.anscl-ipts are localized i n different patterns during

Drosophila embryogenesis. Dev. Bid. 131: 345-355. D. W . . 1983 The tubulins: from DNA to RNA to CLEVELAND, protein and back again. Cell 3 4 330-332. CROSSLEY, G., 1978 T h e nrorphology anddevelopment of the Drosophila muscular system, pp. 499-560 i n The Genetics and Biology ofDrosophila, Vol. 2b, edited by M. ASHBURNER and T. R. F. WRIGHT.Academic Press, New York. CUPPLES,G. C . , and R. E. PEARLMAN, 1986Isolation andcharactet-izarion of the actin gene from Tetrahymenathermophila. Proc. Natl. Acad. Sci. USA 83: 5160-5164. AcaDAVIDSON, E. H., 1986 GeneActivityinEarlyDevelopment. demic Press, New York. FYRBERG, E. A , , I(. L. KINDLE and N. DAVIDSON, 1980 T h e actin genes of Drosophila: adispersed multigene family. Cell 19: 365-378. E. A., J. W . MAHAFFEY, B. J. BONDand N. DAVIDSON, FYRBERG, 1983 'Transcripts o f the six Drosophila actin genes accumulate i n a stage and tissue specific nlanner. Cell 33: 1 15-12.?, GALLWIT%, D., and R. SEIDEL, 1980 Molecular cloning of the actin gene fromSaccharomyces cerevisiae. Nucleic Acids Res. 8: 104:31059. KARLICK, C. C., M. D. COUTUand E. A. FYRBERG,1984 A nonsense mutation within the Act88F actin gene disrupts myofibril formation in Drosophila indirect flight muscles. Cell 38: 71 1-719. KIEHART,D. P., M. S . MALLORY,D. CHAN,A . S. KETCHUM,R. A . LAYMON,B. NGUYEN and I.. S. B. GOLDSTEIN, 1989 Identification of the gene for nonmuscle myosin heavy chain: Drosophila myosin heavy chains are encoded by a gene family. EMBO J. 8: 913-922. K. LEMEUR,M., N. GLANVILLE,J.L. MANDEL,P. GERLINGER, PALMITER and P. CHAMBON, 198 1 T h e ovalbumin gene Family: hormonal controlof X and Y gene transcription and mRNA accumulation. Cell 23: 56 1-57 1. LOUKAS, M., and F. C. KAFATOS, 1986 T h e actin loci in the genus Drosophila: establishment of chromosomal homologies among distantly related species by in situ hybridization. Chromosorna 94: 297-308. MANIATIS,T., E. F. FRITSCHand J. SAMBROOK,1982 Molecular Cloning. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. MCKEOWN,M., W. C. TAYLOR,K. KINDLE,R. A. FIRTEL,W . BENDERand N.DAVIDSON, 1978 Multipleactingenes in Dictyostelium. Cell 15: 789-800. T., and J. A.COOPER, 1986 Actin andactin-binding POLLARD, proteins. A criticalevaluationofmechanisms andfunction. Annu. Rev. Biochem. 55: 987-1035. ROZEK,C . E., and N. DAVIDSON, 1983Drosophila has one myosin I1eavy-chain gene with three developmentally regulated transcripts. Cell 32: 23-34. U . RDEST, E. ZULAUF and B. J. McSANCHEZ,F., S. L. TOBIN, CARTHY, 1983 Two Drosophila actin genes in detail. J. Mol. Biol. 163: 533-551. T., 1987 Isolation of differentiallyexpressedgenes. SARGENT, Methods Enzymol. 152: 423-432. SAUL,S. H., 1986 Genetics of the mediterranean fruit fly. Agric. Zool. Kev. 1: 73-108. SOUTHERN, E., 1975 Detection of specific sequences among DNA fragments separated by gel electrophoresis. J . Mol. Biol. 96: 503-51 7. VIGOREAUX, J. O., and S. L. TOBIN, 1987 Stage-specific selection of alternative transcriptional initiation sites fronr the 5C actin gene of D. melanogaster. Genes Dev. 1: 1 16 1- 1 171. Communicating editor: V. G. FINNERTY