Molecular cloning and characterization of Drosophila genes and their expression during embryonic development and in primary muscle cell cultures

DEVELOPMENTAL

BIOLOGY

90,272-283

(1982)

Molecular Cloning and Characterization of Drosophila Genes and Their Expression during Embryonic Development and in Primary Muscle Cell Cultures’ ROBERT Department

of Biological Received

V. STORTI’ University

of Illinois

31, 1981; accepted

in revised

Chemistry, July

AND ALICE

E. SZWAST

Medical form

Center,

November

Chicago,

Illinois

60612

18, 1981

Recombinant cloned genomic DNA fragments homologous to cultured muscle cell cDNA from Drosophila melanogaeter have been isolated and partially characterized. Each of the eight analyzed clones has been shown by hybrid-selected translation and polyacrylamide gel electrophoresis to encode at least one polypeptide. Three clones hybrid-select two proteins. The developmental pattern of expression of each clone has been determined by hybridization of mRNA isolated from staged embryos, myogenic cultures, and Schneider cells to EcoRI digestion fragments of the cloned DNA. The pattern of hybridization shows that some EcoRI fragments hybridize to transcripts that are developmentally regulated during embryogenesis and in muscle cell cultures as well as transcripts that are always expressed. Each clone has been mapped by in situ hybridization and shown to hybridize to a different single chromosomal locus on salivary gland polytene chromosomes. One clone hybridized to a single chromosomal locus and to the chromocenter. INTRODUCTION

or differences in programmed changes of gene expression. In order to gain more meaningful insights into the control of gene expression during development it will, therefore, be necessary to study the regulation of specific genes that are involved in specific cell determinations during embryogenesis. We recently set out to isolate, by recombinant DNA cloning techniques, developmentally regulated genes from Drosophila melanogaster in order to study the expression of specific genes and their mRNAs during development. Moreover, we have focused our attention on muscle genes in order to study parameters that determine the differentiation of a specific cell type. Myogenesis is particularly well suited for these studies since muscle cells from Drosophila can be studied in primary cell culture and undergo well-defined morphological changes concomitant with the coordinate synthesis and assembly of muscle proteins (Seecof et al, 1971, 1973; Storti et al, 1978; Fyrberg and Donady, 1979). We report the isolation and partial characterization of eight cloned genomic DNA fragments from Drosophila. The hybrid-selected proteins encoded by the genes of these cloned fragments, the pattern of their expression during embryonic development and muscle development in primary cell culture, and the chromosomal distribution of these genes are reported.

Growth and development during early embryogenesis are determined by the expression of previously synthesized and stored maternal mRNA and newly synthesized embryonic mRNA. Until recently, however, it has been possible to study these early events in detail for only a few highly abundant repeated or specialized genes such as 5s DNA, ribosomal DNA, histone, or tubulin genes (see review by Davidson (1976)). The parameters that are used to study mRNA during development often involve a comparative analysis of complex mRNA populations by kinetics of hybridization, rates of synthesis or translational efficiencies (Davidson and Hough, 1971; Turner and Laird, 1973; Levy and Manning, 1981). Although these experiments only measure the average population of mRNAs, it is clear from these studies that early development is controlled by complex transcriptional and post-transcriptional events. Models have been proposed (Britten and Davidson, 1969; Caplan and Ordahl, 1978). It is even more difficult to study gene regulation and/or to interpret the results of studies concerned with the control of gene expression that occur beyond the first few hours of development because these embryos consist of heterogeneous populations of many cell types. During development each cell type is probably determined by different responses to local stimuli ’ This study was supported by a research grant, GM 27611, from the National Institutes of Health and BRSG and CRB grants from the University of Illinois Medical Center. R.V.S. is a recipient of a NIH Research Career Development Award. ’ To whom correspondence should be addressed. 0012-1606/82/0402’72-12$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

272

MATERIALS

Preparation of Primary

AND METHODS

Myogenic Cell Cultures

The method for preparing muscle cell cultures from gastrula stage embryos of D. melanogaster has been

STORTIANDSZWAST

Cloning

and Characterization

described in detail (Storti et aZ., 1978). Briefly, gastrula stage embryos of the P2 line of Oregon R were collected at 3-4 hr after egg laying and homogenized to dissociate the cells. The supernatant of a low-speed centrifugation was plated on 60-mm protamine-coated tissue culture petri dishes. Under these conditions myogenic cells preferentially attach. Fusion of myogenic cells begins at 6 hr after plating and increases rapidly for the next 18 hr. These cultures have been shown to be at least 85% pure myogenic cells as determined by the number of nuclei incorporated into multinucleated myotube cells during cell fusion (Storti et ab, 1978). Synthesis of Muscle Complementary DNA Hybridization to Non-Muscle Cell RNA

(cDNA)

and

Total cytoplasmic mRNA was isolated by phenolchloroform extraction of Triton X-lOO-lysed primary cell cultured myotube cells (16-20 hr postplating) as described by Spradling et ah (1977). Poly(A) mRNA was fractionated by oligo(dT) cellulose chromatography (Spradling et al, 1977). Schneider L-2 cell poly(A) mRNA was isolated by phenol-chloroform extraction of SDS-lysed cells and oligo(dT)-cellulose chromatography as described previously (Scott et al., 1979). The procedure for synthesis of high specific activity [32P]cDNA, complementary to mRNA from myotube cells, was similar to tha.t described by Friedman and Rosbash (1977) except that unlabeled deoxynucleotides were present at 200 PM a:nd [ol-32P]dATP and [w~‘P]CTP (both greater that 2000 Ci/mmole, Amersham) were used carrier free. Synthesis was in 50 mM Tris-Cl (pH 8.3), 60 mM NaCl, 16 mM K2HPOI, 8 mM MgC12, 5 pg/ ml oligo(dT), 5 mM dithiothreitol, and 100 pg/ml actinomycin D. Myotube poly(A) mRNA was at 50 pg/ml and avian myloblastosis reverse transcriptase was at 560 units/ml. After incubation at 37°C for 1 hr, the myotube RNA was hydrolyzed with alkali and the [32P]cDNA was purified 'by Sephadex G-50 chromatography and ethanol precipitation. Muscle-specific [32P]cDNA sequences were enriched by exhaustive hybridization of myotube [32P]cDNA to excess RNA (log C,t > 2.5) from Schneider L-2 cells. Hybridization was in 0.4 M Na2HP04, 0.2% SDS, pH 7, for 22 hr with a 105-fold1 excess of Schneider cell total RNA. The single-stranded muscle-enriched [32P]cDNA was separated from the hybrid Schneider mRNA/ [32P]cDNA by hydroxyapatite chromatography and the muscle-enriched [32P]cDNA was collected by ethanol precipitation in the presence of 10 pg/ml carrier yeast tRNA. In some experiments the unhybridized, myotubeenriched [32P]cDNA from the first hybridization was further enriched for muscle nucleotide sequences by a second cycle of hybridization to Schneider cell mRNA.

Screening

of Drosophila

Genes

273

of Genomic Library

The Drosophila genomic DNA library was obtained from Dr. T. Maniatis (Maniatis et al., 1978). The recombinant DNA library consists of randomly sheared 1520 Kb Drosophila embryonic DNA fragments inserted with EcoRI linkers into Charon 4 X phage. The library was screened using the in situ plaque hybridization technique of Benton and Davis (1977). Seven loo-mm petri dishes, each containing approximately 2000 hphage plaques, were transferred to nitrocellulose filters and prehybridized according to Maniatis et al. (1978). Filters were prehybridized and hybridized in 4~ SET [1X SET = 0.15 M NaCl, 0.03 M Tris-Cl (pH 8), 2 mM EDTA], 10X Denhardt’s solution (1X Denhardt’s solution = 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll), 0.1% SDS, 50 pg/ml Eschetichia coli DNA, 1.25 mM sodium pyrophosphate, and 5 x 106-1 X lo7 cpm muscle-enriched [32P]cDNA. Hybridization was at 68°C for 16 hr. Filters were washed as described and exposed to Kodak XR5 X-ray film with intensifying screens at -70°C (Maniatis et al, 1978). Plaques corresponding to positives on the autoradiogram were picked and resuspended in SM buffer [O.l M NaCl, 0.05 M Tris-Cl (pH 7.5), 10 mM MgS04, 0.01% gelatin] and rescreened. The first screen produced 705 plaques showing hybridization. After two cycles of screening, 285 purified single plaques showed hybridization to the r2P]cDNA muscle probe and were stored at 5°C in 1.0 ml SM buffer with chloroform. Hybrid-Selection

Translation

Hybrid-selection translation was performed according to Ricciardi et al. (1979). Phage recombinant DNA was isolated by the phage dilution method of Blattner which accompanies the Charon phage Drosophila library. Approximately 5 pg of phage recombinant DNA, isolated from lo-ml cultures, was heat-denatured, spotted on one-half of a lo-mm-diameter nitrocellulose filter, air-dried, and baked for 2 hr at 80°C in vacua. Hybridization was for 2-4 hr at 37°C in 100 ~1 of 50% formamide, 0.75 M NaCl, 2 mM EDTA, 0.1 M Tris-Cl (pH 7.5), 0.2% SDS, and approximately 5 Kg poly(A)containing mRNA made from 12- to 24-hr embryos. Embryo RNA was made as described above for Schneider cell RNA. Following hybridization the filters were washed five times in 150 mM NaCl, 15 mM NaCeH507, 0.5% SDS at 55°C and two times in 2 mM EDTA, 10 mMTris-Cl (pH 7.2) at 55°C. The filters were boiled for 1 min in the presence of 2 mM EDTA (pH 7.0) and 10 pg of calf liver tRNA in order to dissociate the hybrid RNA. The RNA was collected by ethanol precipitation and translated in a micrococcal nuclease-treated rabbit reticulocyte lysate cell-free protein synthesizing system

274

DEVELOPMENTALBIOLOGY VOLUME90.1982

in the presence of 40 PCi [35S]methionine as described previously (Scott et ab, 1979). After translation the reactions were treated with 2 pg of pancreatic RNase for 5 min at 37°C and 4 ~1 was electrophoresed on one- or two-dimensional polyacrylamide gels as described previously (Storti et aZ., 1978). Restriction Endow&ease Digestion Electrophoresis of DNA

and Agarose

Gel

DNA was isolated from C&l-purified phage (Maniatis et al,, 1978) and digested with the restriction endonuclease EcoRI. The enzyme was purchased from Boehringer-Mannheim and digested according to their direction. DNA was electrophoresed in horizontal 1% agarose gels in 0.04 M Tris (pH 8.3), 0.02 M CH3COONa, 2 mM EDTA, 5% glycerol. Restriction fragments were visualized by ethidium bromide staining under ultraviolet light and transferred to nitrocellulose filter paper according to Southern (1975). Filters were baked at 80°C for 2 hr in vacua. Filters were prehybridized in 5~ SSC (1X SSC = 0.15 M Nacl, 0.015 M NaCGH507), and 10X Denhardt, 0.1% SDS, 1.25 mM sodium pyrophosphate, and 50 pg/ml salmon sperm DNA for 4 hr or overnight at 68°C. Hybridization was in 2-5 ml of the same fresh prehybridization buffer at 68°C for 16-20 hr. In later experiments the prehybridization and hybridization was in the same salt buffer as above made 50% in formamide and incubated at 42°C. Hybridization was for 40 hr. Filters were washed two times for 20 min each in 2~ SSC at room temperature and three times for 20 min each in 5 mM Tris-Cl (pH 8.2), 1 mM EDTA, 0.1% SDS, 1.25 mM sodium pyrophosphate, 1X Denhardt at 60°C and rinsed in 0.1X SSC at room temperature. Filters were air-dried and exposed to Kodak XR-5 or XAR-5 film with intensifying screens at -70°C. The hybridization probe was either [32P]cDNA prepared from embryonic or myogenic mRNA as described above or 5’ in vitro 32P-labeled mRNA. In vitro 5’-labeled RNA was prepared by brief alkali treatment and labeling with [Y~~P]ATP (~2000 Ci/mmole, New England Nuclear) by T4 polynucleotide kinase according to Maizels (1976). All experiments involving recombinant DNA were performed under containment conditions specified by the NIH recombinant DNA guidelines. In Situ Hybridization

[3H]cRNA was prepared from Drosophila recombinant DNA genomic clones and hybridized in situ to squashes of larval salivary gland polytene chromosomes according to Pardue and Gall (1975). Squashes were exposed to emulsion for l-3 weeks for short exposures and l-3 months for long exposures.

Screening

RESULTS of Recombinant DNA Genomic Library

To isolate muscle genes a cDNA probe enriched for muscle nucleotide sequences was prepared. The strategy for preparing a [32P]cDNA probe enriched for fused muscle (myotube) sequences involved hybridizing total myotube [32P]cDNA to excess Schneider cell mRNA. Total poly(A) mRNA was isolated for ZO-hr myotube culture cells and served as template for high specific activity [“P]cDNA. In order to remove the non-muscle cell cDNA nucleotide sequences, the [32P]cDNA was hybridized to an excess amount of Schneider cell total mRNA. Schneider cells are non-muscle tissue culture cells originally of embryonic origin (Schneider, 1972). By this method the single-stranded muscle cDNA sequences were separated from the non-muscle cDNA sequences that were bound in hybrids. Hybridization was greater than COT 2.5 in order to hybridize homologous repetitive and less abundant rare sequences. In several experiments, 63 to 76% of the ZO-hr myotube muscle cDNA hybridized to Schneider cell RNA. The remaining single-stranded muscle [32P]cDNA was fractioned and recovered by hydroxyapatite chromatography. The muscle-enriched [32P]cDNA was used as a probe to screen approximately 15,000 X-phage genomic LWsophiZa recombinant DNA plaques (Maniatis et aZ., 1978). Approximately 700 plaques showed hybridization and were rescreened. A total of 285 plaques showed hybridization to the muscle [32P]cDNA after two cycles of hybridization. Identification

of Recombinant

DNA

Clones

The method of hybrid-selected translation was used to identify the protein(s) encoded by each clone (16). Briefly, DNA extracted from individual cloned phage was bound to nitrocellulose filter paper hybridized to total mRNA from 12- to 24-hr embryos. Muscle cell fusion during embryogenesis begins at about 9-11 hr of development (Poulson, 1950). Thus RNA 12- to 24-hr embryos should contain a full complement of myotube mRNAs. This was verified by a comparison of the cellfree translation products by two-dimensional gel electrophoresis directed by embryo and myotube culture mRNA (not shown). The hybrid-selected RNA was recovered by boiling, ethanol precipitated, and translated in a rabbit reticulocyte lysate cell-free protein synthesizing system. The product(s) of translation was determined by polacrylamide gel electrophoresis and corresponds to the protein encoded by the cloned gene. The hybrid-selected products of seven of these clones electrophoresed in one- and two-dimensional gels are shown in Fig. 1 and 2. The XDm203 cloned DNA hybrid-selects two proteins,

STORTI AND SZWAST

Clming

and Characterization

of Drosophila

Genes

275

FIG. 1. Hybrid-selected identification of the proteins encoded by recombinant genomic DNA clones. Autoradiogram of [35S]methioninelabeled reticulocyte lysate translation products electrophoresed in a 12% polyacrylamide gel. The mRNA in each translation was obtained by hybrid-selection of embryo RNA to filter-bound cloned DNA. E is the product of an endogenous translation with no mRNA added. Lanes 1-8 are the hybrid-selected products of hDm203,85, and 85 electrophoresed for an additional 1 hr, and 217,227,231,236, and 270 filter-bound DNA, respectively. The arrows indicate the hybrid-selected products.

29,000 and 18,000 daltons (Fig. 1.1. and 2.2). Each has an isoelectric point (pl) of about 5.0. XDm85 also hybrid selects two proteins. These two proteins are 35,000 and 36,000 daltons and have pI values of about 5. The two proteins are only slightly resolved from each other when electrophoresed in SDS alone and under our standard electrophoresis conditions (Fig. 1.2) but are well separated when electrplhoresis is continued for longer times (Fig. 1.3). Both proteins are also well resolved when electrophoresed in gels containing urea-SDS (not shown). XDm217 hybri.d-selects a single protein of 20,000 daltons and an approximate pI of 4.0 (Figs. 1.4 and 2.1). Clones XDm22’7 and XDm231 each selects a very basic protein that only slightly enters the pH 310 ampholine gradient of a second-dimension isoelectric focusing gel. These proteins have molecular weights of 33,00@ and 23,500, respectively (Figs. 1.5 and 1.6). The

two-dimensional gel of the XDm231 product is shown in fig. 2.4. Clone XDm236 hybrid-selects two proteins (Fig. 1.7); they are 29,000 and 27,500 daltons and have pI values of about 5.2 and 5.4, respectively. The twodimensional gel is shown in Fig. 2.3. XDm270 hybridselects a low-molecular-weight protein of about 12,000 daltons and pI of about 6. This protein, which appears as a faint band with the exposure shown in Fig. 1.8, is apparently encoded by a low abundant mRNA or has a low methionine content. XDmllA hybrid-selects a protein with the same or very similar one-dimensional gel electrophoretic mobility as the 18,000 dalton of XDm203 (not shown in Fig. 1 but identified in Fig. 3.2). A composite identification of the proteins hybrid-selected by these clones among the total translation products of embryo RNA electrophoresed in a two-dimensional gel is shown in Fig. 3.

276

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VOLUME 90. 1982

FIG. 2. Autoradiogram of [%]methionine-labeled hybrid-selected translation products electrophoresed in two-dimensional polyacrylamide gels. Panels 1-4 contain the hybrid-selected translation products of clones XDm217, 203, 236, and 231, respectively. The arrows indicate the hybrid-selected products. E represents the endogenous translation products labeled only in panels 1 and 4. The three additional basic endogenous products in panel 4 do not focus in the range pH 4-6 of first-dimension gels in panels 1-3. Second dimensions are electrophoresed in 12% polyacrylamide gels. The major endogenous product at the bottom of panel 4 is rabbit globin. The three faint spots in the upper-left portion of panel 3 are translation products of nonspecifically bound actin mRNA.

Expression of Cloned Genes during Embryonic Development One of our objectives in this work was to assemble a collection of cloned genes encoding identifiable proteins whose expression could be studied during development in embryos and myogenesis in cell culture. Accordingly, the transcriptional expression of each clone was determined by hybridizing mRNA from different stages of embryonic and myogenic development to re-

striction endonuclease-digested Southern blots of each cloned DNA. Hybridization to restriction endonucleasedigested DNA has the advantage of determining both the pattern of hybridization and distribution of transcripts throughout the entire length of the cloned fragment. Briefly, each clone was digested with the restriction endonuclease EcoRI. Because the library was construtted by ligating Drosophila DNA and XDNA at internal EcoRI XDNA sites, the restriction endonuclease

STORTI AND SZWAST

Cloning

and Characterization

of Drosophila

Genes

277

FIG. 3. Composite identification of hybrid-selected translation products displayed among the total [35S]methionine-labeled reticulocyte translation products of embryo RNA. RNA from 11 to 24-hr embryos was translated in a rabbit reticulocyte lysate and the [?S]methioninelabeled products were electrophoresed in pH 4-6 first-dimension and 12% second-dimension polyacrylamide gels. The translation products numbered l-8 correspond to the hybrid-selected products of clones XDm203, llA, 85, 217, 227, 231, and 270, respectively.

digestion products of each recombinant clone consist of 19% and 10.9-Kb X anid the inserted Drosophila DNA fragments. The digested DNA fragments were separated by agarose gel electrophoresis and blotted to nitrocellulose filter paper (Southern, 1975). [32P]cDNA or 5’-32P-labeled alkali-treated mRNA was prepared from RNA made from embryos of early and late stages of development, and from early (myoblast) and late (myotube) myogenic cultures, and hybridized to the Southern blots of digested cloned DNAs. The results of hybridization of the digested cloned DNAs to embryos r2P]clDNA are shown in Fig. 4. Figures 4A,l-8, and B,l-8, show the hybridization pattern of 4 to 7- and 17 to 2O-hr embryonic [32P]cDNA, respectively, to duplicate sets of filters containing identical amounts of digested cloned DNA fragments. Two generalizations can be made from a comparison of the hybridization of these different RNAs to the DNA fragments shown in Figs. 4A and B. First, there are quantitative and qualitative differences in the patterns of hybridization of the two different stages of embryo mRNAs to each of the individual cloned DNA fragments. Second, while some clones show single EcoRI

fragments complementary to cDNA, others show multiple bands of hybridization. For instance, DNA from XDm203 (lanes A-l and B-l) shows no detectable hybridization to early embryo cDNA but shows strong hybridization of late embryo cDNA to one of three closely migrating EcoRI fragments in the 1.6-Kb region of the gel. We have not determined which of the three ethidium bromide-stained fragments corresponds to the single band of hybridization. XDmllA shows some hybridization to early but somewhat stronger relative hybridization to late embryo cDNA (lanes A-2 and B2). The broad band of hybridization is coincident with two closely migrating ethidium bromide bands of 5.6 and 5.8 Kb seen in the ethidium bromide-stained gel. Similarly, hDm85 shows hybridization to both early and late embryo cDNAs (lanes A-3 and B-3), although hybridization is also stronger to late embryo cDNA. There are, also, two closely migrating DNA fragments in this region of the gel. More recent experiments with better resolved fragments and restriction enzyme map analysis indicate that early cDNA hybridizes only to a 7.1Kb fragment while late cDNA hybridizes to the 7.1-Kb and a 7.3-Kb fragment (Bautch and Storti, unpublished

278

DEVELOPMENTAL

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90, 1982

FIG. 4. Expression of cloned genes during embryogenesis. Autoradiogram of embryo [32P]cDNA hybridized to Southern blots of EcoRIdigested cloned DNAs. One microgram of each DNA was digested with EcoRI restriction endonuclease and the DNA fragments were separated in a 1% agarose gel. The sizes of selected fragments in kilobase pairs is indicated for reference. The [a*P]cDNA probe was made from RNA extracted from 4 to 7-hr embryos (A) and 17 to 20-hr embryos (B) and hybridized to duplicate sets of filters. Approximately 8 X 10” cpm (specific activity, approximately 5 X lo7 cpm) was hybridized to each filter. Lanes 1-8 are DNA from clones XDm203, llA, 85, 217, 227, 231, 236, and 270, respectively. Autoradiography was for 8 hr. The gels in A and B were electrophoresed at 1 V/cm for 15 hr.

observations). hDm217 (lanes A-4 and B-4) shows three bands of hybridization to both stages of embryo cDNA. The fastest migrating 2.1-Kb fragment shows the strongest hybridization to early embryo cDNA while the 6.4Kb fragment is the strongest in late embryos. These differences are reproducible and occur when both [32P]cDNA probe or 5’-labeled [32]RNA (not shown) is used as a probe. Clone XDm22’7 has two DNA fragments that hybridize the cDNA from both stages of development (lanes A-5 and B-5). Both fragments, however, are larger than the 19.8- and 10.9-Kb h end fragments and suggest that rearrangement of DNA or loss of an RI site may have occurred during the cloning or phage growth. There are no other fragments detected in the stained gels. Clone XDm231 is particularly interesting in its pattern of expression (lanes A-6 and B-6). This clone selects a single protein of 23,500 daltons from late embryo mRNA (Fig. 1.6 and 3.6). cDNA from early embryos shows strong hybridization to one or both fragments in the 2.6 to 2.9-Kb region of the gel and additional hybridization in order of intensity to the 2.1-, 1.4-, 3.4-, and 1.2-Kb fragments. The latter two fragments are more easily seen on longer exposures of the X-ray film. Late embryo cDNA, on the other hand, shows hy-

bridization to only the 2.6 to 2.9-Kb-region fragments. The autoradiograms of Fig. 4 were exposed for 8 hr. The 2.1-, 1.4-, 3.4-, and 1.2-Kb fragments are still not visible at exposures of 72 hr. Clones hDm270 and XDm236 each shows a single DNA fragment complementary to both early and late embryo cDNA. XDm270, however, shows the same amount of hybridization to cDNA from both stages while XDm236 shows stronger relative hybridization to cDNA made from early embryos. hDm236 hybrid-selects two proteins. Expression of Cloned Genes during Non-Muscle Schneider Cells

Myogenesis

and in

Embryos consist of a heterogeneous mixture of several different cell types. Since the probe used to screen the recombinant DNA library was made from RNA from myogenic cultures the pattern of expression of each clone was, therefore, analyzed in myogenic cultures in order to correlate the expression of the cloned genes with muscle differentiation. It has been shown previously that Drosophila cultures consist predominantly of myogenic cells (>85%). Differentiation in Drosophila myogenic cultures has also been correlated

STORTI AND SZWAST

Clorting

und

with the induced or increased synthesis of mRNA and protein (Storti et ah, 1978; Bernstein et al.. 1980). Accordingly, filters similar to those described above were hybridized to [32P]cDNA or [32P]mRNA made from 3-hr myoblast cultures and 20-hr myotube cultures. Cell fusion begins in culture at about 6 hr after plating (Storti et al., 1978). The results of this analysis are shown in Fig. 5. It can be seen that although there are some quantitative differences in the patterns of hybridization between cultured myogenic cell cDNA and embryo cDNA to individual EcoRI fragments, the general patterns of hybridization are qualitatively very similar. XDm203A, llA, and 85, for instance, show reduced or barely detectable hybridization to 2-hr myoblast cDNA and considerably stronger hybridization to myotube cDNA (compare Figs. 5A-1, 2, 3, with B-l, 2, 3). The hybridization of myotube cDNA in Fig. 5B-1 to the 1.6Kb region of 203A is indicated by an arrow. This band is more prominant on l~onger exposures. These differences in hybridization are similar to those observed previously for early and late embryo cDNA. Clone XDm217 shows hybridization of both stage myogenic

Characterization

of Drosophila

Genes

279

cDNAs to the single 4.9-Kb fragment (Figs. 5A-4 and B-4). This is different from embryo cDNA, however, which showed additional hybridization to 2.1- and 6.‘7Kb fragments. We could not detect hybridization to the 2.1- and 6.4-Kb fragments with longer exposures. Clones 227, 236, and 270 have hybridization patterns similar to those for embryo cDNA except that XDm270 shows somewhat stronger hybridization to early myoblastcultured cDNA than late myotube cDNA, unlike embryo cDNAs which showed approximately equal amounts. Since 10-20s of the cells in our primary myogenic cultures are of nonmuscle origin we also determined the RNA hybridization profile to each clone using RNA made from Schneider cells. Schneider cells are of embryonic origin and have been in culture for several years (Schneider, 1972). In this way we could determine which transcripts are preferentially expressed by the myogenie cells. mRNA was extracted from Schneider cells and its cDNA hybridized to filters as described above. Figure 6 shows the results of these experiments. Clones XDm203A, llA, and 217 show no hybridization to Schneider cell cDNA at this or longer exposures of the

FIG. 5. Expression of cloned genes during myogenesis. Same protocol as in Fig. 4 except that [“P]cDNA was made from RNA extracted from 3-hr myoblast cultures (.A) and 20-hr myotube cultures (B). Approximately 4 X lo6 cpm was hybridized to each filter and autoradiography was for 40 hr. Lane assignments are the same as in Fig. 4. Arrow indicates the position of hybridization to the 1.6Kb-region fragment seen more clearly on longer exposures. The gel in A was electrophoresed at 1 V/cm for 15 hr. The gel in B was electrophoresed at 1 V/cm for 10 hr.

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in situ hybridization site of [H]cRNA from each clone to squashes of larval salivary gland chromosomes. Each clone hybridizes to a different single chromosomal locus with the exception of XDm236D. XDm236D hybridizes to 46D-F of chromosome 2R and in addition, shows extensive hybridization over the chromocenter region. We have not observed hybridization to any additional bands for any of the clones with longer exposures of the squashes although in some cases the increased background made this determination difficult. Additionally, small regions of chromosome homology might not have been detected because of the sequence complexity of the cloned probes. The loci homologous to each clone are widely distributed among the chromosomes with no apparent clustering. DISCUSSION

FIG. 6. Expression of cloned genes in Schneider cells. Same protocol as in Fig. 4 except that [32P]cDNA was made from RNA extracted from log-phase cultures of Schneider cells. Approximately 5 X lo6 cpm was hybridized and autoradiography was for 18 hr. Lane assignments are the same as in Fig. 4. The arrow indicates a band of hybridization to a XDm85 DNA fragment. The gel was electrophoresed at 1 V/cm for 15 hr.

film (lanes 1,2,4, respectively). There is a slight amount of hybridization to the 7.1-Kb fragment of XDm85 (lane 3, arrow). There is strong hybridization to clones XDm22’7,231,236, and 270. This is similar to the pattern seen in early and late embryos and myogenic cells and indicates that these transcripts may represent general “household-” type RNAs. hDm231, however, also shows hybridization to the 2.1-Kb fragment shown previously to occur only in early embryos and myoblast cultures and not in late embryos or myotube cultures. The properties of each clone and its pattern of expression are summarized in Table 1. Chromosomal Mapping of Cloned DNA

One of ability to mosomal cordingly, encoding

the distinct advantages of Drosophila is the the chrodirectly map by in situ hybridization loci homologous to cloned DNA genes. Acwe have determined the chromosomal locus each cloned DNA fragment. Table 1 lists the

In this work we set out to isolate developmentally regulated recombinant DNA cloned muscle genes. Of the entire set of clones derived from our inital screen, the clones reported here were selected for analysis because of their expression during embryogenesis and muscle development in culture or because of their possible identity with known muscle contractile proteins. Our current work, for instance, indicates that the two proteins encoded by XDm85 have properties similar to those of chicken a- and P-tropomyosin. Similarily, clones hDm231, 227, 203, and 11A each hybrid-selects abundant proteins with two-dimensional gel electrophetic mobilities similar to those of vertebrate skeletal muscle troponins and myosin light chains. We are currently in the process of confirming the identity of these proteins. Of more immediate concern to this work, however, is that each clone not only encodes at least one identifiable protein but also hybridizes to transcripts that show general cellular and/or muscle related expression. One of our goals is to construct a library of genes that show constitutive and developmental regulation. The hybrid-selection translation method employed here is useful for identifying proteins encoded by abundant mRNA. Rare mRNA and their products, however, are poorly selected by this method. This is probably due to a loss in recovery of the RNA which in part results from RNA degradation that occurs during the hybridization and recovery steps and insufficient sensitivity in detecting small amounts of RNA by cell-free translation. This possibility is suggested by an analysis of eight clones, not reported here, that were picked at random from the clones that were negative in hybrid-selection to late embryo mRNA. Southern blots of seven of these clones showed hybridization to either early and/or late embryo mRNA indicating that these clones

STORTI AND SZWAST

Cloning

CHARACTERISTICS

and Characterization

TABLE 1 AND EXPRESSION

of Drosophila

OF CLONED

281

Genes

GENES Expression“

Clone

Protein encoded WW

Map

position

Chromosome

203

29,000 18,000

3L

11A

18,000

85

36,000 35,000

3R

217

20,000

227

Locus

RI fragment Wb)

Late embryo

Myoblast

Myotube

KC

1.5-1.7*

0

++

0

+

0

5.6-5.Bb

++

+++

+

++

0

88F

7.3 7.1

0 +

+ +

0 +

+ +

0 +

2R

47F-48D

6.4 4.9 2.1

+ + ++

+++ ++ +

0 + 0

+ + 0

0 0 0

33,000

3L

63A-C

>20 >15

+++ +++

++ ++

+++ +++

++ ++

++ ++

231

23,500

2L

32C-D

3.4 2.6-2.9* 2.1 1.4 1.2

+ +++ ++ + +

0 ++ 0 0 0

+ +++ ++ + +

0 ++ 0 0 0

+ +++ + + +

236

27,500 29,000

270

12,000

2R Chromocenter 3L

62C-D

Early embryo

46D-F

7.6

++

++

+

+

++

69F

6.3

++

++

++

+

++

a The assigned values are arbitrary. 0 indicates fragments hybridized to different probes. * It was not determined which ethidium-stained

no detectable fragment(s)

hybridization, of this

are transcribed. These c.lones may, therefore, contain genes encoding less abundant or rare late embryo mRNAs. Moreover, we have only used mRNA from 12 to 24-hr embryos for our selections. Additional proteins might be hybrid-selecteld with early embryo RNA or enriched muscle RNA. Of the clones analyzed, XDm203, llA, 85, and 217 show either total or partial stage specificity and developmental regulation. E.ach hybridizes to transcripts that are abundant in late embryos or myotube cultures and are reduced or absent in early embryos, myoblast cultures, or Schneider cells. XDm231, on the other hand, expresses two types of transcripts. One type is expressed in all cells. XDm.227, 236, and 270 show similar levels of expression in all developmental stages and cell types. Therefore, these transcripts may be constitutively regulated. The method we have used for determining transcription of the cloned genes is quantitative. Each of the clones analyzed had the same amount of DNA blotted to nitrocellulose filters and was hybridized in excess to the same amount of radioactively labeled cDNA or mRNA probe. This meth[od has been shown to be quantitative for analysis of cDNA clones. The amount of

group

+ indicates corresponded

relative

amount

to the single

band

of hybridization

of the EcoRI

of hybridization.

hybridization to each clone has been shown to measure the relative amount of homologous mRNA present at each stage of development or tissue type (Biessmann et aZ., 1979, 1981; Kafatos et al., 1979). In addition, the pattern of hybridization of the restriction fragments can also be helpful in determining the distribution of additional transcripts if they exist. For instance, clone XDm217 hybrid-selects a single protein. However, the changes in the pattern of EcoRI restriction fragment hybridization to RNA from early and late embryos, muscle cultures, and Schneider cells suggest that there may be at least two transcribed gene regions on this 14-Kb Drosophila fragment. Similarly, XDm231 contains at least five EcoRI fragments that show stage-dependent differences in their pattern of expression. Five fragments are expressed in early embryos and at least two in myoblast cell cultures and undifferentiated Schneider cells. We have not positively identified the 1.2-, 1.4-, and 3.4-Kb coding fragments in myoblast cultures or Schneider cells. These differences may represent quantitative levels of control in these cell types or, alternatively, additional developmentally regulated transcripts. The 2.6 to 2.9-Kb-region fragment, however, is the only fragment expressed in late

282

DEVELOPMENTAL

BIOLOGY

stage embryos or myotube culture cells. It is entirely possible that these results, and those from other clones, could be accounted for by devising models of complex developmentally regulated mRNA processing or rearrangements. However, the simplest explanation of these results is that the cloned fragments encode genes for independently regulated transcripts. A more definitive conclusion on this point, however, will be derived more directly by restriction enzyme mapping analysis of each clone. This analysis will determine the location and boundaries of each transcribed region. Also, the different transcripts will be more directly determined by hybridization of each clone or subfragments of each to electrophoretically separated mRNA. These experiments are currently in progress for several of the cloned fragments. Indeed, preliminary restriction enzyme mapping data now available on two of the clones show that XDm85 contains three separate transcribed genes (Bautch and Storti, unpublished observation) while XDm217 has two separate and distinct transcribed regions and a portion of a third (Grice and Storti, unpublished observation). Recently, Scherer et al. (1981) reported a similar analysis of restriction endonucleasedigested genomic DNA fragments hybridized to staged embryo RNA and also concluded that some genomic fragments may contain multiple transcribed regions. If our findings of multiple closely linked transcripts are also correct for other clones, this could account for why we have identified clones in our original screen with no apparent muscle specificity in their expression, such as XDm227,231, and 236. The EcoRI fragments of these clones may contain DNA regions homologous to both constitutive and muscle transcripts. Therefore, developmentally regulated expression would not be observed by blot hybridization analysis. Further restriction mapping analysis will be necessary to determine whether muscle genes are present in these cloned fragments. This may have important implications for others, who attempt to screen and isolate genomic clones for developmentally regulated genes by differential hybridization of mRNA or cDNA made from different developmental stage or cell types. For instance, if two genes are linked on the same genomic DNA fragment but each is expressed differently they will appear to show constitutive or lack of stage- or tissue-specific expression by a plaque or dot blot hybridization. The in situ hybridization results indicate that each clone is complementary to a single and different chromosomal locus. Unlike the other clones reported, clone XDm236 also shows strong hybridization to the chromocenter. This suggests regions of homology with centromeric CY-and/or /3-heterochromation (Hilliker et al., 1980) which has been shown to be located in this region of the genome. The map positions of all of the clones

VOLUME

90, 1982

are widely dispersed throughout the genome except for XDm203A and 227. These two clones are relatively close to each other on the distal arm 3L. XDm236D and 217 are also relatively close to each other on the proximal portion of chromosome 2R. Each pair, however, is separated by several bands. Any similarities in the expression of the clones reported here, therefore, do not appear to be related to genetic organization. From the in situ hybridization analysis each clone appears to be a single copy or at most repetitive at a single locus. However, because of the sequence complexity of the cloned probes, multiple chromosomal sites might not be detected if complementary to only a small region of the cloned insert. Small repetitive DNA fragments or copialike elements (Finnegan et aZ., 1978) might not be detected by this hybridization. The in situ hybridization results, therefore, do not completely rule out the possibility that the transcripts which are homologous to the cloned fragments might originate from homologous sequences elsewhere in the genome. This question will be answered more definitively by the Southern blots of total genomic digest. We would like to give special thanks to Dietmar Mischke for this assistance and initial stages of this work. We also thank a generous supply of Drosophila embryos help in learning in situ hybridization.

Drs. Mary Lou Pardue use of facilities during Dr. Anthony Mahowald and Dr. Ralph Sinibaldi

and the for for

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ST~RTI AND Szw~sT

Cloning

and Characterization

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