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Volume 270, Number 9, Issue of March 3, 1995 pp. 4375-4382
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Analysis of the Paramyosin/Miniparamyosin Gene
MINIPARAMYOSIN IS AN INDEPENDENTLY TRANSCRIBED, DISTINCT PARAMYOSIN ISOFORM, WIDELY DISTRIBUTED IN INVERTEBRATES (*)

(Received for publication, July 12, 1994; and in revised form, November 4, 1994)

Miguel Maroto (§), ,  Juan J. Arredondo (§), ,  Marta San Román (¶), ,  Roberto Marco,  Margarita Cervera (**)

From the Departamento de Bioquímica & Instituto Investigaciones Biomédicas, CSIC, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Miniparamyosin, a distinct Drosophila melanogaster paramyosin isoform of 60 kDa, is shown here to be encoded by the same gene as paramyosin. The gene, located at 66D14, spans over 12.8 kilobases (kb) and is organized into 10 exons, 9 of which code for the paramyosin transcripts. An exon, located between exons 7 and 8, codes for the 5`-end of the miniparamyosin, and the two proteins share the two last exons of the gene. Mapping of the 5`-ends of these transcripts indicates that the paramyosin and miniparamyosin mRNAs arise from two overlapping transcriptional units; the miniparamyosin transcription initiation site is located inside a paramyosin intron, 8 kb downstream of the one used for paramyosin transcription. The existence of two different promoters and the conserved and nonconserved features of their sequences suggest a very complex regulation of these two muscle proteins. In fact, while paramyosin is expressed at two distinct stages of development as most other Drosophila muscle proteins, miniparamyosin appears late in development, being present only in the adult musculature. The absence of exon 1B, the specific exon of miniparamyosin, in the nematode Caenorhabditis elegans, as well as additional lines of evidence support the lack of miniparamyosin in this particular organism. However, it is present in most invertebrate species examined, including different arthropod, annelid, mollusc, and echinoderm species.

INTRODUCTION

The regulated expression of the muscle proteins is the main characteristic of myogenesis. Differentiation of a specific muscle type is achieved by a combination of mechanisms including the expression of distinct muscle proteins (1) and the expression of type-specific isoforms of the muscle proteins produced by differentially spliced transcripts or posttranscriptional modification(2, 3) . In Drosophila melanogaster, the majority of the muscle proteins seem to be encoded by a single gene, and the generation of the diversity is mainly produced by alternative splicing(4) . This diversity is often increased by the choice of alternative polyadenylation sites or posttranslational modifications(4, 5, 6, 7) . The occurrence of muscle type- and stage-specific isoforms has been maintained throughout evolution, suggesting specific roles for the different isoforms(3, 8) . However, the functional significance of expressing specific isoforms in different muscles remains an unsolved issue.

The identification of the different components of Drosophila thick filaments is a necessary step in exploiting the advantages of Drosophila for studies of muscle structure and function(4, 6, 9) . Our laboratory has been involved in the last few years in the study of the thick filament organization in Drosophila, characterizing some of the regulatory properties of muscle proteins (10, 11, 12) . The understanding of how assembly of thick filaments in the muscle fibers takes place, the molecular mechanisms involved, and the specific roles of each thick filament component remain to be clarified. Although myosin is the major component of the thick filament, paramyosin and a few additional minor proteins have also been biochemically identified as components of the thick filament core in invertebrates(13, 14) . Paramyosin, a coiled coil alpha-helical fibrillar dimer, is a structural component of invertebrate thick filaments (13, 14, 15, 16, 17, 18, 19, 20) . There is no vertebrate homolog of paramyosin(15, 16, 17, 18, 19, 20) . In Caenorhabditis, paramyosin is required for proper assembly of the body wall musculature(19) . Mutant analysis indicates that interaction of myosin with paramyosin and maintenance of the proper stoichiometry of both proteins are also necessary for assembly and determination of the thick filament length(21, 22, 23, 24) . In insects, paramyosin has been studied mainly in flight muscles(15, 16, 25) . The flight musculature, a distinct insect muscle type, has been one of the experimental systems where, at least historically, the structure and organization of the contractile tissues has been analyzed(26) . D. melanogaster paramyosin was identified and cloned in our laboratory(10, 11) , where it was shown that it has a similar sequence and molecular weight similar to those of other invertebrate paramyosins. Drosophila paramyosin was especially abundant in non-fibrillar musculature and relatively less abundant in the fibrillar indirect flight muscles(11) . Several isoforms of approximately the same molecular mass of about 107 kDa were identified in Drosophila(11) . More recently, through the identification of a cDNA, miniparamyosin, a distinct paramyosin isoform of lower molecular weight was described in Drosophila. Miniparamyosin, an isoform not previously identified in any invertebrate system, was described as exclusively present in certain types of muscles in the adult fly(27) .

The role of different forms of paramyosin in regulating the properties and complex phenomenology of the thick filaments in invertebrate muscles made it interesting to study in more detail. In this article, we present the genomic clones that code for the complete sequence of paramyosin and miniparamyosin together with their intron/exon structure. The evidence supports the idea that all isoforms are coded in D. melanogaster by a single gene. Nevertheless, paramyosin and miniparamyosin are regulated from different promoter sequences located several kilobases apart. Their transcription initiation sites and the nucleotide sequence of the promoters have been determined. The analysis of these sequences has shown the existence of several putative regulatory sites, some of which are consensus binding sites for muscle-specific transcription factors. Furthermore, by producing antibodies specific for the paramyosin and miniparamyosin isoforms we have been able to show that miniparamyosin is widely distributed among phylogenetically distant invertebrates.

MATERIALS AND METHODS

Isolation of the Genomic Clones

The genomic DNA library ( DASH) was screened following a standard protocol(28) . Three paramyosin cDNA probes were used: probe K (1-256-bp (^1)fragment) covering the paramyosin 5`-end region, probe X300 (1014-1320-bp fragment), and probe HP (3432-3652-bp fragment) from the paramyosin 3`-end region. Positive clones were purified and subcloned into pBluescript II. Clones were mapped for positions of restriction endonuclease cleavage and by Southern analysis with paramyosin cDNA probes and oligonucleotides derived from the paramyosin and miniparamyosin cDNA sequences(10, 27) .

Sequencing and PCR

Sequencing of the ends of each clone was done (T3 and T7 primers) as described(29) . Based on the paramyosin and miniparamyosin cDNA sequences, several oligonucleotides covering distinct regions of the cDNAs were synthesized and used as primers. PCR fragments were amplified through 30 cycles (94 °C/1 min, 50 °C/1 min, 72 °C/1 min) using Taq polymerase (Boehringer Mannheim).

Mapping of 5`-Termini by Primer Extension

Primer extensions were conducted basically as described by Heberlein(30) . The primers used were as follows: 5`-P3 (5`-TTC GCG TAT CTA GTA TTA TGA TTT GTG TGT-3`) and 5`-P1 (5`-CTA TAC AGC ACC GCA CGC AG-3`) complementary to nucleotides 107-136 and 70-89, respectively, of the paramyosin cDNA sequence; and mP5 (5`-CTG CAA TAT CTT AAG AGT GTC GCG TTT GAT-3`) and mP6 (5`-TCT TGT CGA GTT CGT TAT CCA ATT CAA TC-3`) complementary to nucleotides 69-98 and 109-138, respectively, of the miniparamyosin cDNA sequence. Oligonucleotides were end-labeled with 50 µCi of [-P]ATP and annealed at 60 °C for 90 min with 100 µg of total RNA from late pupae (200-210 h). The annealed primers were extended with 10 units of reverse transcriptase (Boehringer Mannheim) at 42 °C for 90 min. The same primers were used on cloned genomic DNA templates to generate dideoxy sequencing ladders that were compared with the extension products of the primers to determine the paramyosin and miniparamyosin transcription initiation sites. The primer extensions were electrophoresed on a 6% polyacrylamide-urea sequencing gel.

Nuclease S1 Protection Assay

Assays were carried out as described previously(31) . In the paramyosin assay, a 665-bp genomic fragment was amplified by PCR with the primers 5P (5`-CTT ATA TGT ATG TCA GTT CGA-3`) and 5`-P3 (described above). In the miniparamyosin assay, the 642-bp genomic PCR fragment was amplified using the primers 5mP (5`-CTG CCA CTG CTG CCA ACC GAG-3`) and mP5 (described above).

Preparation of Specific Anti-paramyosin and Anti-miniparamyosin Polyclonal Antibodies

Three antibodies were prepared. One of the antibodies was made against the polypeptide encoded by a specific exon of the paramyosin (exon 5), and the other two antibodies were raised against the polypeptide encoded by exon 1B, the specific exon of miniparamyosin. A 594-bp PCR fragment from exon 5 of the paramyosin (corresponding to nucleotides 1271-678 of the paramyosin cDNA) was cloned into the expression plasmid pT7-7. Following induction with isopropyl-beta-D-thiogalactopyranoside, a 198-amino acid polypeptide (23.2 kDa) was obtained, and inclusion bodies were purified. A 309-bp PCR fragment from exon 1B of miniparamyosin (corresponding to nucleotides 123-431 of the miniparamyosin cDNA) coding for a 103-amino acid polypeptide (11.7 kDa) was cloned in two different plasmids: pUEX3 and pMALc. The fusion proteins of 128.5 kDa and 53.5 kDa, respectively, were synthesized after induction, and inclusion bodies were purified. The three polypeptides were run on SDS-polyacrylamide gels and cut from the gels, and the bands were used to immunize the rabbits(32) . Specific antibodies against the polypeptide expressed by exon 1B were obtained by affinity purification (28) . Antisera corresponding to each miniparamyosin fusion protein were purified by adsorption with the other miniparamyosin fusion protein immobilized on strips of nitrocellulose. The specificity of the three antibodies was tested. They recognized only one band, paramyosin or miniparamyosin, as expected, by Western blot in thorax samples (data not shown). The two miniparamyosin antibodies recognized the protein with the same specificity and intensity. Since the titer was higher, this work was done using the antibody obtained with the pUEX3 construct. The three antibodies gave a similar sarcomeric band pattern by immunofluorescence analysis with isolated Drosophila muscle fibers.

SDS-Polyacrylamide Gels and Immunoblot Analysis

Dissected muscles or whole animals were homogenized in 50 mM Tris, pH 7.0, 140 mM ClNa, 0.5% Triton X-100, 5 mM EDTA, 10 mM EGTA, 0.1 mg/ml soybean trypsin inhibitor, 0.1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml antipain. Insoluble cytoskeletal protein samples from various stages of Drosophila development were prepared as described previously (11) . Electrophoresis and immunoblot analysis were done as described previously(11) . The amount of protein loaded in each lane was about 20 µg.

RESULTS

The Structure of the D. melanogaster Paramyosin/Miniparamyosin Gene

After screening of a DASH genomic library using paramyosin cDNA probes from the 5`, 3`, and middle region of the gene (see ``Materials and Methods''), three different genomic clones were isolated. The positives clones, containing inserts of 16, 17, and 17.5 kb, were analyzed. All clones share the same restriction sites in the overlapping regions and cover a region of 25 kb (Fig. 1a). The genomic DNA clones were partially sequenced, and it was found that the gene spans 12.8 kb and comprises 10 exons and 9 introns (Fig. 1a). Paramyosin is coded by 9 exons, all except exon 1B. This exon, located in an intron following exon 7 of paramyosin, codes for the 5`-end of miniparamyosin. Thus, the paramyosin/miniparamyosin gene codes for two proteins that share the two last exons, 8 and 9, of the gene. Furthermore (Fig. 1b), the positions of the intron-exon boundaries were defined precisely. The donor and acceptor splice sites correlate with the splice junction consensus for Drosophila(33) .

Figure 1: Genomic structure and organization of the paramyosin/miniparamyosin gene in the 66D14 locus. a, in the upper part the three analyzed genomic clones and the restriction map of the clones (H, HindIII; E, EcoRI) are shown. Black squares indicate cDNA probes (K, X300, and HP) used and their positions with respect to the genomic clones. In the lower part a schematic representation of the paramyosin/miniparamyosin gene is shown with the numbered exons in black boxes. A portion of this organization appears in a figure of a recent review(6) . In the lower part the complete restriction map of the DNA region is presented (B, BamHI; H, HindIII; E, EcoRI; X, XhoI; Xb, XbaI; Hc, HincII; P, PstI). b, the nucleotide sequences of the intron-exon boundaries of the genomic clones. The donor and acceptor splice sites correlate with the splice junction consensus for Drosophila(33) . Numbers in brackets specify the nucleotides in each exon. The approximate size of the introns is at the right of the figure in bp or kb (indicated). The positions of the boundaries are indicated by the number(s) appearing above each sequence. Numbering starts at the initial nucleotide of the complete paramyosin and miniparamyosin (italics) cDNAs.


Paramyosin and Miniparamyosin Are Regulated from Two Different Promoters 8 kb Apart

Paramyosin and miniparamyosin present at least two transcripts each, differing in the choice of alternative polyadenylation sites(10, 27) . The 5`-ends of the paramyosin and miniparamyosin transcripts were mapped by primer extension analysis and nuclease S1 protection assays (Fig. 2). Assays were performed with total late pupae RNA because both the paramyosin and miniparamyosin transcripts are present at this developmental stage(10, 27) . The results in both assays indicate the existence of two transcription initiation sites for paramyosin mRNA, with the main one corresponding to the already published cDNA sequence. Slightly longer cDNAs with sequences corresponding to the minor site of transcription initiation at nucleotides (-35 to -37) have not yet been identified (10, 27) . In contrast, only one transcription start site for miniparamyosin has been detected. According to the mapping of the 5`-end of the miniparamyosin mRNAs (Fig. 2, a and b), this transcription start site is located 45 bp upstream of the published cDNA sequence (Fig. 2c).

Figure 2: Identification of the transcription initiation sites of the paramyosin amd miniparamyosin mRNAs. a, primer extension analysis of the paramyosin and miniparamyosin transcription units (see ``Materials and Methods''). D. melanogaster late pupae total RNAs were annealed with oligonucleotides complementary to paramyosin (PM, upper panel) and miniparamyosin (mPM, lower panel) and extended with reverse transcriptase. The same primers were used with the appropriated genomic clones to generate the dideoxy sequencing ladders shown in the insets. The transcription initiation start sites are indicated by arrows. Numbering starts at nucleotide +1 corresponding, respectively, to the major initiation starts of the paramyosin and miniparamyosin transcripts as detected in these assays. b, nuclease S1 protection assay with genomic fragments amplified by PCR (see ``Materials and Methods''). Two different fragments corresponding to the 5`-regions of exon 1A (for paramyosin) and exon 1B (for miniparamyosin) were used. c, the sequences of the genomic regions corresponding to the paramyosin (PM) and miniparamyosin (mPM) transcription initiation sites are presented. Arrows indicate the transcriptional starting sites. The starting nucleotides of the published paramyosin and miniparamyosin cDNAs (10, 27) are indicated by bent arrows. As can be seen, 45 nucleotides were missing in the miniparamyosin cDNA.


In an effort to identify conserved sequences that may also serve a regulatory function, we have cloned and sequenced the 5`-flanking regions of the paramyosin and miniparamyosin transcriptional initiation sites in D. melanogaster and Drosophila virilis. These two Drosophilidae species diverged more than 50 million years ago and therefore are useful to detect evolutionarily conserved regulatory features. In Fig. 3, the nucleotide sequences of the regions extending about 350 and 450 nucleotides for paramyosin and miniparamyosin, respectively, upstream of the main transcription initiation sites are presented. The nucleotide sequences reveal a number of potential regulatory elements (boxed or underlined in Fig. 3). The comparison with the homologous D. virilis sequences allows the preliminary identification of the functionally significant features in the sequence (boxed in Fig. 3). The pentanucleotide TCAGT, a consensus sequence for the eucaryotic promoter cap site (34) and for the regulatory initiator element(35) , is present in the sequenced paramyosin and miniparamyosin transcriptional initiation sites. It is highly conserved between the two Drosophilidae and provides support for the likelihood of a functional role of these motifs. Furthermore, paramyosin and miniparamyosin proximal promoters in D. melanogaster show AT-rich regions between -25 and -40 from the main transcriptional initiation sites. Interestingly, the comparison with the D. virilis sequences suggests distinct roles for these putative TATA boxes. Whereas the AT-rich region of the paramyosin promoter is exactly conserved in D. virilis, validating the likelihood of it serving as a functional element, the corresponding region of the miniparamyosin promoter is not conserved in D. virilis. In addition, the CG and CCAAT elements, found to be critical in numerous eucaryotic promoters, were also detected. The paramyosin proximal promoter has a putative CCAAT element at position -45 that could be recognized by CCAAT transcriptional factors (36) and is conserved in the two Drosophilidae species. An equivalent CCAAT element is not found in the miniparamyosin promoter. Instead, several GC boxes that could bind the general transcriptional factor Sp1 (37) are present at positions -45 and -52 in both Drosophilidae miniparamyosin promoters. Even though two GC boxes at -121 and -136 are present in the paramyosin promoter, they are not conserved in the D. virilis sequence, which suggests they have no functional role. Similarly, the GC element present at -113 in the miniparamyosin promoter is also absent in the D. virilis sequence. Therefore, in spite of being present in both D. melanogaster promoters, it is doubtful that these GC boxes contribute to the regulation of these proteins.

Figure 3: Comparison of the paramyosin and miniparamyosin promoters of D. melanogaster and D. virilis. The upper panel (a) illustrates the regions extending 340 and 357 nucleotides upstream of the transcriptional initiation sites of the D. melanogaster and D. virilis paramyosin promoters, respectively. The lower panel (b) illustrates regions extending 440 and 453 nucleotides upstream of the initiation starts of the D. melanogaster and D. virilis miniparamyosin promoters, respectively. The initiation starts are indicated by black circles, and +1 corresponds to the main ones. Putative regulatory elements are boxed when conserved and underlined when unconserved in the corresponding D. virilis sequence. Underneath are the names of the corresponding putative DNA-binding elements, namely, E-box, the consensus binding site for the MyoD family; CCAATT, the consensus sequence for the CCAATT binding elements; A-T, the A-T-rich regions; and G-C, the putative GC boxes.


The possibility of identifying cis-acting DNA elements responsible for muscle-specific transcription of the paramyosin/miniparamyosin gene was also examined. The analysis of the currently available sequence in the two Drosophilidae species reveals the conservation of several E-boxes in both promoters. The sequence CANNTG, or E-box, has been proposed as the consensus binding site of muscle-specific transcription factors of the MyoD family(38) . The paramyosin promoter has only one conserved E-box at -263, and the miniparamyosin sequence has two E-boxes, at -59 and -152, all numbered from the transcription initiation sites. On the other hand, two E-boxes at -61 and at -296 in the D. melanogaster paramyosin promoter are not conserved in D. virilis; neither is the E-box at -417 in the miniparamyosin promoter.

Miniparamyosin Expression during Drosophila Development

To confirm that the two proteins are independently expressed in Drosophila, the developmental time course of the miniparamyosin expression was determined by Western analysis using the specific antibody against miniparamyosin produced in our laboratory (see ``Materials and Methods''). Miniparamyosin was first detected in third instar larvae (90-100 h of development), and its levels decreased during early pupal stages. In late pupae (170-180 h of development), the levels increased progressively, reaching maximal levels at adulthood. Western analysis of thorax samples from 20-, 30-, and 45-day-old flies indicates that the amount of miniparamyosin remains constant during aging (data not shown). In contrast, paramyosin was detected at approximately 10-12 h of embryonic development and remained relatively constant. The amount of paramyosin decreases only slightly at pupal stages, in contrast to the levels of mRNA(10, 27) .

The Genomic Organization of the D. melanogaster Paramyosin Gene Is Different from That of Caenorhabditis elegans

The only paramyosin gene for which the detailed sequence is known is in C. elegans(39) . As can be seen in Fig. 4, conservation of the exon and intron organization of the two paramyosin genes is very limited. Only two exons (namely, exons 2 and 3 of D. melanogaster that correspond to exons 3 and 4 of the nematode) are conserved and share the same splice sites. In addition, exon 4 of D. melanogaster corresponds to exons 5 and 6 in C. elegans. It is noteworthy that the sequence of exon 1B, the specific exon of the miniparamyosin, is not present in any region (exons or introns) of the C. elegans paramyosin gene sequence. In fact, exon 7 and 8 of the D. melanogaster paramyosin gene, the flanking exons of exon 1B, are linked in the same exon of the C. elegans paramyosin gene (Fig. 4). Thus, in contrast to paramyosin, either miniparamyosin is not generally expressed in invertebrates or it is transcribed from a still unidentified gene in C. elegans.

Figure 4: Exon 1B of the D. melanogaster miniparamyosin is not included in the C. elegans paramyosin gene. A comparison of D. melanogaster and C. elegans(39) genomic organizations is shown. Boxes represent exons, and lines represent introns. Interrupted lines mark the unidentified C. elegans 5`-genomic region (according to data bank information). Genomic regions coding for homologous peptides are indicated by lines joining the two genomic regions.


To explore this alternative, we have used the polyclonal antibodies against specific regions of paramyosin/miniparamyosin gene (see ``Materials and Methods''). The presence of a similar cross-reactive protein was investigated by immunoblotting analysis of extract from several representative members of different invertebrate phyla (namely, Diptera (D. melanogaster, D. virilis, and Calliphora), Hymenoptera (Formica), Coleoptera (Coccinella), Orthoptera (Locusta and Blatta), and a member of the Apterygota, the silverfish (Lepisma)) and from representative members of other arthropod classes (Arachnida (Araneus), Crustacea (Astacus) and Chilopoda (Scalopendra)). In parallel, muscle extracts from additional representative protostome invertebrates were also processed and tested (namely, Mollusca (Mytilus and Helix), Annelida (Lumbricus), and Nematoda (Caenorhabditis)). In addition, muscles from a deuterostome invertebrate, the sea urchin Sphaerochinus, and a mammal, Rattus, are included. In Fig. 5, Western blots made with three different antibodies (namely, anti-miniparamyosin (anti-exon 1B), anti-paramyosin (an antibody made against the purified protein(11) ), and anti-exon 5 antiserum (specific to paramyosin in D. melanogaster) are presented (see ``Materials and Methods''). The miniparamyosin antiserum recognizes a protein of lower molecular mass than paramyosin (50-80 kDa) in all preparations except those from Caenorhabditis and Rattus. Interestingly, the miniparamyosin polyclonal antibody also cross-reacts with paramyosin in all the analyzed species except in Diptera (the two Drosophilae species and Calliphora) and Caenorhabditis (Fig. 5b). In Fig. 5c, the reaction with the antiparamyosin antibody made against the purified protein (11) recognizes paramyosin in all the invertebrate species. The reaction in the case of Caenorhabditis is very weak and is not visible in the figure. The absence of paramyosin in mammalian muscles is well known. In Fig. 5d, the anti-exon 5 antiserum recognizes paramyosin in all invertebrate species tested, except in Coccinella where it recognizes myosin instead. It is worth noting that in more evolutionarily distant invertebrates, the anti-exon 5 antiserum increasingly recognizes myosin in addition to paramyosin.

Figure 5: Differential expression of miniparamyosin in species representative of a wide range of protostome phyla. Whole extracts were prepared from representative members of the following insect classes; Diptera, flies (D. melanogaster and virilis) and blowflies (Calliphora); Hymenoptera, ants (Formica); Coleoptera, ladybugs (Coccinella); Orthoptera, locusts (Locusta) and cockroaches (Blatta); and Apterygota, silverfishes (Lepisma) were prepared. In parallel, muscle extracts from other arthropod classes (Chilopoda, centipede (Scalopendra); Arachnida, spider (Araneus); and Crustacea, crayfish (Astacus)) as well as from other representative protostome invertebrate phyla (namely, Annelida, earthworm (Lumbricus); snail (Helix); and Mollusca, mussel (Mytilus)) were tested. About 20 µg of protein corresponding to each sample were run in 10% SDS-polyacrylamide gels (a), and Western blots were cross-reacted with the the following antibodies: D. melanogaster anti-miniparamyosin (b), antiparamyosin (c), and anti-exon 5 of paramyosin (d). In the right part of the figure, the molecular weight of the markers and the positions of myosin (M), paramyosin (PM), and miniparamyosin (mPM) are indicated.


DISCUSSION

By using a probe against its 3`-end, it was previously shown that D. melanogaster paramyosin is coded by a single gene(10, 27) , since this region is shared with miniparamyosin. It was thought that both proteins were encoded by the same gene. Our studies on the genomic organization of the paramyosin/miniparamyosin gene support this conclusion. The gene located at 66D14 (10) spans 12.8 kb and is organized into at least 10 exons and 9 introns. The available nucleotide sequence of the genomic DNA has allowed the identification of the exact positions of the splice junctions. All exons identified could be ascribed to the paramyosin sequence except for exon 1B (in between exon 7 and 8 of the paramyosin). This exon, together with exons 8 and 9, codes for miniparamyosin.

The comparative analysis of the paramyosin and miniparamyosin promoters in D. melanogaster and D.virilis suggests interesting differences. Only the paramyosin promoter shows a putative TATA box, the conserved AT-rich region, but both promoters show the same initiator/cap site sequence. The main initiation start sites for miniparamyosin and paramyosin mRNAs show an identity of 8-11 nucleotides and share the initiator consensus sequence, TCAGT(34, 40) . This initiation promoter and related sequences are present in several Drosophila muscle genes studied to date(41, 42, 43, 44) . Recent work has shown that in genes lacking TATA boxes the initiator element plays a role functionally analogous to the TATA element. Through its interaction with the TFIID complex, it is capable of directing the basal transcription by RNA polymerase II and of determining the precise site of transcription initiation(35, 45) . In promoters that contain a TATA box, the initiator element greatly enhances promoter strength (45) . Therefore, the initiator elements present in the two promoters driving the expression of the paramyosin and miniparamyosin may play different roles. The decision as to whether the conservation of the AT-rich region at -39 in the paramyosin promoter is attributable to its serving as putative TATA box or as an alternative transcription initiation site (see above) must wait until further experimental evidence is obtained. In addition, the paramyosin promoter has a conserved CCAAT box within an appropriate distance of the transcriptional start. The CCAAT element can be recognized by different transcriptional factors in different cells; in some cases, they produce transcription stimulation, whereas in others, they result in repression (46) . The role of this element in this promoter may be interesting to study, since most D. melanogaster muscle promoters do not contain CCAAT boxes in spite of the fact that they drive patterns of expression very similar to the paramyosin promoter. With regard to the differences between these two promoters, an additional evolutionary conserved element, the GC motif, is found in the miniparamyosin promoter of both Drosophilidae but not in the D. virilis paramyosin promoter. Complete clarification of the relative importance of the different features of these inititation sequences in different types of muscle and developmental stages must await the results of in vivo and in vitro expression studies. Nevertheless, the results presented in this and earlier work (10, 27) fully support the concept that the D. melanogaster paramyosin/miniparamyosin gene is expressed from two overlapping transcriptional units that encode two proteins, paramyosin and miniparamyosin, using two promoters and a combination of different origins of transcription and alternative polyadenylation sites.

A number of eucaryotic genes have now been characterized containing two promoters (for example, see (47) ). Among D. melanogaster muscle genes, a similar situation to that of the paramyosin/miniparamyosin gene has been detected in the tropomyosin II gene(42) . This gene encodes for both a specific muscle isoform and a cytoplasmic isoform. The first exon of the cytoplasmic form is located between exons 3 and 4 of the muscle-expressed isoform. Both genes have an internal promoter that drives the expression of a smaller protein with a different expression pattern; miniparamyosin is only expressed in the adult musculature, whereas the smaller form of tropomyosin II is a cytoplasmic isoform. In contrast with the tropomyosin II gene, both paramyosin and miniparamyosin promoters are muscle-specific, whereas the promoter used by the cytoplasmic tropomyosin II isoform is a more generalized housekeeping type promoter. In addition, the paramyosin/miniparamyosin gene promoters are regulated differentially. The paramyosin promoter is active throughout development in developing muscle cells of the embryo, larva, and adult. This pattern is equivalent to most muscle proteins in D. melanogaster including myosin, the main component of the thick filament(6) . In contrast, the miniparamyosin promoter is active mainly in the adult musculature. The protein has been detected transiently in third instard larvae, becomes fully expressed in adults, and maintains its expression in older adults. It is interesting to point out that the expression of the two proteins is switched on both at the mRNA and polypeptide levels (27, 11, this paper). Different myogenic programs are known to exist, controlling the expression of muscle-specific genes. The activation of most of the skeletal muscle-specific genes characterized to date requires a functional binding site for one of the known betaHLH myogenic factors. The sequence CANNTG, or E-box, constitutes an integral component of the positive regulatory elements, mainly enhancers, of muscle-specific genes. Since not all the muscle types express MyoD, it is likely that additional factors, like MEF-2, M-CAT, or CarG factors, are also involved in the differential muscle regulation(48, 49, 50) . Moreover, recent data suggest that the interaction of MyoD and MEF-2 plays a central role in the regulation of the muscle expression. In Drosophila, MyoD and MEF-2 homologs have been isolated(51, 52, 53) . The study of the cis-elements involved in the regulation of the expression of two muscle proteins encoded by the same gene, such as paramyosin and miniparamyosin, could provide an interesting paradigm of the myogenic programs such as those that control the expression level of different proteins in different types of Drosophila muscles at distinct stages of development.

The organization of the C. elegans gene, the only well characterized paramyosin gene, was compared with that of the D. melanogaster gene. In C. elegans, the gene is organized into 11 exons, which encode for a protein with high homology to the D. melanogaster paramyosin(10) . When the respective splice junctions were analyzed, a greater degree of conservation in the splicing sites was found in the amino-terminal region of the genes. In the rest of the sequence, the arrangement of exons differs (Fig. 4). In particular, exon 5 in D. melanogaster (the one that was chosen to raise the specific paramyosin antibody) has its homologous peptide encoded by exons 7, 8, and part of 9 of C. elegans. Furthermore, the sequence of exon 1B, the specific exon of the miniparamyosin, was not found in any known region (exons or introns) of the C. elegans paramyosin gene. No space exists for an exon 1B homolog in C. elegans. The exons that flank 1B, exons 7 and 8, as well as part of exon 5 and exon 6 in D. melanogaster, correspond in C. elegans to a single exon (exon 9). This lack of conservation of the exon/intron organization of paramyosin contrasts with the situation in many of the muscle genes, for example in the tropomyosin II gene, in which the exon organization has been conserved in all species examined so far including vertebrates and invertebrates(42) . The high variation in the genomic organization of this gene is substantiated by the available sequence of Drosophila virilis, which indicates that there are even variations between the paramyosin splicing sites in these two Drosophilidae. (^2)Several lines of evidence indicate that miniparamyosin is absent in the pseudocoelomate C. elegans. First, there is a lack of a similar sequence in the gene, insofar as the genetic organization is presently known. Second, Northern analysis failed to identify additional mRNAs to the paramyosin ones in C. elegans(54) . Our antibodies and, more interestingly, the C. elegans anti-paramyosin polyclonal antibody also failed to detect a second isoform of different molecular weight in this organism (13, this paper).

The evolutionary diversification of paramyosin in invertebrate muscle, including the presence of miniparamyosin, has been approached through the use of antibodies specific for the two isoforms. Both types of proteins are present in all invertebrate species investigated except C. elegans. Whereas slight variations in the molecular mass of paramyosin were known (10, 11, 39; see Fig. 5, c and d), the molecular mass of miniparamyosin shows a much wider range of variation (from 50 to 80 kDa; see Fig. 5b). The miniparamyosin antiserum recognizes a single protein with a similar mobility as miniparamyosin only in Diptera. In all other species analyzed, the antiserum recognizes two proteins, paramyosin and the putative miniparamyosin. This result suggests that the D. melanogaster miniparamyosin-specific protein domain, exon 1B, may be expressed not only in the miniparamyosin but also in the paramyosin of the other species. In Annelida and Mollusca, the cross-reaction with the paramyosin was stronger with the miniparamyosin antibody than with a paramyosin-specific antibody (prepared with a peptide encoded by exon 5), suggesting that the specific function of the protein domain coded by exon 1B is in some species included in the paramyosin gene. This is in accordance with the much greater variability in the exon organization of the paramyosin/miniparamyosin genes mentioned in the preceding paragraph. In light of this, it is interesting that the anti-exon 5 antibody, specific to paramyosin, is able to cross-react not only with paramyosin but with myosins of several invertebrate species. Coccinella provides an extreme example because only the myosin cross-reaction occurs. This suggest that a similar exchange of functions may be occurring between parts of myosin and paramyosin in these species. Furthermore, in the case of the deuterostome invertebrates, such as Echinodermus, the results suggest that a miniparamyosin isoform may be present in addition to the already known paramyosin. In conclusion, we suggest that the patterns of antibody cross-reactivity obtained reflect different specializations of the paramyosin gene complex such that, in Diptera (possibly through the evolution of a separate regulatory control) miniparamyosin has acquired a structure and function distinct from those of paramyosin. In any case, the widespread occurrence of miniparamyosin in invertebrate muscles and the complexity of the transcriptional regulation of the two distinct isoforms encoded by a single gene in Drosophila species point to the importance of the functional role of paramyosin and miniparamyosin in producing the structural and functional diversity of invertebrate muscles. Future work, currently under way in our laboratory, will contribute to the clarification of these important questions.

FOOTNOTES

*
This work has been supported by Grants PB91-0098 and ESP91-627 from the DGICYT (Spanish Ministry of Education and Science) and Plan Nacional de Investigación Científica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X79485 [GenBank]and X79484[GenBank].

§
Predoctoral fellows from the Universidad Autónoma de Madrid with funds provided by the European Space Agency.

Supported by a contract from the Fundación de la Universidad Autónoma de Madrid with funds of the Plan Nacional del Espacio.

**
To whom correspondence should be addressed. Tel.: 34-1-397-5409; Fax: 34-1-585-4587.

(^1)
The abbreviations used are: bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction.

(^2)
J. J. Arredondo et al., unpublished results.

ACKNOWLEDGEMENTS

We heartily thank Dr. Hanh Nguyen and Dr. Terri Williams for critical reading of the manuscript and corrections of the English version. Antonio Fernández and Elena Fraga expertly prepared the photographic material. Juan Roberto Marco and Lorenzo Marco helped in gathering several of the invertebrates used in this work. Helpfully, Dr. H.Y. Marthy provided the echinoderm muscle sample, and Dr. Mercedes Martínez provided the nematodes.

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