Multiple actins in Drosophila melanogaster.

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.

The Drosophila melanogaster tropomyosin II gene produces multiple proteins by use of alternative tissue-specific promoters and alternative splicing.

The structure of the Drosophila melanogaster tropomyosin II (TmII) gene has been determined by DNA sequencing of cDNA clones and the genomic DNA coding for the gene. Two overlapping transcriptional units produce at least four different tropomyosin isoforms. A combination of developmentally regulated promoters and alternative splicing produces both muscle and cytoskeletal tropomyosin isoforms. One promoter is a muscle-specific promoter and produces three different tropomyosin isoforms by alternative splicing of the last three 3' exons. The second promoter has the characteristics of a housekeeping promoter and produces a cytoskeletal tropomyosin isoform. Several internal exons along with a final 3' exon are alternatively spliced in the cytoskeletal transcript. The intron-exon boundaries of the TmII gene are identical to the intron-exon boundaries of all vertebrate tropomyosin genes reported, but are very different from the intron-exon boundaries of the D. melanogaster tropomyosin I gene. The TmII gene is the only reported tropomyosin gene that has two promoters and a quadruple alternative splice choice for the final exon. Models for the mechanism of D. melanogaster tropomyosin gene evolution are discussed.

Alternative splicing of a Drosophila tropomyosin gene generates muscle tropomyosin isoforms with different carboxy-terminal ends.

The muscle tropomyosin I (mTm I) gene from Drosophila melanogaster has been analyzed and shown to express a complex transcription unit consisting of two sets of tissue-specific mRNAs. A 1.3- and 1.6-kilobase set of mRNAs is expressed during myogenesis in embryos, and in myogenic cell cultures. The mRNAs encode a 34,000-dalton muscle tropomyosin isoform. The same mTm I gene expresses a different set of 1.7- and 1.9-kilobase mRNAs in thoracic flight muscle tissue of the adult. The thorax RNAs encode a new tropomyosin isoform resolved on two-dimensional gels. The structure of the gene has been determined, and we show that the embryonic and thoracic mRNAs are generated by alternative splicing. The alternate exon splicing patterns determine a different 27 amino acids at the carboxy-terminal end of the two tropomyosin isoforms. These results show that the carboxy-terminal domain of tropomyosin is highly regulated in determining tropomyosin function. The results also show that contractile protein isoforms can be generated by single as well as multiple genes.

A muscle-specific intron enhancer required for rescue of indirect flight muscle and jump muscle function regulates Drosophila tropomyosin I gene expression.

The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless and jumpless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of our analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70-Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes. One of the intron domains is required for expression in the indirect flight muscle of the adult. The function of the second domain is unknown, but it could regulate the level of expression or be required for expression in other muscle.

Drosophila P element-enhanced transfection in mammalian cells.

We constructed a gene transfer vector containing the herpes simplex virus type 1 thymidine kinase (TK) gene flanked by Drosophila P element terminal repeats (W. R. Engels, Annu. Rev. Genet. 17:315-344). This vector was introduced into mouse LTK- cells and enhanced the frequency of stable transformation to the TK+ phenotype by approximately 50-fold relative to a similar plasmid lacking the P element terminal repeats.

Small differences in Drosophila tropomyosin expression have significant effects on muscle function.

The effects of promoter deletions on Drosophila tropomyosin I (TmI) gene expression have been determined by measuring TmI RNA levels in transformed flies. Decreases in RNA levels have been correlated with rescue of flightless and jumpless mutant phenotypes in Ifm(3)3 mutant transformed flies and changes in muscle ultrastructure. The results of this analysis have allowed us to identify a region responsible for 20% of maximal TmI expression, estimate threshold levels of TmI RNA required for indirect flight and jump muscle function, and obtain evidence suggesting that sarcomere length may be an important determinant of flight muscle function.

In vitro transcription analysis of the Drosophila tropomyosin and other muscle genes.

Nuclear transcription extracts were prepared from embryos of Drosophila melanogaster to study the in vitro transcription of the tropomyosin genes. Several non-muscle gene promoters, including the non-muscle promoter of the Tropomyosin II gene, were shown to be efficiently transcribed in vitro. The Tropomyosin I gene and the muscle promoter of the Tropomyosin II gene, as well as two other contractile protein muscle genes, were not transcribed in vitro. The embryonic extract did, however, contain developmental-specific proteins that bound to the muscle enhancer regulatory region of the Tropomyosin I gene.

Nucleotide sequence of a cDNA clone encoding a Drosophila muscle tropomyosin II isoform.

A cDNA clone was sequenced that contains the entire coding region for the muscle tropomyosin II isoform from Drosophila. The cDNA clone is 1253 nucleotides (nt) long and contains an 88-nt 5'-leader sequence and a 310-nt 3'-untranslated sequence. The muscle tropomyosin II isoform consists of 285 amino acids and is 60% homologous with the previously reported muscle tropomyosin I isoform Drosophila and 55% homologous with rabbit muscle tropomyosin.

One- and two-dimensional polyacrylamide gel analysis of the heat shock proteins of the virilis group of Drosophila.

The heat shock proteins of the virilis group of Drosophila are analyzed by one- and two-dimensional polyacrylamide gel analysis. This group consists of the two closely related but distinct virilis and montana phylads. The analysis reveals that some of the heat shock proteins are highly conserved among the two phylads while others are not. The 83-, 72-, and 69-kdalton proteins comigrate in all species examined. There is, however, a noticeable trend toward greater molecular weight variability in the smaller heat shock proteins. In general, the heat shock protein patterns within each phylad follow the proposed phylogenetic relationships with some exceptions. D. ezoana and D. littoralis, both members of the montana phylad, exhibit heat shock protein patterns more similar to those of the virilis phylad. The data also demonstrate that the montana phylad has almost two times the heat shock allele members that the virilis phylad has. It is also shown that F1 and F2 hybrid flies of crosses between Drosophila species having different patterns of heat shock proteins show Mendelian segregation of alleles. After several generations of inbred growth, however, the pattern of heat shock protein synthesis in reciprocal hybrids each resembles that of the paternal parent. The implications of these findings are discussed.

Structure and DNA sequence of the tropomyosin I gene from Drosophila melanogaster.

The muscle tropomyosin I gene of Drosophila melanogaster undergoes alternative splicing in different muscles of the fly to generate two isoforms of the same protein. We report here the structural analysis and DNA sequence of the tropomyosin I gene. The gene spans 5 kilobase of DNA and is comprised of five exons and four introns. Exon 4 is alternatively spliced in RNA of different muscle, resulting in two isoforms of the same protein. The gene lacks a "TATA" box homology at the map position; it is usually found in the vast majority of eukaryotic genes characterized thus far. Instead, a series of three alternating TG stretches are located upstream from the site of initiation of transcription. The gene encodes a 5' untranslated leader of 103 base pairs, and the 3' untranslated region comprises between 30 and 50% of the transcripts. The DNA sequence is extremely G + C rich in the protein coding regions of the gene, and A + T rich in the non-coding, flanking, and intron regions. The DNA sequence upstream of the acceptor sites in the two introns which are subject to alternative splicing displays a stretch of homology which is noted. The 3' untranslated region of the fifth exon contains multiple polyadenylation sites. The 284 amino acid protein encoded by the gene is split by introns between residues 198/199 and 257/258. These sites correlate closely with two important functional domains in the tropomyosin molecule. A comparison of the first 257 amino acids and the carboxyl-terminal 27 amino acids of the Drosophila and vertebrate tropomyosins together, shows two distinct and mutually exclusive classes for these domains. The functional significance of the Drosophila tropomyosin isoforms is discussed.

Developmental regulation of the Drosophila Tropomyosin I (TmI) gene is controlled by a muscle activator enhancer region that contains multiple cis-elements and binding sites for multiple proteins.

Developmental gene regulation in vertebrate somatic muscles involves the cooperative interaction of MEF2 (myocyte-specific enhancer-binding factor 2) and members of the b-HLH (basic helix-loop-helix) family of myogenic factors. Until recently, however, nothing was know about the factors that control the developmental regulation of muscle genes during embryogenesis in Drosophila. The Drosophila Tropomyosin I (TmI) gene contains a proximal and distal muscle enhancer within the first intron that regulates its expression in embryonic/larval and adult muscles. We have recently shown that the 355-bp proximal enhancer contains a binding site for the Drosophila homologue of vertebrate MEF2 and that MEF2 acts cooperatively with a basal level muscle activator region to direct high level muscle expression in transgenic flies. The 92-bp muscle activator region, however, does not contain any consensus E-box (CANNTG) binding site sequences for b-HLH myogenic factors, suggesting the MEF2 may interact with other factors to regulate muscle genes in Drosophila. In this study we have used mutation analysis and germ-line transformation to analyze cis-acting elements within the muscle activator region that regulate its expression in transgenic flies. We have identified a 71-bp region that is sufficient for low basal level temporal- and muscle-specific expression in the embryo, larva, and adult. Substitution mutations within the muscle activator region have identified several cis-element regions spanning 60-bp that are required for either full or partial muscle activator function. An analysis of proteins that bind to this region by gel mobility shift assay and copper nuclease footprinting has allowed us to identify the sites in this region at which multiple proteins complex and interact. We propose that these cis-elements and the proteins that they bind regulate muscle activator function and together with MEF2 are capable of regulating high level muscle expression.

Identification of a cytoplasmic tropomyosin gene linked to two muscle tropomyosin genes in Drosophila.

A Drosophila cytoplasmic tropomyosin gene has been identified and is located on the same genomic DNA clone as two Drosophila muscle tropomyosin genes previously identified. A positive hybrid-selection translation assay using the subcloned gene and RNA from non-muscle cell sources yielded a protein with a size (Mr, 31,000) and isoelectric point (5.0) similar to vertebrate cytoplasmic tropomyosin. A modified protocol for the purification of vertebrate cytoplasmic tropomyosin was used to partially purify cytoplasmic tropomyosin from the Drosophila Kc cell line. The Kc cell protein was identified as a cytoplasmic form of tropomyosin on the basis of its size, isoelectric point, and crossreactivity with a polyclonal vertebrate antitropomyosin antibody in a two-step binding assay. The Kc cell cytoplasmic tropomyosin comigrates in two dimensions with the hybrid-selected in vitro translation product of the region 3 gene, and both proteins show a mobility shift in NaDodSO4/urea/polyacrylamide gels that is characteristic of vertebrate tropomyosins. The cytoplasmic tropomyosin gene hybridizes to both Drosophila muscle tropomyosin genes under decreased stringency conditions. This cross-hybridization spans several internal restriction endonuclease sites in each muscle tropomyosin gene and indicates an overall partial homology among the three Drosophila tropomyosin genes. These results show that Drosophila tropomyosins are encoded by a closely linked family of differentially regulated genes.

Developmental regulation of the Drosophila tropomyosin II gene in different muscles is controlled by muscle-type-specific intron enhancer elements and distal and proximal promoter control elements.

Transcriptional control of the Drosophila tropomyosin II gene muscle promoter has been investigated by expressing TmII promoter lacZ reporter gene constructs in P-element-mediated transformed flies. A TmII/lacZ reporter gene containing 243 bp of upstream sequence, the first exon, the first intron, and 72 bp of the second exon was expressed in all muscles of embryos, larvae, and adults. Deletion of upstream sequences between -243 and -22 bp only reduced the levels of transgene expression in muscle while deletion of the intron eliminated expression. Analysis of deletions within the first intron indicated that a 454-bp muscle enhancer region, from +167 to +621, was required for high levels of transgene expression in all larval and adult muscles. When this region was deleted low levels of expression still occurred in larval and adult somatic and visceral muscles; however, there was no detectable expression in adult indirect flight and jump muscles. The 454-bp muscle enhancer region was also able to drive muscle-specific expression when placed upstream of a heterologous hsp70 promoter; however, three subfragments of the 454-bp region were unable to drive expression of the hsp70 promoter, suggesting that this region may contain multiple interacting cis-acting elements. Interestingly, the 454-bp region was inactive when placed upstream of a TmII promoter construct containing upstream DNA and most of the first exon but was active when additional exon and intron DNA was included, indicating that additional promoter elements are located in this region. Thus TmII transcription is controlled by multiple muscle type-specific cis-acting control elements and upstream and downstream promoter control elements.

Chick cytoplasmic actin and muscle actin have different structural genes.

Actins isolated from embryonic chick brain and muscle differ in mobility when subjected to electrophoresis in gels containing urea and sodium dodecyl sulfate. Experiments were carried out to determine whether these actins are products of different structural genes and differ in primary amino acid sequence, or whether they are products of the same structural gene but are different because of post-translational modification. Messenger RNA from brain and muscle tissue was used to direct cell-free protein synthesis in wheat germ extracts. The synthesized actins were identified by conversion from globular to fibrous actin and by two-dimensional chromatographic analysis of tryptic peptides. The differences in electrophoretic mobility of brain compared to muscle actin were maintained in the cell-free protein synthetic products. Therefore, these mobility differences were not due to post-translational modification. It was concluded that brain and muscle actin are coded by different messenger RNAs and therefore arise from different structural genes. In addition, messenger RNA from 13- and 16-day embryonic thigh muscle directed the synthesis of both brain- and muscle-type actins, suggesting that muscle cell differentiation involves the regulation of at least two different actin genes.

Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.

Characterization of a Drosophila cDNA clone that encodes a 252-amino acid non-muscle tropomyosin isoform.

We report here the isolation and DNA sequence of a cDNA clone encoding a 252-amino acid non-muscle or cytoskeletal tropomyosin (cTm) isoform from Drosophila. The Drosophila cTm shows considerable homology with vertebrate cTm throughout the middle portion of the molecule. The amino-terminal end of the molecule, however, shows less homology and contains five more amino acids than the equine platelet and human tropomyosins. There is also a proline at position 6 in the Drosophila protein. The carboxyl-terminal 27 amino acids also show little homology with vertebrate non-muscle tropomyosins. This is a region of the molecule that shows considerably diversity among other Drosophila tropomyosins and vertebrate tropomyosins. A comparison of the DNA sequence of the cTm cDNA and a previously reported muscle tropomyosin II cDNA sequence shows regions of identical DNA sequence alternating with regions of nonidentical sequence, suggesting that both mRNAs are produced by alternate splicing of the same gene.