Functional nonequivalence of Drosophila actin isoforms.

We show that different Drosophila actin isoforms are not interchangeable. We sequenced the six genes that encode conventional Drosophila actins and found that they specify amino acid replacements in 27 of 376 positions. To test the significance of these changes we used directed mutagenesis to introduce 10 such conversions, independently, into the Act88F flight muscle-specific actin gene. We challenged these variant actins to replace the native protein by transforming germline chromosomes of a Drosophila strain lacking flight muscle actin. Only one of the 10 reproducibly perturbed myofibrillar function, demonstrating that most isoform-specific amino acid replacements are of minor significance. In order to establish the consequences of multiple amino acid replacements, we substituted portions of the Drosophila Act88F actin gene with corresponding regions of genes encoding other isoforms. Only one of five constructs tested engendered normally functioning flight muscles, and the severity of myofibrillar defects correlated with the number of replacements within the chimeric genes. Finally, we completely converted the flight muscle actin-encoding gene to one specifying a nonmuscle isoform, a change entailing a total of 18 amino acid replacements. Transformation of flies with this construct resulted in disruption of flight muscle structure and function. We conclude that actin isoform sequences are not equivalent and that effects of the amino acid replacements, while minor individually, collectively confer unique properties.

Genetic dissection of Drosophila myofibril formation: effects of actin and myosin heavy chain null alleles.

We used null mutations of Drosophila actin and myosin genes to investigate two aspects of myofibril assembly. First, we eliminated all actin or myosin in flight muscles to evaluate contributions of thick and thin filaments to sarcomere formation. Results demonstrate that thick and thin filament arrays can assemble independently but that both are essential for sarcomeric order and periodicity. Second, we examined how filament stoichiometry affects myofibril assembly. We find that heterozygotes for actin (Act88F) or myosin heavy chain (Mhc36B) null alleles have complex myofibrillar defects, whereas Mhc36B-/+; Act88F-/+ double heterozygotes have nearly normal myofibrils. These results imply that most defects observed in single heterozygotes are due to filament imbalances, not deficits, and suggest that thick and thin filament interactions regulate myofibrillar growth and alignment.

Arthrin, a myofibrillar protein of insect flight muscle, is an actin-ubiquitin conjugate.

Flight muscles of some insects contain a myofibrillar protein termed arthrin, which is closely related to actin (mw 43,000). Here we demonstrate that arthrin (mw 55,000) is ubiquitinated actin. We show that in Act88FM342, a flightless Drosophila mutant wherein the Act88F actin gene specifies a glu93----lys replacement, isoelectric points of both actin III and arthrin are shifted, revealing that both are encoded by the same gene. Arthrin reacts with an anti-ubiquitin antibody, which demonstrates that its extra mass results from ubiquitin ligation. Approximately one-seventh of myofibrillar actin is stably ubiquitinated, suggesting that there may be one arthrin molecule per actin-tropomyosin-troponin cooperative unit. Arthrin formation lags several hours behind that of actin III, implying that ubiquitination coincides with some aspect of myofibril assembly.

Two missense alleles of the Drosophila melanogaster act88F actin gene are strongly antimorphic but only weakly induce synthesis of heat shock proteins.

We have characterized two extant mutations of the flight muscle-specific act88F actin gene of Drosophila melanogaster. Both defective alleles were recovered from flightless mutants isolated previously (K. Mogami and Y. Hotta, Mol. Gen. Genet. 183:409-417, 1981). By directly sequencing the mutant alleles, we demonstrated that in act88FIfm(3)2 a single G-C to A-T transition converted arginine-28 to cysteine and that in act88FIfm(3)4 a single A-T to T-A transversion changed isoleucine-76 to phenylalanine. We showed that the actins encoded by either allele were strongly antimorphic. Mutant alleles effectively disrupted myofibril structure and function in the flight muscles of strains having the diploid complement of wild-type act88F genes. However, unlike antimorphic actins encoded by three previously characterized act88F alleles, neither that encoded by act88FIfm(3)2 nor that encoded by act88FIfm(3)4 was a strong inducer of heat shock protein synthesis.

Genetic rescue of muscle defects associated with a mutant Drosophila melanogaster tropomyosin allele.

We describe a genetic transformation system which should prove useful for investigating tropomyosin assembly and function. Muscle abnormalities associated with a defective tropomyosin allele were corrected by integrating the wild-type gene into germ line chromosomes. The transformation protocol permits application of directed mutagenesis techniques in investigations of contractile regulatory mechanisms.

5'-flanking sequence required for regulated expression of a muscle-specific Drosophila melanogaster actin gene.

We have functionally tested derivatives of a muscle-specific Drosophila melanogaster actin gene in which 5'-flanking sequences have been deleted or rearranged. From our results we conclude that approximately 1,000 nucleotides of 5'-flanking sequence are required for wild-type levels of mRNA accumulation during flight muscle development. Derivatives having 875 or 865 nucleotides of upstream sequence could be expressed normally, but were prone to influence by flanking foreign DNA sequences. Derivatives retaining 600 or fewer nucleotides of flanking DNA did not direct detectable levels of mRNA accumulation. The sequence residing between -919 and -640 could be inverted and yet retain normal function. Deletion of this sequence reduced mRNA accumulation markedly, but did not affect its spatial localization, suggesting that elements which confer tissue specificity reside close to the point of transcription initiation.

Two Drosophila melanogaster tropomyosin genes: structural and functional aspects.

We compared the structure and function of the two Drosophila melanogaster tropomyosin genes. The most striking structural aspect was their size disparity. Codons 1 through 257 of gene 2 occupied 833 nucleotides and contained only one intron, whereas the corresponding region of gene 1 occupied 17.5 kilobases and was interrupted by eight introns. The intron-exon arrangement of gene 1 reflected evolutionary expansion of tropomyosin via 42- and 49-residue duplications, which are probably actin-binding domains. Functionally, gene 1 was considerably more complex than gene 2; it was active in both muscle and nonmuscle cell lineages, had at least five variable exons, and specified a minimum of five developmentally regulated isoforms. Two of these isoforms, which accumulated only in flight muscles, were unprecedented fusion proteins in which the tropomyosin sequence was joined to a carboxy-terminal proline-rich domain.

An insertion within a variably spliced Drosophila tropomyosin gene blocks accumulation of only one encoded isoform.

We have characterized an aberrant allele of a variably spliced Drosophila tropomyosin gene. The allele was recovered from the flightless Ifm(3)3 strain, which has been shown to have structurally and functionally abnormal indirect flight muscles. The transcribed portion of the mutant gene is interrupted by an 8,8 kb insertion of middle repetitive DNA having a structure typical of copia-like Drosophila mobile elements. The insertion is positioned so as to interrupt an exon sequence in one splicing mode and, simultaneously, an intron in the alternate mode. As a consequence of the insertion the allele fails to direct synthesis of the flight muscle-specific tropomyosin isoform, but remains capable of specifying a second isoform, which accumulates in nonfibrillar Drosophila muscles.

The flightless Drosophila mutant raised has two distinct genetic lesions affecting accumulation of myofibrillar proteins in flight muscles.

We have used a combination of histological, molecular, and genetic techniques to investigate the flightless Drosophila mutant raised. Electron microscopy of indirect flight muscles of raised homozygotes confirms that they are grossly abnormal, lacking thin filaments and Z discs. These defects correspond to aberrant protein accumulation in thoraces, where several myofibrillar components are reduced or absent. Utilizing the germ-line transformation technique we demonstrate that one genetic lesion associated with the raised phenotype resides within the act88F actin gene, which, as a result, fails to specify normal mRNA accumulation during thoracic muscle differentiation. We also provide evidence for a distinct second genetic lesion, which apparently eliminates proper posttranslational modification of two myofibrillar proteins, one of which is actin.

A nonsense mutation within the act88F actin gene disrupts myofibril formation in Drosophila indirect flight muscles.

We have investigated the molecular basis of muscle abnormalities in the flightless Drosophila mutant lfm(3)7. This EMS-induced, semi-dominant allele was isolated by Mogami and Hotta (1981) and was shown to disrupt the organization of myofibrils in indirect flight muscles. Here we demonstrate that lfm(3)7 contains a nonsense mutation within codon 355 of the act88F actin gene. A single G greater than A transition converts a tryptophan (TGG) codon to an opal (TGA) terminator, thus deleting the carboxy-terminal 20 amino acids of an actin isoform that accumulates only in thoracic flight muscles. The truncated actin polypeptide is stable, and retains antigenicity to at least two anti-Drosophila actin monoclonal antibodies. We suggest that abnormalities in lfm(3)7 flight muscles result from incorporation of the mutant actin isoform into assembling myofibrils.

Organization of contractile protein genes within the 88F subdivision of the D. melanogaster third chromosome.

We have investigated contractile protein gene arrangement within the 88F subdivision of the Drosophila melanogaster third chromosome. We show that at least five antigenically related myofibrillar proteins, three of which accumulate only within indirect flight muscles, are encoded by a 20 kilobase chromosome segment located 140 kilobases proximal to the act88F actin gene. The chromosome segment includes two transcribed regions, each of which directs the synthesis of multiple mRNAs. RNA blot-hybridization experiments and DNA sequencing suggest that overlapping transcripts are generated by differential RNA splicing. The nucleotide sequence also establishes that two of the encoded proteins are tropomyosin isoforms, and two are related to tropomyosin. The arrangement of myofibrillar genes parallels the distribution of third chromosome mutations that disrupt indirect flight muscle formation.

Transcripts of the six Drosophila actin genes accumulate in a stage- and tissue-specific manner.

We have surveyed expression of the six Drosophila actin genes during ontogeny. Unique portions of cloned actin genes were used to monitor levels of respective mRNAs in developmentally staged whole organisms and dissected body parts. We find that each gene is transcribed to form functional mRNA, which accumulates with a distinct pattern. Two of the genes, act5C and act42A, are expressed in undifferentiated cells and probably encode cytoplasmic actins. Act57A and act87E are expressed predominantly in larval, pupal, and adult intersegmental muscles; act88F in muscles of the adult thorax; and act79B in the thorax and leg muscles. These composite data define three main patterns of actin gene expression which are correlated with changing Drosophila morphology, particularly muscle differentiation and reorganization.

The actin genes of Drosophila: protein coding regions are highly conserved but intron positions are not.

The entire set of six closely related Drosophila actin genes was isolated using recombinant DNA methodology, and the structures of the respective coding regions were characterized by gene mapping techniques and by nucleotide sequencing of selected portions. Structural comparisons of these genes have resulted in several unexpected findings. Most striking is the nonconservation of the positions of intervening sequences within the protein-encoding regions of these genes. One of the Drosophila actin genes, DmA4, is split within a glycine codon at position 13; none of the remaining five genes is interrupted in the analogous position. Another gene, DmA6, is split within a glycine codon at position 307; at least two of the Drosophila actin genes are not split in the analogous position. Additionally, none of the Drosophila actin genes is split within codon four, where the yeast actin gene is interrupted. The six Drosophila actin genes encode several different proteins, but the amino acid sequence of each is similar to that of vertebrate cytoplasmic actins. None of the genes encodes a protein comparable in primary sequence to vertebrate skeletal muscle actin. Surprisingly, in each of these derived actin amino acid sequences in the initiator methionine is directly followed by a cysteine residue, which in turn precedes the string of three acidic amino acids characteristic of the amino termini of mature vertebrate cytoplasmic actins. We discuss these findings in the context of actin gene evolution and function.

The actin genes of Drosophila: a dispersed multigene family.

We have initiated a study of the organization and expression of the actin genes of D. melanogaster. Using actin gene-specific probes from both chicken and Dictyostelium sources, a clone--denoted lambda DmA2--containing a Drosophila actin gene has been isolated from a representative library of Drosophila genomic DNA cloned in the lambda bacteriophage vector, Charon 4. Southern blotting experiments reveal that there is only one actin structural gene contained in the 17.5 kb Drosophila insert of lambda DmA2 and that the sequences immediately flanking the structural gene are single copy. Observations by electron microscopy of the R loop structures formed by hybridizing total cytoplasmic poly(A)+ RNA from Drosophila embryos to an appropriate subcloned segment of lambda DmA2 indicate that the gene consists of an approximately 70-170 nucleotide leader sequence encoding the 5' portion of the mature mRNA, a 1.65 kb intervening sequence not present in the mRNA and a 1.55 kb sequence containing the major portion of the gene. Using genomic blots with actin-specific probes derived from lambda DmA2, we show that there are six actin genes per haploid Drosophilia genome. They direct the synthesis of three major size classes of mRNA. Using in situ hybridization, the six genes have been localized to six widely dispersed sites on the polytene chromosomes; the locus for lambda DmA2 is 5C on the X chromosome. In vitro translation of mRNA selected hybridization by a DNA segment specific to lambda DmA2 suggests that this particular gene codes for one of the cytoplasmic actin polypeptides.

Isolation and partial characterization of Drosophila myoblasts from primary cultures of embryonic cells.

We describe a method for preparing highly enriched cultures of Drosophila myoblasts from a heterogeneous cell population derived from gastrulating embryos. Enriched cultures are prepared by plating this heterogeneous population of cells in medium from which much of the free calcium is chelated by ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA). Adhesion of myoblasts to tissue culture plastic is better than that of other cell types when plated in this medium. Data concerning cell identity, timing of S phase, and fusion kinetics document the degree of enrichment for myogenic cells and illustrate their synchronous differentiation in vitro.