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.

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.

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.