The total sequence of the Drosophila melanogaster gene encoding the myosin light chain dissociated by alkali (MLC-ALK) has been determined. By sequence comparisons with an MLC-ALK cDNA clone and by S1 nuclease analyses, the pattern of introns and exons within the gene has been deduced. There are multiple polyadenylylation signals that can account for most of the observed heterogeneity in the lengths of mRNAs. In the 3' half of the gene, there are two alternative splicing patterns which result in mRNAs that translate to give proteins with two alternative 14 amino acid carboxyl-terminal sequences. There is developmental regulation of the selection of the above splicing sites. One splicing pattern produces an mRNA that translates into a protein used for both larval and adult musculature, whereas the other splicing pattern is used for the latter stage only.

Abstract. We have used a combination of classical genetic, molecular genetic, histological, biochemical, and biophysical techniques to identify and characterize a null mutation of the myosin light chain-2 (MLC-2) locus of Drosophila melanogaster. Mlc2 e3a is a null mutation of the MLC-2 gene resulting from a nonsense mutation at the tenth codon position. Mlc2 E3s confers dominant flightless behavior that is associated with reduced wing beat frequency. Mlc2 E3a heterozygotes exhibit a 50% reduction of MLC-2 mRNA concentration in adult thoracic musculature, which results in a commensurate reduction of MLC-2 protein in the indirect flight muscles. Indirect flight muscle myofibrils from Mlc2 ~38 heterozygotes are aberrant, exhibiting myofilaments in disarray at the periphery. Calcium-activated Triton X-100-treated single fiber segments exhibit slower contraction kinetics than wild type. Introduction of a transformed copy of the wild type MLC-2 gene rescues the dominant flightless behavior of Mlc2 E3s heterozygotes. Wing beat frequency and single fiber contraction kinetics of a representative rescued line are not significantly different from those of wild type. Together, these results indicate that wild type MLC-2 stoichiometry is required for normal indirect flight muscle assembly and function. Furthermore, these resuits suggest that the reduced wing beat frequency and possibly the flightless behavior conferred by Mlc2 e3s is due in part to slower contraction kinetics of sarcomeric regions devoid or partly deficient in MLC-2.F ORCE production in virtually all types of muscle requires the formation of mechanically strained, elastic cross-bridges between myosin-and actin-containing filaments. These cross-bridges are composed of the globular heads of the myosin heavy chain subunits and their associated light chains (the myosin alkali light chain and the myosin light chain-2 (MLC-2) t, the regulatory light chain). The myosin cross-bridge projects out from the thick (myosin) filament and attaches to an adjacent thin (actin) filament, activating an actomyosin Mg2 § which provides the chemical energy required for muscle contraction (Adelstein and Eisenberg, 1980). The cyclic making and breaking of these cross-bridges, together with a conformational change within the myosin molecule, causes the actin and myosin filaments to slide past each other enabling the muscle to shorten against an external load (Huxley, 1969 Two independent systems regulate the actomyosin ATPase cycle and contraction: a thin filament control system regulated by the troponin-tropomyosin complex, and a thick filament control system modulated by MLC-2 (Lehman and Szent-Gyorgyi, 1975;Sweeney and Stull, 1990, and references therein). The sophisticated molecular and genetic manipulations possible in Drosophila provides a powerful approach with which to investigate the structure-funodon relationships of these regulatory proteins (Peckham et al., 1990;Fyrberg and Beall, 1990). Our efforts have been directed toward defining the role of the MLC-2 protein.Her...

A chimeric lambda DNA molecule containing the myosin alkali light-chain gene of Drosophila melanogaster was isolated. The encoded amino acid sequence was determined from the nucleic acid sequence of a cDNA homologous to the genomic clone. The identity of the encoded protein was established by two criteria: (i) sequence homology with the chicken alkali light-chain proteins and (ii) comparison of the two-dimensional gel electrophoretic pattern of the peptides synthesized by in vitro translation of hybridselected RNA to that of myosin alkali light-chain peptides extracted from Drosophila myofibrils. There is only one myosin alkali light-chain gene in D. melanogaster; its chromosomal location is region 98B. This gene is abundantly expressed during the development of larval as well as adult muscles. The Drosophila protein appears to contain one putative divalent cation-binding domain (an EF hand) as compared with the three EF hands present in chicken alkali light chains.Myosin light chains are proteins which occur abundantly and in a defined stoichiometry in myofibrils. They are members of an evolutionarily related group of calciumbinding proteins known as the troponin C superfamily, which includes calmodulin, troponin C, and the myosin alkali and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) light chains. The primary amino acid sequence has been determined for at least one vertebrate example of each of these polypeptides (2). The principal sequence homology between these proteins resides in the putative Ca-+-binding domains, which are known as EF hands (14). The roles of all of these proteins, except for the myosin alkali light chain, in muscle function have been determined (10. 13, 29; R. A. Murphy, M. 0. Askoy, P. F. Dillon, W. T. Gerthoffer, and K. E. Kanim, Fed. Proc. 42:51-57, 1983).The skeletal muscle myosin alkali light chains are so named because of the high pH required to dissociate them from the myosin heavy chain (39). For vertebrate muscles, they are sometimes called MLC-1 and MLC-3. The two skeletal muscle alkali light chains of mammals and chickens, which have molecular weights of about 21,000 (MLC-1) and 17,000 (MLC-3), are virtually identical in sequence over their C-terminal 141 residues, but diverge in sequence at the amino terminus. MLC-1, depending upon the tissue from which it is isolated, has an additional alanine-proline-or alanine-lysine-rich sequence of 40 amino acids at its amino terminus. There is evidence that in rats the two proteins are encoded by a single gene (L. Garfinkel, R. Gubits, B. NadalGinard, and N. Davidson, manuscript in preparation). At one time, these peptides were thought to be essential for the actin-activated adenosine triphosphatase activity of myosin (16,32,38)

Recombinant DNA clones encoding the Drosophila melanogaster homolog of the vertebrate myosin light-chain-2 (MLC-2) gene have been isolated. This single-copy gene maps to the chromosomal locus 99E. The nucleotide sequence was determined for a 3.4-kilobase genomic fragment containing the gene and for two MLC-2 cDNA clones generated from late pupal mRNA. Comparison of these sequences shows that the gene contains two introns, the positions of which are conserved in the corresponding rat sequence. Extension of a primer homologous to the mRNA reveals two start sites for transcription 12 nucleotides apart. The sequence TATA is not present ahead of the mRNA cap site. There are two major sites of poly(A) addition separated by 356 nucleotides. The protein sequence derived from translation of the cDNA sequence shows a high degree of homology with that for the DTNB myosin light chain (MLC-2) of chicken. A lower degree of sequence homology was seen in comparisons with other evolutionarily related calcium-binding proteins. RNA blots show high levels of expression of several transcripts during the developmental time stages when muscle is being produced. In vitro translation of hybrid-selected RNA produces two polypeptides which comigrate on two-dimensional gels with proteins from Drosophila actomyosin, although the cDNA sequence reveals only one 26-kilodalton primary translation product.

Abstract. Using a combination of molecular and genetic techniques we demonstrate that Ifm(2)2 is an