Characterization of the myosin light-chain-2 gene of Drosophila melanogaster.

Abstract

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.

Transcriptional regulation of the Drosophila melanogaster muscle myosin heavy-chain gene

“…Each has a large first intron that interrupts the 5′ noncoding region or the first two codons of the gene. This structure is present in both Drosophila (Basi and Storti, 1986;Falkenthal et al, 1984;Geyer and Fyrberg, 1986;Marin et al, 2004;Mas et al, 2004;Parker et al, 1985) and vertebrate muscle genes (Baldwin et al, 1985;Chang et al, 1985;Fornwald et al, 1982;Nabeshima et al, 1984;Strehler et al, 1986). Transcriptional enhancer elements are located in the first intron of a number of such muscle genes (Konieczny and Emerson, 1987;Marin et al, 2004;Mas et al, 2004;Meredith and Storti, 1993;Ng et al, 1989;Yutzey et al, 1989).…”

“…Hanke and Storti (1988) noted that a 26 bp element beginning at -76 of the Drosophila Mhc gene is quite similar to sequences upstream of the Drosophila tropomyosin I and II genes. This sequence could serve as a position-dependent enhancer in all three genes.Other Drosophila muscle genes are interrupted by an intron near their 5′ ends (Basi and Storti, 1986;Falkenthal et al, 1984;Geyer and Fyrberg, 1986;Marin et al, 2004;Mas et al, 2004;Parker et al, 1985), and these introns often contain transcriptional activation sequences. The ß3-tubulin gene has a visceral muscle-specific enhancer element in its first intron (Gasch et al, 1989), as well as a somatic muscle element (Hinz et al, 1992).…”

Myosin light chain-2 mutation affects flight, wing beat frequency, and indirect flight muscle contraction kinetics in Drosophila.

“…Likewise, pCasJW3 was constructed by inserting a 5.9-kb BclI/HindIlI fragment containing the entire MLC-2 transcription unit plus 3.2 kb of 5' flanking sequences and ,x~650 bp of 3' flanking sequences into CaSpeR. The MLC-2 gene is the only transcription unit present in the sequences used to construct these transformation vectors (Parker et al, 1985;Toffenetti et al, 1987).…”

“…In Drosophila, sarcomeric MLC-2 is encoded by a single gene (Mlc2) which maps to polytene chromosome bands 99E1-3 (Parker et al, 1985;Toffenetti, et al, 1987). Mlc2 encodes a single protein isoform which is expressed in all muscle types throughout development.…”

“…Likewise, pCasJW3 was constructed by inserting a 5.9-kb BclI/HindIlI fragment containing the entire MLC-2 transcription unit plus 3.2 kb of 5' flanking sequences and ,x~650 bp of 3' flanking sequences into CaSpeR. The MLC-2 gene is the only transcription unit present in the sequences used to construct these transformation vectors (Parker et al, 1985;Toffenetti et al, 1987).Microinjection and P element-mediated gem'dine transformation were performed using a stable genomic source of P element transposase, P[ry + A2-3] (99B), as described previously (Rohextson ct el., 1988). Chromosome linkage was determined for a number of independent transformants, and either a balanced lethal or a homozygous stock for each transformed line was established using standard genetic techniques.…”