Flightin, a novel myofibrillar protein of Drosophila stretch-activated muscles.

The indirect flight muscles of Drosophila are adapted for rapid oscillatory movements which depend on properties of the contractile apparatus itself. Flight muscles are stretch activated and the frequency of contraction in these muscles is independent of the rate of nerve impulses. Little is known about the molecular basis of these adaptations. We now report a novel protein that is found only in flight muscles and has, therefore, been named flightin. Although we detect only one gene (in polytene region 76D) for flightin, this protein has several isoforms (relative gel mobilities, 27-30 kD; pIs, 4.6-6.0). These isoforms appear to be created by posttranslational modifications. A subset of these isoforms is absent in newly emerged adults but appears when the adult develops the ability to fly. In intact muscles flightin is associated with the A band of the sarcomere, where evidence suggests it interacts with the myosin filaments. Computer database searches do not reveal extensive similarity to any known protein. However, the NH2-terminal 12 residues show similarity to the NH2-terminal sequence of actin, a region that interacts with myosin. These features suggest a role for flightin in the regulation of contraction, possibly by modulating actin-myosin interaction.

HeT-A, a transposable element specifically involved in "healing" broken chromosome ends in Drosophila melanogaster.

Eight terminally deleted Drosophila melanogaster chromosomes have now been found to be "healed." In each case, the healed chromosome end had acquired sequence from the HeT DNA family, a complex family of repeated sequences found only in telomeric and pericentric heterochromatin. The sequences were apparently added by transposition events involving no sequence homology. We now report that the sequences transposed in healing these chromosomes identify a novel transposable element, HeT-A, which makes up a subset of the HeT DNA family. Addition of HeT-A elements to broken chromosome ends appears to be polar. The proximal junction between each element and the broken chromosome end is an oligo(A) tract beginning 54 nucleotides downstream from a conserved AATAAA sequence on the strand running 5' to 3' from the chromosome end. The distal (telomeric) ends of HeT-A elements are variably truncated; however, we have not yet been able to determine the extreme distal sequence of a complete element. Our analysis covers approximately 2,600 nucleotides of the HeT-A element, beginning with the oligo(A) tract at one end. Sequence homology is strong (greater than 75% between all elements studied). Sequence may be conserved for DNA structure rather than for protein coding; even the most recently transposed HeT-A elements lack significant open reading frames in the region studied. Instead, the elements exhibit conserved short-range sequence repeats and periodic long-range variation in base composition. These conserved features suggest that HeT-A elements, although transposable elements, may have a structural role in telomere organization or maintenance.

HeT DNA: a family of mosaic repeated sequences specific for heterochromatin in Drosophila melanogaster.

HeT DNA is a complex family of repeated DNA found only in pericentric and telomeric heterochromatin. In contrast to other DNA families that have been specifically associated with heterochromatin, HeT DNA is not principally a family of tandemly repeated elements. Much of the HeT DNA family appears to be a mosaic of several different classes of large sequence elements arranged in a scrambled array; however, some elements of the family can be found in tandem repeats. In spite of the variable order of the different elements in HeT DNA, the sequence homology between different members of each class of element is extremely high, suggesting that the members are evolving in a concerted fashion. Sequence analysis suggests that some elements in the HeT family may make up a novel family of heterochromatin-specific transposable elements and that the mosaic organization of the elements may be produced by retroposition and other mechanisms involved in the transposition of mobile elements. We suggest that such mechanisms may be a general feature for the maintenance of chromosome structure.

Addition of telomere-associated HeT DNA sequences "heals" broken chromosome ends in Drosophila.

Stocks of D. melanogaster X chromosomes carrying terminal deletions (RT chromosomes) have been maintained for several years. Some of the chromosomes are slowly losing DNA from the broken ends (as expected if replication is incomplete) and show no telomere-associated DNA added to the receding ends. Two stocks carry chromosomes that have become "healed" and are no longer losing DNA. In both stocks the broken chromosome end has acquired a segment of HeT DNA, a family of complex repeats found only at telomeres and in pericentric heterochromatin. Although the HeT family is complex, the HeT sequence joined to the broken chromosome end is the same in both stocks. In contrast, the two chromosomes are broken in different places and have no detectable sequence similarity at the junction with the new DNA. Sequence analysis suggests that the new telomere sequences have been added by a specific mechanism that does not involve homologous recombination.

Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster.

Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-band components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies alpha:1-4 recognize a 90-100-kD protein which we identify as alpha-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate alpha-actinins. Monoclonal antibodies P:1-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A- and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (approximately 400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized alpha-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z- and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila alpha-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.

Quantitation of muscle-specific mRNAs by using cDNA probes during chicken embryonic muscle development in ovo.

The emergence of abundant-class mRNAs specific for contractile muscle proteins and their distribution between polysomal and free mRNP fractions were studied in skeletal muscle excised from chicken embryos during the transition from myoblasts (day 9) to myotubes (day 18). Muscle-specific cDNA was selectively prepared by hybridizing cDNA to template RNA (polysomal poly(A)+ mRNA) from day-14 embryos followed by isolation of the abundant class, which represents approximately 20% of total mRNA. The specificity of the cDNA probe for this class was confirmed by the differential degree of hybridization to cytoplasmic RNA from cultured myotube and myoblast cells and by its inability to hybridize with mRNA from nonmuscle cells such as liver. Except for muscle from day-9 embryos, the concentrations of the abundant-class muscle-specific mRNAs were higher in polysomes than in free mRNP fractions. Furthermore, the levels of these mRNAs in polysomes increased 12-fold from day 9 (myoblast) to day 14 (intermediate) with a further 3.6-fold increase from day 14 to day 18 (myotube). In contrast to this 45-fold net increase in the polysomal level of these mRNAs from day 9 to day 18, the levels in the free mRNP fraction showed only a 3-fold decrease during this period. Because the amount of mRNA lost from the mRNP fraction is much less than the net increase in the polysome fraction, mRNP does not serve as a reservoir of untranslated muscle-specific mRNA for transfer to polysomes. Consequently, the emergence of muscle-specific polysomal mRNA for contractile proteins during myogenesis in ovo appears to be regulated primarily by transcriptional control.