Identification and characterization of Drosophila melanogaster paramyosin.
Paramyosin, a major structural component of thick filaments in invertebrates has been isolated, purified and characterized from whole adult Drosophila melanogaster extracts and a specific polyclonal antibody against it has been prepared. Paramyosin has been identified on the basis of several criteria, including molecular weight, alpha-helicity, species distribution, capability of fiber formation in vitro and sequence. We have used the immunopurified polyclonal antibody to isolate eight clones from a lambda gt11 expression library of Drosophila 1 to 22 h embryo cDNA. The largest clone (pJV9) has been sequenced and encodes the coiled-coil region of D. melanogaster paramyosin that is 47% identical to Caenorhabditis elegans paramyosin. Indirect immunofluorescence in semi-thin sections of adult flies show fluorescence mainly in tubular muscle. Freshly prepared tubular myofibrils decorated with the immunoabsorbed antibody show the A region in the sarcomere as the specific localization of paramyosin. The amount of paramyosin in tubular synchronous muscles of insects appears to be five times higher than in fibrillar insect muscles. There are at least three paramyosin isoforms as shown by isoelectrofocusing separation. The more acidic and less abundant form is phosphorylated as shown by 32P in vivo labeling experiments in adult flies. The developmental pattern of expression of Drosophila paramyosin is presented. This mesoderm-specific protein, immunologically undetectable during gastrulation and early phases of germ band formation, progressively increases during organogenesis to the adult stage. Interestingly, it is also expressed as a major maternal product in the insoluble cytoskeletal fraction of the mature oocyte.
Vinós
J
J
Departamento de Bioquímica de la U A M, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
Domingo
A
A
Marco
R
R
Cervera
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics / Q000502:physiology*; D004625:Embryo, Nonmammalian / Q000502:physiology; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D014335:Tropomyosin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D014335:Tropomyosin