Molecular cloning and expression of sea urchin embryonic ciliary dynein beta heavy chain.
The determination of the structure and the expression of dynein during embryonic development are central to the understanding of dynein function. As an important first step toward these objectives, cDNAs encoding portions of sea urchin ciliary dynein were identified by antibody screening of a sea urchin cDNA expression library. Because of the complete lack of protein sequence data, it was first necessary to prove the identity of the dynein cDNAs. Of the five cDNA inserts initially cloned, one, designated P72A1, was characterized extensively. Four independent criteria demonstrated that P72A1 encoded a portion of a dynein heavy chain. (1) The beta-galactosidase-P72A1 fusion protein affinity-purified dynein-specific antibodies from crude antiserum. (2) Two other antisera to dynein, raised independently of the antiserum used to screen the cDNA library, reacted with the fusion protein. (3) A new antiserum raised against the fusion protein reacted with authentic dynein heavy chain on Western blots and stained embryonic cilia by indirect immunofluorescence microscopy. (4) Two new antisera, elicited against opposite ends of the P72A1 open reading frame, each reacted with authentic dynein heavy chain protein. Western blot analyses of dissociated dynein heavy chains revealed that P72A1 encoded a portion of the beta heavy chain. Epitope mapping experiments confirmed the identity of P72A1 as part of the beta heavy chain and also demonstrated that P72A1 encoded epitopes of the carboxyl-terminal fragment B domain of the dynein beta heavy chain. Northern blot analyses of poly(A)+ RNA revealed that P72A1 hybridized with a large RNA species ca. 12.5 kb in length. The dynein mRNA concentration increased during embryonic development. Dot blot analyses of RNA isolated at various times after embryo deciliation demonstrated that the dynein beta heavy chain mRNA accumulated rapidly in response to deciliation. The accumulation was similar to but not identical with the induction of tubulin mRNA in response to the same stimulus.
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
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Foltz
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Asai
D000251:Adenosine Triphosphatases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002874:Chromosome Mapping; D002923:Cilia / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004398:Dyneins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D015870:Gene Expression; D007106:Immune Sera / Q000276:immunology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012617:Sea Urchins / Q000196:embryology / Q000378:metabolism*
D000939:Epitopes; D007106:Immune Sera; D012333:RNA, Messenger; D004247:DNA; D000251:Adenosine Triphosphatases; D004398:Dyneins