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The unique catalytic subunit of sperm cAMP-dependent protein kinase is the product of an alternative Calpha mRNA expressed specifically in spermatogenic cellscAMP-dependent protein kinase has a central role in the control of mammalian sperm capacitation and motility. Previous protein biochemical studies indicated that the only cAMP-dependent protein kinase catalytic subunit (C) in ovine sperm is an unusual isoform, termed C(s), whose amino terminus differs from those of published C isoforms of other species. Isolation and sequencing of cDNA clones encoding ovine C(s) and Calpha1 (the predominant somatic isoform) now reveal that C(s) is the product of an alternative transcript of the Calpha gene. C(s) cDNA clones from murine and human testes also were isolated and sequenced, indicating that C(s) is of ancient origin and widespread in mammals. In the mouse, C(s) transcripts were detected only in testis and not in any other tissue examined, including ciliated tissues and ovaries. Finally, immunohistochemistry of the testis shows that C(s) first appears in pachytene spermatocytes. This is the first demonstration of a cell type-specific expression for any C isoform. The conservation of C(s) throughout mammalian evolution suggests that the unique structure of C(s) is important in the subunit's localization or function within the sperm.
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The 78,000 M(r) intermediate chain of Chlamydomonas outer arm dynein isa WD-repeat protein required for arm assemblyWe have isolated and sequenced a full-length cDNA clone encoding the 78,000 Mr intermediate chain (IC78) of the Chlamydomonas outer arm dynein. This protein previously was shown to be located at the base of the solubilized dynein particle and to interact with alpha tubulin in situ, suggesting that it may be involved in binding the outer arm to the doublet microtubule. The sequence predicts a polypeptide of 683 amino acids having a mass of 76.5 kD. Sequence comparison indicates that IC78 is homologous to the 69,000 M(r) intermediate chain (IC69) of Chlamydomonas outer arm dynein and to the 74,000 M(r) intermediate chain (IC74) of cytoplasmic dynein. The similarity between the chains is greatest in their COOH-terminal halves; the NH(2)-terminal halves are highly divergent. The COOH-terminal half of IC78 contains six short imperfect repeats, termed WD repeats, that are thought to be involved in protein-protein interactions. Although not previously reported, these repeated elements also are present in IC69 and IC74. Using the IC78 cDNA as a probe, we screened a group of slow-swimming insertional mutants and identified one which has a large insertion in the IC78 gene and seven in which the IC78 gene is completely deleted. Electron microscopy of three of these IC78 mutants revealed that each is missing the outer arm, indicating that IC78 is essential for arm assembly or attachment to the outer doublet. Restriction fragment length polymorphism mapping places the IC78 gene on the left arm of chromosome XII/XIII, at or near the mutation oda9, which also causes loss of the outer arm. Mutants with defects in the IC78 gene do not complement the oda9 mutation in stable diploids, strongly suggesting that ODA9 is the structural gene for IC78.
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Flagellin gene transcription in Bordetella bronchiseptica is regulated by the BvgAS virulence control systemThe products of the bvgAS locus activate expression of a majority of the known Bordetella virulence factors but also exert negative control over a class of genes called vrg genes (bvg-repressed genes). BvgAS negatively controls the production of flagella and the phenotype of motility in Bordetella bronchiseptica. In this study flaA, the flagellin gene, was cloned and characterized to facilitate studies of this negative control pathway. An internal flaA probe detected hybridizing sequences on genomic Southern blots of Bordetella pertussis, Bordetella parapertussis, and Bordetella avium, although B. pertussis and B. parapertussis are nonmotile. FlaA is similar to the FliC flagellins of Salmonella typhimurium and Escherichia coli, and flaA complemented an E. coli flagellin mutant. Insertional inactivation of the chromosomal flaA locus eliminated motility, which was restored by complementation with the wild-type locus. Analysis of flaA mRNA production by Northern (RNA) blotting and primer extension indicated that negative regulation by BvgAS occurs at the level of transcription. The transcriptional start site of flaA mapped near a consensus site for the alternative sigma factor, sigma F, encoded by fliA in E. coli and S. typhimurium. Consistent with a role for a fliA analog in B. bronchiseptica, transcriptional activation of a flaA-lacZ fusion in E. coli required fliA and a flaA-linked locus designated frl.frl also efficiently complemented mutations in the flagellar master regulatory locus, flhDC, of E. coli. Our analysis of the motility phenotype of B. bronchiseptica suggests that the Bordetella virulence control system mediates transcriptional control of flaA through a regulatory hierarchy that includes the frl locus and an alternative sigma factor.
