THE DYNEIN GENES OF PARAMECIUM-TETRAURELIA - SEQUENCES ADJACENT TO THE CATALYTIC P-LOOP IDENTIFY CYTOPLASMIC AND AXONEMAL HEAVY-CHAIN ISOFORMS

Paramecium tetraurelia is a unicellular organism that utilizes both ax onemal and cytoplasmic dyneins. The highly conserved region containing the catalytic P-loop of the dynein heavy chain was amplified by RNA-d irected polymerase chain reaction. Eight different P-loop-containing c DNA fragments were cloned. Southern hybridization analysis indicated t hat each fragment corresponds to a separate dynein gene and that there are at least 12 dynein heavy chain genes expressed in Paramecium. Sev en of the eight cloned contain sequence motif A, which is found in axo nemal dyneins, and one contains sequence motif B, which is found in th e dyneins from cell types that do not have cilia or flagella. Two of t he Paramecium dynein genes were further investigated: DHC-6 which cont ains motif A, and DHC-8 which contains motif B. Additional sequencing of the central portions of these genes showed that DHC-6 most closely matches sea urchin ciliary beta heavy chain and DHC-8 is similar to th e cytoplasmic dynein from Dictyostelium. Deciliation of the cells resu lted in a substantial increase in the steady state concentration of DH C-6 mRNA but only a small change in DHC-8 mRNA. Antisera were produced against synthetic peptides derived from sequence motifs A and B. Comp etitive solid-phase binding assays demonstrated that each antiserum wa s peptide-specific. In western blots, the antiserum to motif A reacted with both ciliary and cytoplasmic dyneins. In contrast, the antiserum to motif B reacted with the cytoplasmic dyneins of Paramecium and bov ine brain but did not react with ciliary dynein. Indirect immunofluore scence microscopy showed that the motif B epitope was excluded from ci lia. These results suggest that sequence motif A may be present in bot h classes of dynein but that sequence motif B is restricted to cytopla smic dynein isoforms. This study provides direct evidence that individ ual dynein gene products are spatially segregated in a single cell.