Dynein isoforms in paramecium

Susan Marie Beckwith, Purdue University

Abstract

Dynein is an ATPase that powers the translocation of molecules along microtubules. This microtubule motor molecule was first discovered in cilia where it powers ciliary beating. More recently, a cytoplasmic form of dynein was discovered which participates in movement of organelles, vesicles, and chromosomes during mitosis. My research has focused on the mechanism of dynein functional diversity. My hypothesis is that ciliary and cytoplasmic dyneins are encoded by distinct sets of genes. Dynein is a complex of 9-12 polypeptides. My work has centered on the heavy chains of dynein because they contain the functional ATP-binding sites and the microtubule binding domains. The paramecium expresses both the ciliary and cytoplasmic dyneins in a single cell, and thus provides an excellent experimental system in which to compare the dynein heavy chain isoforms. I performed biochemical and immunological studies which demonstrated that there are multiple dynein protein isoforms. The dynein heavy chains in the cilia are structurally different from each other and from the previously characterized cytoplasmic dynein of paramecium. To determine if the individual isoforms originate from separate genes or from posttranscriptional modification(s), I undertook a molecular analysis of the dynein heavy chain isoforms. Degenerate nucleotide primers were utilized to amplify by RNA-directed polymerase chain reaction (PCR) the highly conserved phosphate-binding loop of Paramecium tetraurelia dynein heavy chains. Southern blot analysis revealed the presence of multiple dynein genes. The total PCR product was cloned and individual inserts were sequenced. Seven cDNAs corresponding to ciliary dynein and an eighth cDNA clone corresponding to the presumptive cytoplasmic dynein were obtained. Individual cloned fragments hybridized with single bands on genomic Southern blots. These results demonstrate that the multiple isoforms of dynein heavy chains expressed in paramecium are derived from separate genes. Northern analyses using one probe specific for a ciliary dynein and one from a presumptive cytoplasmic dynein indicate a difference in the regulation of ciliary versus cytoplasmic dynein heavy chain genes. The mRNA encoding ciliary dynein is induced 5-6X more than the mRNA encoding cytoplasmic dynein after deciliation. Immunological analyses show that the multiple dynein genes are spatially segregated within a single cell, implying a functional specificity.

Degree

Ph.D.

Advisors

Asai, Purdue University.

Subject Area

Biology|Genetics

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