Analysis of the DYH1 gene in Tetrahymena thermophila reveals a novel gene, RBN1, implicated in nuclear function and cortical organization
Abstract
Dyneins are enormous motor proteins that convert chemical energy into directed movement along the microtubules of the cytoskeleton. We have sequenced the majority of the DYH1 gene from the ciliate Tetrahymena thermophila and have found that this gene is a conserved member of the cytoplasmic dynein family. Incomplete knockdowns of DYH1 (KO-1) in the somatic nucleus of Tetrahymena are defective in micronuclear maintenance with some cells lacking micronuclei, whereas others contain multiple mitotic micronuclei. KO-1 cells display chromosome segregation defects, micronuclear spindle defects, and defects in mitotic progression. In our characterization of the DYH1 gene, we discovered a second gene located 380 bp downstream of the DYH1 stop codon. The Tetrahymena RBN1 gene includes an open-reading frame of 900 bp, and is predicted to encode a protein of 300 amino acids. RT-PCR data and Northern data demonstrate that RBN1 is expressed. Database searches identify this gene as an orthologue of a family of genes of unknown function identified in Eubacteria and Eukarya. Sequence analysis suggests that all Rbn1 family members contain a novel ∼250 amino acid domain, the Rbn1 homology domain. However, some family members contain additional domains of unknown function. Complete replacement of the RBN1 gene in the somatic nucleus does not appear to affect the nearby DYH1 locus or DYH1 expression. The ΔRBN1 populations are generally healthy; however, many cells lack micronuclei, while others have small, unorganized micronuclei. Some ΔRBN1 cells display chromosome segregation defects and defects in cell-cycle progression through mitosis like KO-1 cells, however few accumulate mitotic micronuclei. ΔRBN1 cells also display a defect in macronuclear positioning during division that is absent in KO-1 cell populations. Finally, some rapidly dividing ΔRBN1 cells display abnormal cellular morphologies that appear to result from aborted attempts at cell division. These cells often lack micronuclei, lack organized oral structures, and contain an unorganized cortical cytoskeleton. These results suggest that RBN1 affects the organization of the cell cortex and therefore is a regulator of both nuclear function and cortical organization, whereas DYH1 appears to function primarily during micronuclear mitosis.
Degree
Ph.D.
Advisors
Asai, Purdue University.
Subject Area
Cellular biology|Molecular biology
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