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R H Gavin

Publications and source records attributed to R H Gavin.

24 records · Page 2Linked to original sources
PubMedDate not supplied

Characteristics of basal body cartwheel reassembly.

Cartwheel complexes reassembled in a fraction derived by treating isolated oral apparatuses from Tetrahymena with 1.0 M KCl for 12 h. Approximately 40% of the KCl-soluble protein reassembled into cartwheel complexes. The reassembly reaction was protein-concentration dependent, and reassembled cartwheels were stable at 3 degrees C. Sucrose gradient centrifugation resolved 3 high molecular mass protein complexes from the KCl-soluble fraction. Each of the 3 complexes has a different mass, but each contains the same 5 polypeptides, 2 of which are probably tubulins. When these complexes were removed from the KCl-soluble fraction by high speed centrifugation, cartwheel reassembly did not occur. The 5 polypeptides in the high molecular mass complexes were among several other polypeptides resolved from reassembled cartwheels by 2-dimensional gel electrophoresis. The high molecular mass complexes are probably essential for cartwheel formation. The electrophoretic data also show that several polypeptides in the KCL-soluble fraction do not appear to be incorporated into cartwheels. These polypeptides are probably non-essential for cartwheel formation.

Animals↗
PubMedDate not supplied

A PCR screen identifies a novel, unconventional myosin heavy chain gene (MYO1) in Tetrahymena thermophila.

Degenerate primers for two regions of sequence homology in the myosin head domain were used in a polymerase chain reaction screen of Tetrahymena thermophila genomic DNA to amplify a 765 bp fragment that was cloned and sequenced. Based on the presence of conserved, myosin-specific sequences, the 765 bp PCR product was identified as a fragment of a myosin gene, the first to be discovered in ciliated protozoa and herein referred to as MYO1. An inverse polymerase chain reaction strategy was used to obtain additional sequence data that included the entire head domain of MYO1. Alignment of the predicted amino acid sequence of the MYO1 head domain with known myosin sequences identified the ATP-binding site, a phosphorylation site, and other myosin-specific consensus regions. In a northern blot analysis, a 765 bp MYO1-specific probe detected a 6.6 kb transcript under highly stringent hybridization conditions. Phylogenetic analysis revealed that the predicted protein encoded by MYO1 is not a member of any of the previously defined myosin classes and therefore represents a presumptive new myosin class.

Amino Acid Sequence↗
PubMedDate not supplied

MYO1, a novel, unconventional myosin gene affects endocytosis and macronuclear elongation in Tetrahymena thermophila.

Targeted gene disruption was used to investigate the function of MYO1, an unconventional myosin gene in Tetrahymena thermophila. Phenotypic analysis of a transformed strain that lacked a functional MYO1 gene was conducted at both 20 degrees C and 35 degrees C. At either temperature the delta MYO1 strain had a smaller cytoplasm/nucleus ratio than wild type. At 20 degrees C, delta MYO1 populations had a longer doubling time than wild type, lower saturation density, and a reduced rate of food vacuole formation. However, at 35 degrees C, these characteristics were comparable to wild type. Although micronuclear division and cytokinesis appeared normal in delta MYO1 cells, failure of the macronucleus to elongate properly resulted in unequal segregation of macronuclear DNA in cells maintained at either 20 degrees C or 35 degrees C.

Animals↗
PubMedDate not supplied

Expression of GFP-actin leads to failure of nuclear elongation and cytokinesis in Tetrahymena thermophila.

Green fluorescent protein (GFP)-tagged actin was used to investigate the distribution and function of actin in Tetrahymena. A strain that expresses both GFP-actin and endogenous actin was developed by transformation of Tetrahymena thermophila with a ribosomal DNA-based replicative vector. Confocal microscopy of living cells and immunogold electron microscopy confirmed localization of GFP-actin to basal bodies and the contractile ring. Incorporation of the fusion protein into these and other actin-related structures correlated with severe impairment of macronuclear elongation and cytokinesis. At 30 degrees C macronuclear elongation failed to occur in 25% of the transformants despite completion of micronuclear division. At 20 degrees C macronuclear elongation failed to occur in 2% of the population. Arrest of cytokinesis coincided with failure of macronuclear elongation. Arrested cells developed into homopolar doublets with two sets of oral structures. This study indicates a requirement for actin in nuclear elongation and cytokinesis. Although GFP-actin can interfere with the functioning of actin-containing structures, the GFP-actin transformant strain can be used to monitor actin distribution and dynamics and is therefore an important new tool for further studies of Tetrahymena actin.

Actins↗