Biomedical subjects
D Cassidy-Hanley
Publications and source records attributed to D Cassidy-Hanley.
Biolistic transformation of macro- and micronuclei.
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A mutational analysis of conjugation in Tetrahymena thermophila. 1. Phenotypes affecting early development: meiosis to nuclear selection.
Conjugation in the freshwater ciliate Tetrahymena thermophila involves a developmental program that models meiosis, fertilization, and early developmental events characteristic of multicellular eukaryotes. We describe a gallery of five early-acting conjugation mutations. These mutants, cnj1-5, exhibit phenotypes in which specific steps in the conjugal pathway have been altered or eliminated. Specifically, cnj1 and cnj2 fail to condense their micronuclear chromatin prior to each of the three prezygotic nuclear divisions. This results in nuclear division failure, failure to replicate DNA, and failure to initiate postzygotic development. The cnj3 mutant appears to exhibit a defect in chromosome separation during anaphase of mitosis. cnj4 mutants successfully carry out meiosis I, yet are unable to execute the second meiotic division and abort all further development. cnj5 mutants are unable to initiate either meiosis I or meiosis II, yet proceed to execute all subsequent developmental events. These mutant phenotypes are used to draw inferences regarding developmental dependencies that exist within the conjugation program.
Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment.
Mating Tetrahymena thermophila were bombarded with ribosomal DNA-coated particles at various times in development. Both macronuclear and micronuclear transformants were recovered. Optimal developmental stages for transformation occurred during meiosis for the micronucleus and during anlagen formation for the macronucleus. Evidence is given for transient retention of the introduced plasmid. Genetic and molecular tests confirmed that sexually heritable transformation was associated with integration at the homologous site in the recipient micronuclear chromosome.
A method for mapping germ line sequences in Tetrahymena thermophila using the polymerase chain reaction.
A method for mapping DNA sequences to specific germinal chromosomes in the ciliated protozoan Tetrahymena thermophila has been developed. This mapping technique (PCR mapping) utilizes the polymerase chain reaction and template DNA derived from nullisomic strains to directly assign micronuclear DNA sequences to specific micronuclear chromosomes. Using this technique, a number of unique sequences and short repetitive sequences flanked by unique sequences have been mapped to four of the five germinal chromosomes.
A simple, efficient technique for freezing Tetrahymena thermophila.
We have developed a simple, efficient procedure for the long term freezing of Tetrahymena thermophila in liquid nitrogen. This technique yields excellent recovery of viable cells with all strains tested and does not require the use of a controlled rate low temperature freezer. To optimize the freezing technique, we have examined the effects of varying a number of parameters, including the physiological state of the cells prior to freezing, the time of exposure to cryoprotectant, and the rate of freezing and thawing. The frequency of viable cell recovery following freezing using this technique has been tested for a variety of different cell lines.