Date of Award
Dissertation - Restricted
Doctor of Philosophy (PhD)
Ciliated protozoa contain two types of nuclei, germ line micronuclei (Mic) and transcriptionally active macronuclei (Mac). During sexual reproduction (conjugation), the parental Mac degenerates and a new Mac develops from a mitotic product of the zygotic Mic. Macronuclear development involves extensive endoreplication of the genome. However, very little is known about its regulation. The gene ASI2 in Tetrahymena thermophila encodes a putative signal transduction receptor and is nonessential for vegetative growth. It is, however, required to complete sexual reproduction. Cells that lack ASI2 in the developing Mac anlagen are arrested in DNA endoreplication and die.
The present study shows that both the parental Mac and the Mac anlagen contribute to the increased level of ASI2 transcripts during conjugation. The parental Mac is the source of ASI2 transcripts early in conjugation, and the Mac anlagen is the source at later stages. Over-expressed Asi2p tagged with HA was present at early stages, and localized at the periphery of a cell, which is a characteristic of signal transduction receptor.
The phenotype of the ASK germ line knockouts was further analyzed by the occurrence of later events during conjugation, including Pddlp distribution and the presence of small scRNA (scRNA), chromosome breakage and telomere addition and de novo DNA methylation. All these events happened similarly in matings between ASI2 germ line knockouts compared to that of wild type cells.
The phenotype of the somatic ASI2 knockouts was analyzed to investigate the function of parental ASI2 by the occurrence of early events during conjugation. Without the parental ASK, various molecular events, including synthesis of Pddlp and scRNAs and methylation of the Mac DNA occurred similarly to that of wild type cells. However, DNA endoreplication was delayed at early stages of the Mac anlagen development.
Mutagenesis study showed that a Tyr to Phe mutation at the predicted phosphorylation site in the parental Mac also affected DNA endoreplication during the Mac anlagen development.
These results suggested that AS 12 specifically regulates endocycling during the Mac anlagen development, instead of a general checkpoint for cell survival.