The initiation of DNA replication is normally under cell cycle control. Mechanisms that prevent reinitiation from a replication origin in a given S phase are not understood. In mammals, errors in replication control can result in spontaneous amplification of segments of the genome. Gene amplification is frequently involved in tumorigenesis and, therefore, of significant medical importance. Because amplification events are spontaneous in mammals, our understanding of amplification mechanisms is limited. In contrast, gene amplification is developmentally-programmed in the protozoan Tetrahymena, providing the opportunity to directly study and dissect the amplification process. The rDNA minichromosome of T. thermophila, encoding the ribosomal RNA genes, has been used extensively as a model for DNA replication and gene amplification. Vegetative replication of the rDNA initiates from a single origin of replication. Cis-acting sequences controlling initiation from this origin have been identified. The vegetative origin is also used to amplify the rDNA. Thus, cell cycle control of this origin is suppressed during gene amplification. During early amplification, the vegetative origin is quiescent. At this time, developmentally-regulated mechanisms promote initiation from other segments of the chromosome. rDNA amplification occurs as part of a developmental program, accompanied by chromosome breakage and DNA rearrangement, similar to mammalian amplification events. Cis-acting mutations affecting excision, rearrangement and amplification of the rDNA have been identified. Their roles in amplification are beginning to be understood. Classical genetic, molecular and physical approaches will be employed to investigate the control of replication and amplification of the rDNA minichromosome. New cis-acting elements controlling the formation and amplification of the rDNA will be identified by DNA sequencing of existing mutants. These mutants will be exploited to identify genes encoding trans- acting factors required for information and amplification of the rDNA. Extragenic suppressors of these recessive-lethal cis-acting mutations will be sought. rDNA replication origins and cis-regulatory determinants will be studied. 2D gel electrophoresis will be used to determine whether early amplification initiates randomly or from specific developmentally- regulated replication origins. Amplification origins will be precisely mapped by 2D methods. Cis-acting elements required for amplification will be localized by deletional analysis of rDNA constructs, using a transient transformation DNA replication assay. These elements should include constitutive DNA replication determinants. Amplification-specific determinants, such as binding sites for factors that suppress cell cycle control, should also be identified. 2D analysis and transformation will be used to further localize the vegetative replication origin and identify the minimal origin of vegetative rDNA replication. Mutants unable to amplify the rDNA will be exploited as DNA transformation recipients for these studies. By understanding the relationship of gene amplification to cell cycle-regulated replication in Tetrahymena, mechanisms that control these processes in higher eukaryotes may become clear.
