Cis-acting sequence determinants for rDNA replication in T. thermophila

Abstract

The rDNA minichromosome of Tetrahymena thermophila raphics. exists as a 21 kb head-to-head palindrome and is a model system for studying DNA replication. Previous molecular and genetic approaches identified cis-acting sequence elements involved in replication control. Two-dimensional (2D) gel electrophoresis studies have determined that cell cycle-controlled (DNA replication initiates at two closely spaced origins within the 5' nontranscribed spacer (5' NTS) (Zhang et a1., 1997). In this thesis, the role of specific conserved sequence elements in replication control was addressed using stable and transient transduction assays. The plasmic PUC 1x1.9 contains a single copy of the 5' NTS and no additional rDNA sequences. Transient transduction experiments with PUC 1x1.9 revealed that a single copy of the 5' NTS is necessary and sufficient to support autonomous DNA replication. Analysis of PUC 1x1.9 deletion derivatives suggested that replicative efficiency was impaired for 5' NTS mutants. Stable transfection experiments with derivatives of the plasmic prD1, which contains a single copy of the (DNA coding region and 5' NTS, confirm these results. Pulse field gel electrophoresis indicated that mutant plastics frequently propagate by integration into the endogenous minichromosome. 2D gel analysis of one such transforming revealed that the integrated plasmic DNA retained weak origin activity. In addition to investigating cis-acting sequences involved in DNA replication control, a single- stranded binding protein (ssA-TIBF) that interacts with Type l elements was examined in T. thermophila and T. pyrformis. The Type 1 elements are cis-acting determinants for DNA replication control (Larson et al., 1986; Yaeger et a1., 1989). In vitro gel mobility shift assays have demonstrated that the properties of SSA-TIBF isolated from T. pyrformis are virtually indistinguishable from those of T. thermophila SSA-TIBF (Yue et al., 1998; this thesis). Cross-species recognition of the Type l element was also observed. However, this study indicates that the T. pyrformis 5' NTS does not function as an origin of replication in T. thermophila cells in vivo. Hence, other species-specific determinants must be involved in DNA replication control.