Two dimensional gel electrophoresis analysis of DNA replication in Tetrahymena pyriformis

Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to digital@library.tamu.edu, referencing the URI of the item.Includes bibliographical references: p. 112-126.Issued also on microfiche from Lange Micrographics.Neutral/neutral two dimensional gel electrophoresis was used to study replication of the Tetrahymena pyriformis RDNA minichromosome. During vegetative growth RDNA is replicated exclusively from origin in the 5' nontranscribed spacer (NTS). Whereas replication fork movement through the rest of the chromosome appears to be continuous, movement through the 5'NTS is not. One replication pausing site was identified. Using neutral/alkaline two dimensional gel electrophoresis, I determined that the replication pausing site is within a nucleosome-free region in the 5'NTS, adjacent to the Type lb element. The Type I repeat is an evolutionarily conserved sequence element found in t h e replication origin and transcriptional promoter region of the RDNA in Tetrahymenid species. Using a gel mobility shift assay, I also determined that a single-stranded Type I element binding activity is present in the T. pyriformis cell extract. This binding activity has features that are very similar to the T. thermophila Type I element binding protein, ssA-TIBF. Quantitative binding experiments demonstrated that the binding site for T. pyriformis ssA-TIBF included sequences both within the conserved Type I element and its flanking region. The binding properties of ssATIBF, coupled with genetic evidence that Type I sequences function as a cis-acting element regulating replication initiation and replication elongation, suggest a possible role for ssA-TIBF in RDNA replication in Tetrahymena