The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila.

Abstract

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10 per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmml mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmml. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4IB-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 104 copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophila.The rRNA genes (rDNA) in the ciliated protozoan Tetrahymena thermophila provide an excellent opportunity for studying DNA replication in a eucaryotic system. In the somatic nuclei of T. thermophila, the rDNA is a relatively short (21-kilobase pair [kb] somally integrated rDNA copy in each haploid genome. During macronuclear development, the rDNA is excised from the chromosome and converted into a 21-kb palindromic molecule. The palindrome is made up of two inversely oriented copies of the micronuclear rDNA sequence. The extrachromosomal rDNA acquires telomeres (1) and is amplified to a copy number of about 104 molecules per macronucleus. These maturation events are completed prior to the onset of vegetative growth, approximately 18 h after conjugation is initiated. The macronuclear rDNA is maintained at a high copy number throughout vegetative cell divisions. Figure 1A diagrams one-half of an rDNA palindrome. The origin of replication is within the 5' nontranscribed spacer (5'NTS), upstream from the transcription initiation site (Fig. 1A) (4). Two nuclease-hypersensitive regions within the 5'NTS (15), domains 1 and 2, have very similar sequences and contain evolutionarily conserved repeated sequence elements (5, 11). By mutational analysis, one of these repeated elements (type I) was implicated in the activation of rDNA replication (8).We have previously shown that rDNA from a T. thermophila inbred strain C3 (C3 rDNA) has a replicative advantage over rDNA from inbred strain B (B rDNA), causing C3 rDNA to completely replace B rDNA in C3/B heterozygotes during vegetative growth (8)....

A single-stranded DNA binding protein that specifically recognizes cis-acting sequences in the replication origin and transcriptional promoter region of Tetrahymena rDNA

“…Accurate initiation of rRNA transcription in both T. pyriformis (Miyahara et al, 1993) (Umthun et al, 1994), this study demonstrates that additional sequences which make a major contribution to ssA-TIBF binding affinity are located outside the conserved element. It should be noted, however, that although ssA-TIBF can distinguish between the C3-and B-rDNA alleles in vitro, several induced C3-rmm mutations are due to a single base pair deletion in the central tract of 11 A residues in copies of the Type 1 repeat (Larson et al, 1986;Yaeger et al, 1989 (Hofmann & Gasser, 1991;Kuno et al, 1990Kuno et al, . 1991Schmidt et al, 199l;Zeidlere/a/., 1993).…”

“…these sequences are identical for the Type lb and Ic repeats ( Figure 1C), and C3-rmm mutations that affect rDNA replication and/ or maintenance in the macronucleus are due to single base pair deletions within one of these two repeats (asterisks in Figure IB) (Larson et al. 1986;Yaeger et al, 1989 (Figure 3). Sequences of the oligonucleotides used in these experiments are shown in Figure 3A.…”

“…One major band was observed migrating as a polypeptide of 24 kDa (Figure 2 (Figure 1). This region was of special interest because it contains the determinant of the replication disadvantage of B-relative to C3-rDNA (Larson et al, 1986;Yaeger et al, 1989). Moreover, in wild-Type C3-rDNA.…”

“…1985;Niles et al, 1981). Asterisks denote Type I elements altered in C3-rmm mutants defective in rDNA replication and/or maintenance in the macronucleus (Larson et al, 1986;Yaeger et al, 1989 (Yao. 1986;Kapler.…”

A Promoter Region Mutation Affecting Replication of the Tetrahymena Ribosomal DNA Minichromosome

“…This single-copy rDNA is amenable to genetic analysis (20,22,27,48). During macronuclear development, each rDNA allele, along with 5Ј and 3Ј flanking DNA sequences, is excised from the micronuclear chromosome and formed into a 21-kb palindromic linear molecule containing two divergently transcribed rRNA genes, in a head-to-head configuration, and newly added telomeric DNA (Fig.…”