Rate of phenotypic assortment in Tetrahymena thermophila

Abstract

During vegetative, asexual reproduction in heterozygous Tetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both. Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus. The primary tools to study assortment are Rf, the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes. Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number. Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development. Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured. Although published Rf values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of Rf. First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf. Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele. Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with approximately 45 assorting units, as has been asserted.

Accumulation of Spontaneous Mutations in the CiliateTetrahymena thermophila

“…Seven to 12 GE lines per MA line resulted in successful backcrosses. We measured fitness after backcross as described above, except that we inoculated single mating pairs into a 96-well plate and incubated them for ,48 hr at 30°to avoid loss of heterozygosity from phenotypic assortment (Doerder et al 1992). The fitness of 4-0-AI1 was also measured in each assay of GE lines before and after backcross.…”

Essential and Nonessential Histone H2A Variants in Tetrahymena thermophila

“…Given the similarity in intensities of the genomic Southern blots of wild-type cells and HTA3 transformants (Fig. 1C), that limit is probably close to the normal level of 45 copies per (G 1 ) macronucleus (10).…”

“…The initial transformants probably have only a fraction of these copies disrupted. Since macronuclei divide amitotically, distributing the multiple gene copies randomly to each daughter nucleus (a process referred to as phenotypic assortment [10,25]), increasing drug pressure selects for cells with increasing copies of the disrupted genes in the macronuclear genome and the concomitant loss of the undisrupted genes. If a gene is nonessential, eventually this process should lead to complete replacement of intact genes by disrupted ones.…”

The Condensin Complex Is Essential for Amitotic Segregation of Bulk Chromosomes, but Not Nucleoli, in the Ciliate Tetrahymena thermophila

“…Thus, it remains possible that condensation of macronuclear chromosomes does occur and is essential for their proper segregation, perhaps because coupling of condensation with topoisomerase II activity is necessary to untangle the mass of chromatin (19). Genetic evidence (14) indicates that there is no chromosome pairing or equal segregation after macronuclear DNA replication. The chromosome fragments do not have centromeres (7), and our study shows that condensin is not required for nucleolar segregation.…”

“…Genetic data (14,33,51) suggest that these chromosomes segregate randomly during amitosis, in contrast to the equal segregation of sister chromatids typical of mitosis. Another interesting feature of the macronucleus is the presence of approximately 90 nucleoli distributed around its periphery (6), each containing approximately 100 copies of the 20-kb chromosome that holds the rRNA genes.…”