Mapping the mating type locus of Tetrahymena thermophila: meiotic linkage of mat to the ribosomal RNA gene.

Tetrahymena thermophila has a multiple mating type system. While a sexually mature cell usually expresses only one mating type, its germline (micronucleus) carries the genetic potential for 5 to 7 mating types. The set of allowed mating types is specified by the mat locus. The choice of which particular mating type is expressed by a cell reflects a somatically inherited, developmentally programmed differentiation of the somatic nucleus (macronucleus). In this work we report that the mat locus maps to the left arm of chromosome 2, as determined by nullisomic deletion mapping. We also report a distance of 29 cM between the mat locus and the ribosomal RNA gene, previously mapped to chromosome 2L. This represents another (rare) case of meiotic linkage in Tetrahymena.

Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences.

A circular minichromosome carrying functional centromere sequences (cen2) from Schizosaccharomyces pombe chromosome II behaves as a stable, independent genetic linkage group in S. pombe. The cen2 region was found to be organized into four large tandemly repeated sequence units which span over 80 kilobase pairs (kb) of untranscribed DNA. Two of these units occurred in a 31-kb inverted repeat that flanked a 7-kb central core of nonhomology. The inverted repeat region had centromere function, but neither the central core alone nor one arm of the inverted repeat was functional. Deletion of a portion of the repeated sequences that flank the central core had no effect on mitotic segregation functions or on meiotic segregation of a minichromosome to two of the four haploid progeny, but drastically impaired centromere-mediated maintenance of sister chromatid attachment in meiosis I. This requirement for centromere-specific repeated sequences could not be satisfied by introduction of random DNA sequences. These observations suggest a function for the heterochromatic repeated DNA sequences found in the centromere regions of higher eucaryotes.

Construction of functional artificial minichromosomes in the fission yeast Schizosaccharomyces pombe.

The centromere DNAs from chromosomes I and III of Schizosaccharomyces pombe have been cloned in an artificial chromosome vector in both budding and fission yeasts. In S. pombe, synthetic linear and circular minichromosomes containing an intact centromere are stable mitotically and behave as independent genetic linkage groups that segregate properly through meiosis. These experiments present a general strategy for the isolation of centromeres from other organisms.

Unidirectional co-stimulation by a non-mating strain of Tetrahymena thermophila.

We report that a fatty acid auxotroph of Tetrahymena thermophila (RH179E1) fails to mate, yet retains the ability to co-stimulate normal cells unidirectionally. Thus, co-stimulation can be analyzed experimentally in the absence of pair formation. We show that the co-stimulation of normal cells of one mating type is sufficient to shorten the waiting period for pair formation of those cells with initiated cells. This is the first evidence that co-stimulation causes a hyperinduction of mating reactivity in T. thermophila, generating in turn a positive feedback mechanism for (presumably) gamone production. Co-stimulation by the variant strain is at a maximum after 3-4 h of exposure when the variant and wild-type cells are at a ratio of 1:1. When mixed with wild-type cells, RH179E1 induces the formation of progeny (at low frequency) which inherit exclusively genetic material of the wild-type cells.

A restriction fragment length polymorphism in the 5' non-transcribed spacer of the rDNA of Tetrahymena thermophila inbred strains B and C3.

The ribosomal DNA of Tetrahymena thermophila is a 21-kb palindromic molecule which replicates autonomously in the macronuclei of this species. In addition to the rRNA coding regions, there are 5' and 3' flanking sequences which are not transcribed (non-transcribed spacer; NTS). The 5' NTS contains a bidirectional origin of DNA replication and promoter elements which direct transcription. We have identified a restriction fragment length polymorphism in the rDNA 5' NTS by comparing the B and C3 inbred strains of T. thermophila. There is a 42-bp region present in the C3 but not in the B strain rDNA; we present evidence that this difference most likely represents a deletion in the B strain rather than an insertion in the C3 strain. We also include a revised version of the nucleotide sequence of the 5' NTS DNA of inbred strain B.