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

Abstract

A circular minichromosome carrying functional centromere sequences (cen2) from Schizosaccharomyces pombe chromosome H 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. (8,9). In contrast, however, the centromere regions of the fission yeast Schizosaccharomyces pombe resemble those of higher eucaryotes in containing several classes of repeated DNA sequences which encompass many kilobases (kb) of DNA on each of the three S. pombe chromosomes (6,7,10,24). Centromeric repeats designated K, L, and B are heterochromatic-like and untranscribed (10). The centromere regions of the three S. pombe chromosomes, including all the centromere-specific DNA sequence repeats, reside on three large genomic SalI restriction fragments of 65, 100, and 150 kb, derived from chromosomes I, II, and III, respectively (10).Recently Gutz et al. (12). DNA transformations of S. pombe were performed as described before (16). Stable Ura+ transformants obtained by site-directed integration of pSp500 (Fig. 1) into SBPD400 were identified as follows. A total of 240 Ura+ transformant colonies were picked onto nonselective medium and subsequently replica-plated for seven rounds of nonselective growth before they were replica-plated to medium selecting for the Ura+ phenotype. Those clones (14 total) that were still Ura+ after several rounds of nonselective growth and that contained sequences hybridizing to pBR322 were characterized further.

A low copy number central sequence with strict symmetry and unusual chromatin structure in fission yeast centromere.

“…Sequences for the outer repetitive areas were reported previously (Nakaseko et al, 1986(Nakaseko et al, , 1987Chikashige et al, 1989) as was restriction site information Clarke and Baum, 1990;Hahnenberger et al, 1991;Murakami et al, 1991). Hence, the total of -60 kb of nucleotide sequences so far determined has uncovered the complex sequence structures present within the centromeres.…”

“…Sequencing of cenl was complete except for the duplicated regions. However, a part of cen2 remains to be determined Clarke and Baum, 1990;Hahnenberger et al, 1991 The properties of the outer regions are in sharp contrast to those of the central regions. The outer repeat units are commonly present in all of the centromeres.…”

“…Functional centromeric DNAs have been isolated and characterized in two organisms, namely, the budding yeast Saccharomyces cerevisiae (Clarke and Carbon, 1985;Murray and Szostak, 1985;Newlon, 1988;Cotterel et al, 1989), which has 16 chromosomes, and the fission yeast Schizosaccharomyces pombe (Clarke et al, 1986;Nakaseko, et al, 1986Nakaseko, et al, , 1987Niwa et al, 1986Niwa et al, , 1989Fishel et al, 1988;Chikashige et al, 1989;Hahnenberger et al, 1989Hahnenberger et al, , 1991Clarke and Baum, 1990;Matsumoto et al, 1990;Murakami et al, 1991), which has three. In budding yeast, a short stretch of DNA (-125 bp) containing a consensus sequences confer centromere function.…”

“…Meiotic recombination within these regions is minimal (Nakaseko et al, 1986;Niwa et al, 1989). Artificial linear or circular chromosomes (>30 kb) containing these sequences segregate normally in both mitosis and meiosis Hahnenberger et al, 1989Hahnenberger et al, , 1991Clarke and Baum, 1990;Matsumoto et al, 1990). Shorter (<10 kb) DNA fragments derived from cen regions, however, fail to confer centromere function (Nakaseko et al, 1986).…”

“…To address this question, we first determined its structure. Although previously studied by restriction mapping Hahnenberger et al, 1989Hahnenberger et al, , 1991Clarke and Baum, 1990;Murakami et al, 1991;Polizzi and Clarke, 1991), little nucleotide sequence information of the central region has been available Kuhn et al, 1991;Murakami et al, 1991;Takahashi et al, 1991). Previous data showed that the central region seemed to consist of two sequence classes, namely, inmost repeat (designated imr) and center (designated cnt) sequences.…”