Abstract. We have examined the chromatin structure of centromere regions from the fission yeast Schizosaccharomyces pombe. The large and complex centromere regions of the S. pombe chromosomes encompass many kilobase pairs of DNA and contain several classes of tandemly repeated DNA sequences. The repeated sequences are further organized into a large inverted repeat flanking a central core, a conserved structural feature among all three centromeres in S. pombe. The nucleosomal configuration of the centromere regions is nonuniform and highly varied. Most of the centromere-specific repeated DNA sequences are packaged into nucleosomes typical of bulk chromatin. However, the central core and coreassociated repeated sequences from the centromere regions of chromosomes I (cerd) and II (cen2), when present in S. pombe, show an altered chromatin structure, with little or no evidence of regular nucleosomal packaging. The atypical chromatin organization of the cen2 central core is not due to transcription, as no transcripts from this region were detected. These same DNA sequences, however, are packaged into nucleosomes typical of bulk chromatin when present in a nonfunctional environment on a minichromosome in the budding yeast Saccharomyces cerevisiae. Because the cen2 central core sequences themselves do not preclude regular nucleosomal packaging, we speculate that in S. pombe they constitute a specialized site of kinetochore protein assembly. The atypical nucleosomal pattern of the cen2 central core remains constant during the cell cycle, with only minor differences observed for some sequences. We propose that the unusual chromatin organization of the core region forms the basis of a higher order structural differentiation that distinguishes the centromere from the chromosome arms and specifies the essential structure for centromere function.T rI~. centromere ofa eukaryotic chromosome is a highly specialized, multi functional region containing the kinetochore, a DNA-protein complex that mediates chromosome attachment to the spindle and participates in the movement of replicated chromatids along the spindle during mitotic and meiotic cell divisions. The centromere also holds sister chromatids together in mitotic metaphase and throughout the first meiotic division, thus ensuring proper segregation of chromosomes to daughter cells. Such a specialized chromosome function implies the existence of a characteristic chromatin structure that confers specificity for carrying out the precise, timed events of mitotic and meiotic cell divisions. Because the centromere functions in the context of a higher order nucleoprotein assembly, understanding the molecular mechanisms of centromere function and the basis of its differentiation from chromosome arms will depend in large part on the elucidation of the chromatin architecture of the centromere region.The chromatin structure of the relatively simple centromeres from the budding yeast Saccharomyces cerevisiae has been well-characterized (Bloom and Carbon, 1982). The functional S. cerevi...

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.Analysis of centromeric DNA from the fission yeast Schizosaccharomyces pombe has revealed the presence of several classes of moderately repetitive DNA sequences (1-4). These DNA sequence repeats are present only in the centromere regions of the three S. pombe chromosomes and have been shown to be transcriptionally silent (1). Thus, with respect to their heterochromatic properties, the centromeres of S. pombe more closely resemble those of higher eukaryotes than the relatively simple centromeres of the budding yeast Saccharomyces cerevisiae (5).In S. cerevisiae, small [150 base pairs (bp)] cloned centromere DNA sequences (designated here CEN) confer mitotic stability upon autonomously replicating (ARS) plasmids and enable them to segregate faithfully through meiosis (6). Previously, no discrete segment of S. pombe DNA had been found to confer mitotic or meiotic stability on ARS plasmids in S. pombe (1-3). In this study, we have developed a minichromosome assay system for centromere function in S. pombe. The S. cerevisiae yeast artificial chromosome (YAC) vector system (7) has been used to clone large restriction fragments from the S. pombe genome in S. cerevisiae. Linear artificial chromosomes containing the S. pombe centromeric regions from chromosomes I and III (designated here ceni and cen3) were obtained in S. cerevisiae and subsequently introduced by transformation into S. pombe and assayed for proper centromere function. Both linear and circular artificial chromosomes were recovered in S. pombe. These minichromosomes are mitotically stable and segregate properly through meiosis, indicating that the cloned fragments contain functional S. pombe centromeres. They are the first synthetic chromosomes found to be fully functional in an organism other than Saccharomyces. The methods described in this study should provide a general approach for the cloning of centromeres from other organisms as well. MATERIALS AND METHODSStrains, Transformations, and Genetic Manipulations. The genotypes of the S. pombe strains are given in the legends to Tables 1 and 2. S. cerevisiae strain AB1380 (a ura3 trpl ade2-1 can1-100 lys2-1 his5) was kindly provided by David Burke (7). DNA transformations of S. cerevisiae (7) and S. pombe (10) and genetic manipulations of S. pombe (11, 12) were performed as described.Enzymes, DNA Isolation, and Field-Inversion Gel Electrophoresis (FIGE). Restriction enzymes were from New England Biolabs, and T4 DNA ligase and calf intestinal alkaline phosphatase were from Boehringer Mannheim Biochemicals. Genomic DNA from S. cerevisiae (8) ...

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In comparing the tryptic peptide maps of the H-2L and H-2D glycoprotein antigens isolated from NP-40 lysates of RADA1 (H-2 alpha) leukemic cells, no more than 37% of the observed arginine-containing tryptic peptides are found to be homologous. Thus, the primary amino-acid sequences of these two antigens are probably less than 90% homologous. This constitutes the strongest evidence to date that the MHC-linked H-2L region encodes H-2L antigens separately from the H-2D region, even though H-2L antigens bear D-end-associated antigenic determinants of the H-2.28 family. The anti-H-2.28 alloantiserum (k X r anti h2) used to precipitate H-2L antigens in this investigation was the NIH contract antiserum D28b. As the tryptic peptide maps also suprisingly revealed, D28b precipitates H-2D antigens as well and, thus, anti-H-2.4 immunoadsorbants were employed to isolate H-2L free of H-2D antigens. In light of the dual specificity of D28b, its reactivity with BALB/c-H-2dm2 mutant cells was re-examined. Even though mutant lymphocytes, which lack H-2L but not H-2D antigens, are not cytotoxically lysed by D28b (as are parental H-2d cells), D28b appears to precipitate H-2D antigens from NP-40 extracts of mutant splenocytes.

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