We have determined the structural organization and functional roles of centromere-specific DNA sequence repeats in cenl, the centromere region from chromosome I of the fission yeast Schizosaccharomyces pombe. ceni is composed of various classes of repeated sequences designated K', K" (dgl), L, and B', arranged in a 34-kb inverted repeat surrounding a 4-to 5-kb nonhomologous central core. Artificial chromosomes containing various portions of the ceni region were constructed and assayed for mitotic and meiotic centromere function in S. pombe. Deleting K' and L from the distal portion of one arm of the inverted repeat had no effect on mitotic centromere function but resulted in greatly increased precocious sister chromatid separation in the first meiotic division. A centromere completely lacking K' and L, but containing the central core, one copy of B' and K" in one arm, and approximately 2.5 kb of the core-proximal portion of B' in the other arm, was also fully functional mitotically but again did not maintain sister chromatid attachment in meiosis I. However, deletion of K" from this minichromosome resulted in complete loss of centromere function. Thus, one copy of at least a portion of the K" (dgl) repeat is absolutely required but is not sufficient for S. pombe centromere function. The long centromeric inverted-repeat region must be relatively intact to maintain sister chromatid attachment in meiosis I.The centromere ensures the proper segregation of eukaryotic chromosomes by supplying at least two distinct functions. This highly specialized DNA region provides the spindle attachment point in both mitosis and meiosis; in addition, it maintains attachment of sister chromatids in the first meiotic division, resulting in their migration together to the same pole.Functional centromeres from the budding yeast Saccharomyces cerevisiae have been isolated and extensively characterized (reviewed in references 4, 8, 12, and 16). The functional centromeric DNA (CEN) from this organism is approximately 125 bp in length (13) and lacks the large blocks of transcriptionally silent pericentric heterochromatin that are characteristic of the centromere regions of higher eukaryotes (30,42 repeated sequences are present only at the three centromere regions, and no polyadenylated transcripts from these regions have been detected (15, 37). The three S. pombe centromeres (ceni, cen2, and cen3) have been cloned on large genomic Sall restriction fragments in yeast artificial chromosome (YAC) vectors, and a minichromosome assay system has been developed to determine the minimum arrangement of DNA sequences necessary for centromere function in this organism (10,20).Recently, a plasmid integration-excision strategy has been used to facilitate chromosome walking through regions containing repetitive DNA (10). This method was used to clone and map over 80 kb of DNA from the centromere region of S. pombe chromosome II (cen2). The cen2 region contains four tandem units of the centromere-specific K, L, and B repeats, two of which are arranged ...

Microfabrication technology was used to develop a system consisting of disposable glass chips containing etched channels, reagents including polymer matrix and size standards, computer‐controlled instrumentation for performing electrophoretic separations and fluorescence detection of double‐stranded DNA, and software for automated data analysis. System performance was validated for separation and quantitation reproducibility using samples varying in amount and size of DNA fragments, buffer composition, and salt concentrations. Several applications of the microfluidic system for DNA analysis have been demonstrated, such as of polymerase chain reaction (PCR) products, sizing of plasmid digests, and detection of point mutations by restriction fragment length polymorphism (RFLP) mapping.

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) ...

Gross variations in the structure of the centromere of Schizosaccharomyces pombe chromosome II (cen3) were apparent following characterization of this centromeric DNA in strain Sp223 and comparison of the structure with that ofcen3 in three other commonly used laboratory strains. Further differences in centromere structure were revealed when the structure of the centromere ofS. pombe chromosome H (cen2) was compared among common laboratory strains and when the structures ofcen2 and cen3 from our laboratory strains were compared with those reported from other laboratories. Differences observed in cen3 structure include variations in the arrangement of the centromeric K repeats and an inverted orientation of the conserved centromeric central core. In addition, we have identified two laboratory strains that contain a minimal cen2 repeat structure that lacks the tandem copies of the cen2-specific block of K-L-B-J repeats characteristic of Sp223 cen2. We have also determined that certain centromeric DNA structural motifs are relatively conserved among the four laboratory strains and eight additional wild-type S. pombe strains isolated from various food and beverage sources. We conclude that in S. pombe, as in higher eukaryotes, the centromere of a particular chromosome is not a defined genetic locus but can contain significant variability. However, the basic DNA structural motif of a central core immediately flanked by inverted repeats is a common parameter of the S. pombe centromere.The centromere (cen) locus is a specialized region of eukaryotic chromosomal DNA that acts in conjunction with specific proteins to mediate chromosome segregation during mitotic and meiotic cell divisions. Centromere functions include maintenance of sister chromatid attachment during meiosis I and interaction, via the spindle attachment site or kinetochore, with mitotic and meiotic spindles, thereby ensuring proper segregation of chromosomes during cell divisions.Centromere DNAs of the budding yeast Saccharomyces cerevisiae have been extensively characterized, are only about 125 bp in length, and contain no repeated DNA sequences (5,8). Although analogous with respect to function, they appear structurally to be quite unlike the centromeres of higher eukaryotes, which in many cases contain extensive regions of heterochromatic repeated DNA, whose role is as yet unclear (2, 25). The genome of the fission yeast Schizosaccharomyces pombe is organized into only three chromosomes and provides an excellent alternate model system for studying the role of the centromere in cell division. The centromeres of S. pombe and those of higher eukaryotes have significant similarities that may be a reflection of their common mode of cell division. S. pombe centromeric regions are large, spanning approximately 40 to 100 kb of DNA, and consist in part of repetitive, nontranscribed DNA sequences that have been shown to play a role in centromere function (4,7,9,12,20). In addition, S. pombe centromeres are organized into a large inverted repeat (4, 7, 12) which...

The gene encoding M 2 , the ion channel-forming protein of influenza virus A, was expressed under the control of an inducible promoter in Saccharomyces cerevisiae. By using single and multicopy plasmids containing GAL promoter-M 2 fusions, a correlation was observed between plasmid copy number and growth in medium inducing M 2 expression. Cells expressing M 2 from multicopy plasmids have reduced growth rates, suggesting that high levels of M 2 are toxic to growth. The addition of amantadine, a compound known to block the ion channel activity of certain M 2 alleles, restores the growth rates to wild-type levels in cells expressing an amantadine-susceptible allele of M 2 but not an amantadine-resistant allele of M 2 , suggesting that M 2 expression in S. cerevisiae results in the formation of functional M 2 ion channels. Measurements of extracellular acidification by microphysiometry suggest that proton efflux in M 2 -expressing cells is altered and that the addition of amantadine permits the reestablishment of the proton gradient. The growth impairment phenotype resulting from M 2 expression was used to develop a high-capacity screening assay which identified a novel inhibitor possessing an antiviral profile similar to that of amantadine.Functional expression of ion channels in heterologous cell types provides a general method for studying the properties of channel function. Expression in Xenopus laevis oocytes has provided numerous insights into ion channel function (17,18,35). More recently, expression of ion channels in Saccharomyces cerevisiae strains defective in K ϩ uptake has served as a means for isolating new genes encoding functional channels (1, 29). However, expression of ion channels in yeast cells is not restricted to the restoration of uptake functions and may result in a variety of phenotypes, depending on the properties of a particular channel.The M 2 protein of influenza virus A is one of three integral membrane proteins contained in the viral lipid envelope. The M 2 protein is a 97-amino-acid polypeptide containing a single membrane-spanning region. M 2 polypeptides associate as disulfide-linked homotetramers to form ion channels (15,34). Direct evidence defining the ion channel function of M 2 has recently been obtained by expression studies in Xenopus oocytes (26, 38) and in in vitro studies (6,28,36). A therapeutic agent for influenza virus A infections, amantadine, has been shown to function by blocking M 2 ion channel activity (7,12,13,26,32,33,37,38). Other biophysical studies have confirmed that the transmembrane portion of the molecule is the binding site and suggest that the mechanism of action is binding of the compound within the channel pore (6,7,27). Inhibition studies with amantadine suggest that M 2 is required at both early and late stages in the infection cycle of the virus. Early in infection, M 2 permits the flow of protons from the endosome into the virion. The resultant decrease in pH facilitates the dissociation of the matrix protein (M 1 ) from viral genomic ribonucleopro...