Abstract
We developed a novel approach to quantitate the heterogeneity of centromere number in yeast, and the cellular capacity for excess centromeres. Small circular plasmids were constructed to contain theCUP1 metallothionein gene,ARS1 (autonomously replicating sequence) and a conditionally functional centromere (GAL1–GAL10 promoter controlled centromere). TheCUP1 gene provided a gene dosage marker, and therefore a genetic determinant of plasmid copy number. Growth of cells on glucose is permissive for centromere function, while growth on galactose renders the centromere nonfunctional and the plasmids are segregated in an asymmetric fashion. We identified “lines” of cells containing increased numbers of plasmids after transformation. Cell lines containing as many as five to ten active centromeres are stably maintained in the absence of genetic selection. Thus haploid yeast cells can tolerate a 50% increase in their centromere number without affecting progression through the cell cycle. This system provides the opportunity to address issues of specific cellular controls on centromere copy number.
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References
Bitoun R, Zamir A (1986) Spontaneous amplification of yeast CEN ARS plasmids. Mol Cell Genet 204:98–102
Bloom KS, Carbon J (1982) Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes. Cell 29:305–317
Bloom KS, Amaya E, Carbon J, Clark L, Hill A, Yeh E (1984) Chromatin conformation of yeast centromeres. J Cell Biol 99:1559–1568
Chlebowicz-Sledziewska E, Sledziewski AZ (1985) Construction of multicopy yeast plasmids with regulated centromere function. Gene 39:25–31
Fitzgerld-Hayes M, Clarke L, Carbon J (1982) Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell 29:235–244
Fogel S, Welch JW, Cathala G, Karin M (1983) Gene amplification in yeast:CUP1 copy number regulates copper resistance. Curr Genet 7:347–355
Futcher B, Carbon J (1986) Toxic effects of excess cloned centromeres. Mol Cell Biol 6:2213–2222
Hartwell LH, Weinert TA (1989) Checkpoints: controls that ensure the order of cell cycle events. Science 246:629–634
Hartwell LH, Dutcher SK, Wood JS, Garvick B (1982) The fidelity of mitotic chromosome reproduction inS. cerevisiae. Recent Adv Ycast Mol Biol 1:28–38
Heiter P, Mann C, Snyder M, Davis RW (1985) Mitotic stability of yeast chromosomes: a colony-color assay that measures nondisjunction and chromosome loss. Cell 40:381–392
Heiter P, Pridmore D, Hegemann JH, Thomas M, Davis RW, Philippsen P (1986) Functional selection and analysis of yeast centromeric DNA. Cell 42:913–921
Hill A, Bloom KS (1987) Genetic manipulation of centromere function. Mol Cell Biol 7:2397–2405
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Johnston M, Davis RW (1984) Sequences that regulate the divergentGAL1–GAL10 promoter inSaccharomyces cerevisiae. Mol Cell Biol 4:1440–1448
Karin M, Najarian R, Haslinger A, Valenzuela P, Welch J, Fogel S (1984) Primary structure and transcription of an amplification genetic locus: theCUP1 locus of yeast. Proc Natl Acad Sci USA 81:337–341
Kenna M, Amaya E, Bloom K (1988) Selective excision of the centromere chromatin complex forSaccharomyces cerevisiae. J Cell Biol 107:9–15
Koshland D, Kent JC, Hartwell LH (1985) Genetic analysis of the mitotic transmission of minichromosomes. Cell 40:393–403
Larionov VL, Kouprina NY, Strunnikov AV, Vlasov AV (1989) A direct selection procedure for isolating yeast mutants with an impaired segregation of artificial minichromosomes. Curr Genet 15:17–25
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
McGrew JB, Diehl B, Fitzgerald-Hayes M (1986) Single base-pair mutations in centromere element III cause aberrant chromosome segregation inSaccharomyces cervisiae. Mol Cell Biol 6:530–538
Murray AW, Kirschner MW (1989) Dominoes and clocks: the union of two views of the cell cycle. Science 246:614–621
Panzeri L, Landonio L, Stotz A, Philippsen P (1985) Role of conserved sequence elements in yeast centromere DNA. EMBO J 4:1867–1874
Resnick MA, Bloom K (1987) Lessons learned from yeast: a molecular and genetic analysis of centromere function. In: Vig B, Sandberg A (eds) Aneuploidy, Part A: incidence and etiology. Alan R. Liss, New York, pp 395–413
Resnick MA, Westmoreland J, Amaya E, Bloom KS (1987) UV-induced damage and repair in centromere DNA of yeast. Mol Gen Genet 210:16–22
Saunders M, Fitzgerald-Hayes M, Bloom K (1988) Chromatin structure of altered yeast centromeres. Proc Natl Acad Sci USA 85:175–179
Snyder M, Saplosky RJ, Davis RW (1988) Transcription interferes with elements important for chromosome maintenance inSaccharomyces cerevisiae. Mol Cell Biol 8:2184–2194
Tschumper G, Carbon J (1980) Sequence of a yeast DNA fragment containing a chromosomal replicator and theTRP1 gene. Gene 10:157–166
Tschumper G, Carbon J (1987)Saccharomyces cerevisiae mutants that tolerate centromere plasmids in high copy number. Proc Natl Acad Sci USA 84:7203–7207
Whittaker S, Rockmill BM, Blechel AE, Malone DH, Resnick MA, Fogel S (1988) The detection of mitotic and meiotic aneuploidy in yeast using a gene dosage selection system. Mol Gen Genet 215:10–18
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Resnick, M.A., Westmoreland, J. & Bloom, K. Heterogeneity and maintenance of centromere plasmid copy number inSaccharomyces cerevisiae . Chromosoma 99, 281–288 (1990). https://doi.org/10.1007/BF01731704
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DOI: https://doi.org/10.1007/BF01731704