Genetic manipulation of centromere function.

Abstract

A conditional centromere was constructed in Saccharomyces cerevisiae by placing the centromere of chromosome III immediately downstream from the inducible GAL) promoter from S. cerevisiae. By utilizing growth conditions that favor either transcriptional induction (galactose-carbon source) or repression (glucosecarbon source) from the GAL) promoter, centromere function can be switched off or on, respectively. With the conditional centromere we were able to radically alter the mitotic transmission pattern of both monocentric and dicentric plasmids. Moreover, it was possible to selectively induce the loss of a single chromosome from a mitoticafly dividing population of cells. We observed that the induction of chromosome HI aneuploidy resulted in a dramatic change in cell morphology. The construction of a conditional centromere represents a novel way to create conditional mutations of cis-acting DNA elements and will be useful for further analysis of this important stabilizing element.Chromosome segregation in most eucaryotic organisms involves the attachment of a spindle apparatus to a specific chromosomal domain, the kinetochore. While the assembly and disassembly of the mitotic spindle can be visualized by a variety of methods, the mechanism by which chromosomes become attached to this apparatus is not understood. The isolation of the DNA sequences required for chromosome segregation in the budding yeast Saccharomyces cerevisiae has provided the first insights at the molecular level into the function of this unique chromosomal domain (7,12,13,18,24,30,34,38).There are two major components involved in chromosomal segregation: the spindle apparatus and the kinetochore. The spindle apparatus provides the framework for chromosome movement in mitosis through spindle fibers that are connected to the chromosomes. The kinetochore includes the structural components at the site of attachment of the spindle fibers and is located within a unique chromosomal domain, the centromere. During mitosis, the centromere region is observed as the primary constriction along the chromosome fiber in higher eucaryotic cells. The centromeric region in the yeast cannot be seen microscopically due to the absence of mitotic chromosome condensation in S. cerevisiae. Examination of the molecular structure of yeast centromeres by nuclease digestion has revealed that centromeric DNA (CEN) is organized into a 220-to 250-base-pair (bp) protected chromatin structure (4). The centromere core particle, including DNA and associated protein, is therefore structurally distinct from the typical 146-bp DNA-histone core particle that constitutes the bulk of eucaryotic chromatin. Centromere DNA, whether in situ, in heterologous chromosomes or in autonomously replicating plasmids introduced into yeast, is always organized into a 220-to 250-bp core complex (3). Furthermore, point mutations at select sites within this region result in both inactivation of centromere function (28) and complete disruption of the protected chromatin core (M. J. Saunders and K. S. ...

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

“…It is unlikely that VP16 disrupts the kinetochore by directly bombarding it with large numbers of transcribing polymerases, as is the case for conditional centromeres in budding yeast (Hill and Bloom, 1987). The level of RNA polymerase II induced by tetR-EYFP-VP16 remained at only about half of that of a typical euchromatic gene.…”

Chromosome bi-orientation on the mitotic spindle

“…Because such minichromosomes cannot be propagated, we needed to establish an induction system by which we can make them from minichromosomes that are propagated stably (figure 2; Dewar et al 2004). The original minichromosome carried two centromeres; the first one was constitutively active and the second one was conditionally inactivated owing to its juxtaposition to the GAL1-10 promoter (Hill & Bloom 1987). The chromosome also possessed a single replication origin flanked by recombination Monotelic attachment: one of the sister kinetochores attaches to microtubules, whereas the other does not attach to any microtubules.…”

“…Moreover, the centromeres (DNA underlying the kinetochore protein complex) have been pinpointed to sequences no longer than 120 bp (Hegemann & Fleig 1993), which allows us to regulate the activity of centromeres. For instance, we can conditionally turn off the centromere activity by transcription from an adjacent GAL1-10 promoter (Hill & Bloom 1987). The behaviour of yeast centromeres can be followed by marking them with arrays of bacterial operators bound by repressor proteins fused to green fluorescent protein (GFP; Goshima & Yanagida 2000;He et al 2000;Tanaka et al 2000;Pearson et al 2001).…”

Hierarchical Inactivation of a Synthetic Human Kinetochore by a Chromatin Modifier

“…Surprisingly, targeting of factors promoting the formation of heterochromatin associated with HP1␣ strongly disrupted the conditional kinetochore, as judged by loss of centromeric proteins, alteration of the pattern of chromatin modifications within the centromere, and destabilisation of the HAC (Nakano et al, 2008). The alphoid tetO HAC system has important differences from the only other conditional centromere system, which was developed for the point centromere of budding yeast (Hill and Bloom, 1987). That centromere is inactivated by being bombarded by transcription from an adjacent strong promoter.…”

“…The alphoid tetO HAC system has important differences from the only other conditional centromere system, which was developed for the point centromere of budding yeast (Hill and Bloom, 1987). That centromere is inactivated by being bombarded by transcription from an adjacent strong promoter.…”