Identification and cloning of the CHL4 gene controlling chromosome segregation in yeast.

Abstract

A collection of chl mutants characterized by decreased fidelity of chromosome transmission and by minichromosome nondisjunction in mitosis was examined for the ability to maintain nonessential dicentric plasmids. In one of the seven mutants analyzed, chl4, dicentric plasmids did not depress cell division. Moreover, nonessential dicentric plasmids were maintained stably without any rearrangements during many generations in the chl4 mutant. The rate of mitotic heteroallelic recombination in the chl4 mutant was not increased compared to that in an isogenic wild-type strain. Analysis of the segregation of a marked chromosome indicated that sister chromatid nondisjunction and sister chromatid loss contributed equally to chromosome malsegregation in the chl4 mutant. A genomic clone of CHL4 was isolated by complementation of the chl4-1 mutation and was physically mapped to the right arm of chromosome IV near the SUP2 gene. Nucleotide sequence analysis of CHL4 clone revealed a 1.4-kb open reading frame coding for a 53-kD predicted protein which does not have homology to published proteins. A strain containing a null allele of CHL4 is viable under standard growth conditions but has a temperature-sensitive phenotype (conditional lethality at 36 degrees). We suggest that the CHL4 gene is required for kinetochore function in the yeast Saccharomyces cerevisiae.

Differential kinetochore protein requirements for establishment versus propagation of centromere activity in Saccharomyces cerevisiae

“…Nuclei were incubated with increasing concentrations of DraI, which recognizes three closely apposed sites within CDEII of CEN3. The endogenous centromere was 1.5-fold more accessible in chl4⌬ than in wild-type cells, consistent with the increase in chromosome loss observed in chl4⌬ cells (Kouprina et al, 1993). In contrast, new GALCEN was 3.5-fold more accessible to DraI digestion.…”

“…The stability of a fraction of centromeres in the absence of Chl4p for many generations is indicative of an epigenetic mechanism in centromere inheritance in S. cerevisiae. CHL4 encodes a 53-kD predicted protein that shares a low degree of homology with the family of bacterial RecA proteins (25% identical and 47% similarity over 350 amino acids; Kouprina et al, 1993). In addition, it has 29% DNA sequence homology to an uncharacterized ORF in S. pombe (Pi022p) and Neurospora crassa.…”

“…To determine whether CHL4 is differentially required for function on new versus established centromeres, we compared the mitotic stability of centromere plasmids introduced into chl4⌬ mutants (new centromeres) versus the mitotic stability of plasmids introduced into wild-type cells and subsequent removal of Chl4p (old or established centromeres). Centromere plasmids are highly unstable in chl4⌬ mutants (45/45 0.08-5% stability; Table II, chl4⌬→plasmid; Maine et al, 1984;Kouprina et al, 1993). In contrast, deletion of CHL4 from centromere plasmid-bearing cells revealed a range of mitotically stable (60-90%, average of 79.2% Ϯ 9.9) and unstable (0.08-5%, average of 0.8% Ϯ 1.1) transformants (Table II, Wt ϩ plasmid→chl4⌬).…”

“…Mutations in kinetochore genes that decrease the fidelity of chromosome segregation display decreased dicentric chromosome breakage as well. Three of the mutants that exhibit a size-dependent segregation phenotype, chl4 , iml3 , and mcm21 , support the propagation of completely intact dicentric plasmids (Kouprina et al, 1993;Sanyal et al, 1998;Poddar et al, 1999;Ghosh et al, 2001). The presence of two weakened kinetochores on a dicentric chromosome may decrease the chance that both centromeres are attached to opposite spindle poles at a given time, thereby suppressing chromosome breakage (Fig.…”

Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome

“…Disruption of the budding yeast Cenp-N gene (CHL4) causes chromosome loss and instability, without affecting viability 31 . However, combining a chl4 deletion with either mutation of Cenp-A (CSE4), or deletion of other kinetochore subunits, results in synthetic growth defects and lethality 9,27 .…”