RESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular , Cromosomas Fúngicos/ultraestructura , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Centrómero/ultraestructura , Mapeo Cromosómico , Clonación Molecular , Proteínas Fúngicas/química , Prueba de Complementación Genética , Secuencias Hélice-Asa-Hélice , Cinética , Mitosis , Datos de Secuencia Molecular , Mutagénesis , Sistemas de Lectura Abierta , Plásmidos , Estructura Secundaria de Proteína , Mapeo Restrictivo , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de AminoácidoRESUMEN
Eight independent chl (chromosome loss) mutants were isolated using yeast haploid strain disomic for chromosome III. In these mutants, chromosome III is lost during mitosis 50-fold more frequently than in the wild-type strains. chl mutants are also incapable of stable maintenance of circular and linear artificial chromosomes. Seven of the eight mutations are recessive, and one is semidominant. Complementation tests placed these mutants into six complementation groups (chl11 through chl16). Based on tetrad analysis, chl12, chl14 and chl15 correspond to mutations in single nuclear genes. Tetrad analysis of the other mutants was not possible due to poor spore viability. Complementation analysis was also carried out between collection of chl mutants and ctf mutants (chromosome transmission fidelity) (Spencer et al., 1990). The chl3, chl4, chl8, chl12 and chl15 mutants were unable to complement ctf3, ctf17, ctf12, ctf18 and ctf4, respectively. Three CHL genes were mapped by tetrad analysis. The CHL3 gene is placed on the right arm of chromosome XII, between the ILV5 (33.3 cM) and URA4 (21.8 cM) loci. The CHL10 gene is located on the left arm of chromosome VI, 12.5 cM from the centromere. The CHL15 gene is tightly linked to the KAR3 marker of the right arm of chromosome XVI (8.8 cM). The mapping data indicate that these three genes differ from other genes known to affect chromosome stability in mitosis. Therefore, the total number of the CHL genes identified (including those described by us earlier) is 13 (CHL1-CHL10, CHL12, CHL14 and CHL15).