Copyright 0 1990 by the Genetics Society of America Mitotic Transmission of Artificial Chromosomes in cdc Mutants of the Yeast, Saccharomyces cerevisiae Robert E. EileenHogant andDouglas Koshlandt *Department of Population Dynamics, Divisionof Reproductive Biology, The Johns Hopkins UniversitySchool of Hygiene and Public Health, Baltimore, Maryland 21205, and ?Departmentof Embryology, The Carnegie Institution of Washington, Baltimore, Maryland 21210 Manuscript received January1 1, 1990 Accepted for publicationMay 2, 1990 ABSTRACT In the yeast, Saccharomyces cerevisiae, cell division cycle (CDC) genes have been identified whose products are required for the executionof different steps in the cell cycle. In this study, the fidelity of transmission of a 14-kb circular minichromosome anda 155-kb linear chromosome fragmentwas examined in cell divisions where specific CDC products were temporarily inactivated with either inhibitors, or temperature sensitive mutations in the appropriate CDC gene. All of the cdc mutants previously shown to induce loss of endogenous linear chromosomes also induced loss of a circular minichromosome anda large linear chromosome fragmentin our study (either 1:0 or 2:O loss events). Therefore, the efficient transmission of these artificial chromosomes depends upon the same trans factors that are required for the efficient transmission of endogenous chromosomes. In a subset of cdc mutants (cdc6, cdc7 and cdcl6), the rate of minichromosome loss was significantly greater than the rate ofloss of the linear chromosome fragment, suggesting that a structural feature of the minichromosome (nucleotidecontent, length or topology) makesthe minichromosome hypersensitive to the level of function of these CDC gene products. In another subset of cdc mutants (cdc7 and cdcl7), the relativerate of 1:O events to2:O events differed for the minichromosome and chromosome fragment, suggestingthat the typeof chromosomeloss event observedin these mutantswas dependent upon chromosome structure. Finally, we show that 2:O events for the minichromosomecan occur by both a RAD52 dependent and RAD52 independent mechanism. These results are discussed in the context of the molecular functionsof the CDC products. N the yeast, Saccharomyces cermisiae, the construc- tide content, size or topology) which ensures a high I tion and genetic analyses of small circular mini- fidelity of transmission during mitotic cell divisions. chromosomes have been instrumental in understand- In the absence of this structural feature, minichro- ing the structural features of chromosomes that are mosomes may be acted upon by the same replication essential for their properreplication and segregation. and segregation machinerythat acts upon endogenous For example, the analysis of minichromosomes has chromosomes but may bepoor substrates for this led to identification and characterizationof cis se- machinery atone or more steps in the cell cycle. quences required for theinitiation of DNA replication Alternatively, the absence of this structural featurein (HSIAOand CARBON1979; STINCHCOMB,STRUHL and minichromosomes may cause them to be transmitted DAVIS 1979)and for the function of centromeres by secondary replication or segregation processes that (CLARKE and CARBON, STINCHCOMB,1980; MANN and are inherently less faithful. DAVIS1982). Circular minichromosomes that contain An understanding of the molecular mechanism of an origin of replication (ARS)and a centromere (CEN) replication and segregation will also require the iden- are transmittedproperly in 99% of cell divisions tification and characterization of the trans acting fac- (CLARKEand CARBON, 1980; FITZGERALD-HAYESet torsthat comprise the replication and segregation al. 1982). However, their rate of loss per cell division machinery. In S. cerevisiae, temperature-sensitive mu- is still several orders of magnitude greater than en- tants have been identified which arrest at different dogenous (ESPOSITOet al. 1982;HARTWELL et al. stages of the cell cycle when they are shifted to the 1982) or large artificial linear chromosomes (MUR- nonpermissive temperature (PRINGLE and HARTWELL RAY, SCHULTES and SZOSTAK 1986; HIETER et al. 1981). A subset of these cell division cycle (cdc) mu- 1985) suggesting that minichromosomes lack a struc- tants identify genes whose products are required for tural feature(s) of endogenous chromosomes (nucleo- the execution of discrete steps in GI (CDC28, CDCI, ' To whom reprint requests should be addressed at the CarnegieInstitu- CDC7), S (CDC2,CDC6, CDC9, CDC17), G2-M tion. (CDCI3, CDCI6, CDC20, CDC23) and late M phase Genetics 125: 763-774 (August, 1990) 764 R. E. Palmer, E. Hogan and D. Koshland (CDC14, CDCl5)of the cell cycle(PRINGLE and HART- effect of cdc mutations on endogenous chromosome WELL 1981). Given their cell cycle phenotype, these transmission (HARTWELLand SMITH1985). CDC genes are likely candidates for encoding trans factors required for proper chromosometransmission. MATERIALS AND METHODS In fact, when the cdc mutants were incubated at a semi-permissive temperature, conditions that partially Strains: All of the cdc mutations used in this study were inactivate the CDC gene product, many of these mu- obtained by back crossing at least five times the original cdc mutations isolated in A364A to strains congenic with the tants lose endogenous chromosome at elevated rates original A364A parent. All other markers were obtained by demonstrating that these CDC products are involved back crossing the mutation from anotherSaccharomyces cere- in ensuring proper chromosome transmission (HART- uisiae strain into theA364A congenic background by at least WELL and SMITH1985). ten back crosses. The haploid strains usedin this study The molecular functions of some of the CDC gene (Table 1) were constructed by standard genetic methods (SHERMAN,FINK and HICKS 1986) and then transformed products have been identified by cytological and bio- (ITO et al. 1983)with the minichromosome, pDK243 (KOSH- chemical analysis of arrested cells, while the activity LAND, KENT and HARTWELL1985). The homozygous cdc of other CDC products have been inferred from amino diploid strains containing the chromosome fragment (Table acid similarities with other better characterized pro- 1) were constructed as follows. First, the chromosome frag- teins. The CDC28 and CDC7 genes encode protein ment,CF352 (Figure l), was constructed in the haploid strain, 45 13-12 1, by the chromosome fragmentation pro- kinases (LORINCZand REED 1984; PATTERSONet al. tocol (GERRING,CONNELLY and HIETER1989; VOLLRATHet 1986); the former shows extensive similarity to the al. 1988). This strain was then crossed with a set of congenic kinase subunit of maturation promoting factor, an haploid cdc strains (Table l), anda set of congenic haploid essential regulator of the G2 to M transition in many cdc strains were obtained that contained the chromosome organisms (ARIONet al. 1988; BEACH,DURKACZ and fragment. These strains were then mated with the haploid cdc strains listed in Table 1 to generate homozygous cdc NURSE1982; GAUTIERet al. 1988). The CDC4 prod- diploids with 1 copy of the chromosome fragment. uct shares homology tothe @-adrenergicreceptor Growth conditions of exponentially growing cells and (YOCHEMand BYERS1987). CDC2 and CDCl7 encode temporarily arrested cells: The cdc mutant strains (Table DNA polymerases (SITNEY,BUDD and CAMPBELL 1) were grown at 23"to saturation in YPD (SHERMAN, FINK and HICKS1986) supplemented with adenine. These cul- 1989; BOULETet al. 1989; CARSON1987), while CDC9 tures were used to inoculate YPD. When cells reached mid- encodes DNA ligase (BARKERand JOHNSTON1983). log phase (5 X lo6 cells/ml), an aliquot was diluted with However, the function of many of the CDC gene water approximately 1000-fold and spread onto YPD plates. products remains to be elucidated. The remaining cells were temporarily arrested by shifting them tothe nonpermissive condition for 3 hr. The cdc In this paper, we examined the effect of temporarily mutants were temporarily arrested by incubating them at reducing the functionof different CDC gene products 36" for 3 hr. Wild-type cells were temporarily arrested by on the mitotic transmission of two artificial chromo- incubating them in the presence of nocodazole or hydrox- somes, a 14-kb circular minichromosome and a 155- yurea (a final concentration of 0.1 mM and 0.1 M, respec- kb linear chromosome fragment. Both an increase or tively) for3 hr at 23" and 36". The time spentat the nonpermissive condition was sufficient to inactivate most of decrease in the ploidy of these test chromosomes could the CDC gene product in the majority of cells as evidenced be detected in individual cell divisions through the by the fact that greater than 90% of the cells were arrested use of a visual assay for chromosome ploidy (KOSH- at the appropriate stage of the cell cycle. After 3 hr at the LAND, KENT and HARTWELL1985; KOSHLAND and nonpermissive condition, arrested cells were diluted with water and plated on YPD plates at 23" where they reentered HIETER1987). In this study, we identified mutations the cell cycleand formeda colony. The dilution and plating in cdc genes that reduce the fidelity of minichromo- were done rapidly to ensure thatarrested cells did not some transmission as evidenced by an increase in the complete division before they were plated. rate of aberrant celldivisions where ploidy of the Half-sector analysis of minichromosome and chromo- some fragment ploidy: The ploidy of the minichromosome