Meiotic Recombination on Artificial Chromosomes in Yeast Lyle

We have examined the meiotic recombination characteristics of artificial chromosomes in Saccharomyces cereuisiae. Our experiments were carried out using minichromosome derivatives of yeast chromosome ZZZ and yeast artificial chromosomes composed primarily of bacteriophage X DNA. Tetrad analysis revealed that the artificial chromosomes exhibit very low levels of meiotic recombination. However, when a 12.5-kbp fragment from yeast chromosome VZZZ was inserted into the right arm of the artificial chromosome, recombination within that arm mimicked the recombination characteristics of the fragment in its natural context including the ability of crossovers to ensure meiotic disjunction. Both crossing over and gene conversion (within the ARC4 gene contained within the fragment) were measured in the experiments. Similarly, a 55-kbp region from chromosome ZZZ carried on a minichromosome showed crossover behavior indistinguishable from that seen when it is carried on chromosome ZZZ. We discuss the notion that, in yeast, meiotic recombination behavior is determined locally by small chromosomal regions that function free of the influence of the chromosome as a whole. S INCE they were originally described in 1983, linear artificial chromosomes have proven to be useful tools for studying telomere structure and function, and mitotic chromosome segregation (DANI and ZAKIAN 1983; MURRAY and SZOSTAK 1983; PLUTA et al. 1984; HIETER et al. 1985; KOSHLAND, KENT and HARTWELL 1985; MURRAY, SCHULTE~ and SZOSTAK 1986; SUROSKY, NEWLON and TYE 1986; MURRAY, CLAUS and SZOSTAK 1988). These studies have in part been successful because artificial chromosomes can be made to mimic the mitotic behavior of natural chromosomes. We have used artificial chromosomes to investigate meiotic chromosome segregation (DAWSON, MURRAY and SZOSTAK 1986). In some respects our studies have been successful for the opposite reason, ie., the artificial chromosomes do not mimic the meiotic behavior of natural chromosomes in every way. Natural chromosomes participate in meiotic crossing over with great consistency, and this crossing over is necessary to insure the disjunction of homologs at meiosis I. In the course of our experiments, we found that artificial chromosomes crossover at unusually low frequencies in meiosis, a characteristic which allowed us to investigate the fate of noncrossover homolog pairs in meiosis. Additionally, we were led to question the nature of this deficiency in recombination. Here we describe the meiotic recombination properties of artificial chromosomes, and of a minichromosome derivative of a natural yeast chromosome. Genetics 131: 541-550 uuly, 1992) Studies in a variety of organisms have demonstrated that on a chromosomal scale, both the number and distribution of crossovers are regulated (JONES 1987; CARPENTER 1988; HAWLEY 1988). Recently, finescale mapping of crossover events in yeast has shown that frequencies can vary dramatically over distances as small as one thousand base pairs (LARKIN and WOOLFORD 1984; COLEMAN et al. 1986; SYMINGTON and PETES 1988; SYMINCTON et al. 1991). Our results suggest that, in yeast, the recombination behavior of the sequences in a particular small interval is largely determined by the sequences in that interval. Effects from the sequences in adjacent intervals were shown to be minimal in our experiments. MATERIALS AND METHODS Strains, media and genetic methods: Media were prepared as described in SHERMAN, FINK and LAWRENCE (1979). Saccharomyces cereuisiae strains are described in Table 1. DD35, DDll7, DD97 and DD114 were derived from the same parental strain and are isogenic except for differences evident in the genotypes presented in Table 1. DL98, DL1 12 and DL72 are isogenic strains derived from the strains used by NICOLAS et al. (1 989). Chromosome VZZZ of DL98 and DL1 12 has a 2060-bp HpaI-HpaI deletion which removes the entire ARC4 coding region. This deletion was introduced to eliminate ectopic interactions between the ARC4 alleles on chromosome VZZZ and the artificial chromosomes. Recombination data from all yeast linear plasmids (YLps; these constructs may also be referred to as artificial chromosomes), 72-kbp derivatives of chromosome ZZZ (mini I l l s ) and chromosome constructs were obtained by tetrad analy542 L. 0. Ross et al .