Recombination of plasmids into the Saccharomyces cerevisiae chromosome is reduced by small amounts of sequence heterogeneity.

Abstract

As a model system for studying the properties of mitotic recombination in the yeast Saccharomyces cerevisiae, we have examined recombination between a recombinant plasmid (introduced into the S. cerevisiae cell by transformation) and homologous chromosomal loci. The recombinant plasmids used in these experiments contained S. cerevisiae rRNA genes. We found that the frequency of integrative recombination is sensitive to small amounts of sequence heterogeneity. In addition, the frequency and specificity of these recombination events are affected by the lengths of the interacting homologous DNA sequences.Repeated genes are found in all eucaryotes. Although the physical properties of many types of repeated genes have been studied in detail, much less information is available concerning genetic interactions among repeated sequences. Below, we describe a model system that we have used to examine mitotic recombination of the repeated rRNA genes of Saccharomyces cerevisiae.The S. cerevisiae rRNA genes are repeated approximately 100 times per haploid genome (19) and are located in a single tandem array on chromosome XII (13). Each rRNA gene contains about 9 kilobases (kb) of DNA and encodes four species of rRNA; the 25S, 18S, 5.8S, and 5S species (2,17). The 25S, 5.8S, and 18S rRNAs are transcribed as a 35S precursor RNA which is later processed (21). The 5S rRNA is encoded on the opposite DNA strand and is separated from the 35S rRNA species by nontranscribed spacer sequences (9,22). The positions of the rRNA transcripts within the rRNA gene are shown in Fig. 1.Most laboratory strains of S. cerevisiae have rRNA genes with seven EcoRI sites per repeat (2,3,16,17). This type of rDNA is called "form I" and the seven fragments produced by EcoRI treatment of this DNA are labeled A through G in order of decreasing size. An S. cerevisiae strain containing a variant type of rDNA ("form II") has also been previously described (16). is formed which is equal in size to the sum of the EcoRI fragments B and E. In a recent study (M. McMahon, S. Smolik-Utlaut, and T. Petes, unpublished data), a number of other differences have been detected. Most of these changes occurred in the nontranscribed spacer and involved small deletions and insertions (Fig. 1). DNA sequencing of a 424-base-pair TaqI fragment surrounding the 5S rRNA gene showed only seven differences between form I and form II: three point mutations and four single base insertions. In heteroduplexes formed between form I and form II genes, no insertion or deletion loops were detectable by electron microscopy (S. Smolik-Utlaut, Ph.D. thesis, University of Chicago, Illinois, 1982).As described below, we examined the effects of the form I-form II heterogeneity on recombination in S. cerevisiae. Hinnen et al. (7) showed that when recombinant plasmids containing S. cerevisiae DNA were transformed into S. cerevisiae cells, these plasmids recombined with homologous chromosomal sequences. To test the effects of the form I-form II heterogeneity on this type of recombination, we trans...

Influence of homology size and polymorphism on plasmid integration in the yeast CYC1 DNA region

“…These results show that reciprocal recombination between a plasmid molecule and a yeast chromosome is aected by very low sequence divergence. A similar conclusion was reached by Smolik-Utlaut and Petes (1983) who demonstrated that circular plasmids containing yeast rDNA genes preferentially integrate into identical sequences in a yeast diploid strain heterozygous for the rDNA region present on chromosome XII. In the present study, using plasmids with a known number and distribution of point mutations, we found that the inhibitory eect of these mutations is not cumulative since comparable values were observed with one, two and six mutations.…”

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

“…Integration occurs at a single breakpoint and produces a duplication of the homologous sequences (9,17). Further studies have shown that the recombination frequency between donor DNA and the genome is a function of the number of bp of homology between the plasmid and genomic sequences (24).…”

A mobile group I intron from Physarum polycephalum can insert itself and induce point mutations in the nuclear ribosomal DNA of saccharomyces cerevisiae.

“…In our experiments, fewer than 60 bp surrounding the intron insertion site were identical in yeast rDNA on one hand and in the donor plasmid that carried Pp LSU3 and surrounding Physarum rDNA on the other. Since the frequency of homologous recombination in rDNA and in other genes is proportional to the extent of sequence homology (1,50) and since 60 bp of homologous DNA is near the lower size limit for recombination in yeast cells (52), the frequency of intron homing could probably be increased by using a plasmid engineered to contain much larger regions of identical flanking sequences. (42,54).…”