We describe a general method for analyzing the genetic fine structure of plasmid-borne genes in yeast. Previously we had reported that a linearized plasmid is efficiently rescued by recombination with a homologous restriction fragment when these are co-introduced by DNA-mediated transformation of yeast. Here, we show that a mutation can be localized to a small DNA interval when members of a deletion series of wild-type restriction fragments are used in the rescue of a linearized mutant plasmid. The resolution of this method is to at least 30 base pairs and is limited by the loss of a wild-type marker with proximity to a free DNA end. As a means for establishing the nonidentity of two mutations, we determined the resolution of two-point crosses with a mutant linearized plasmid and a mutant homologous restriction fragment. Recombination between mutations separated by as little as 100 base pairs was detected. Moreover, the results indicate that exchange within a marked interval results primarily from one of two single crossovers that repair the linearized plasmid. These approaches to mapping the genetic fine structure of plasmids should join existing methods in a robust approach to the mutational analysis of gene structure in yeast.

We describe a convenient method for constructing new plasmids that relies on interchanging parts of plasmids by homologous recombination in Saccharomyces cerevisiae. A circular recombinant plasmid of a desired structure is regenerated after transformation of yeast with a linearized plasmid and a DNA restriction fragment containing appropriate homology to serve as a substrate for recombinational repair. The free ends of the input DNA molecules need not be homologous in order for efficient recombination between internal homologous regions to occur. The method is particularly useful for incorporating into or removing from plasmids selectable markers, centromere or replication elements, or particular alleles of a gene of interest. Plasmids constructed in yeast can subsequently be recovered in an Escherichia coli host. Using this method, we have constructed an extended series of new yeast centromere, episomal and replicating (YCp, YEp, and YRp) plasmids containing, in various combinations, the selectable yeast markers LEU2, HIS3, LYS2, URA3 and TRP1.

The molecular products of DNA double strand break repair were investigated after transformation of yeast (Saccharomyces cerevisiae) with linearized plasmid DNA. DNA of an autonomous yeast plasmid cleaved to generate free ends lacking homology with the yeast genome, when used in transformation along with sonicated non-homologous carrier DNA, gave rise to transformants with high frequency. Most of these transformants were found to harbor a head-to-head (inverted) dimer of the linearized plasmid. This outcome of transformation contrasts with that observed when the carrier DNA is not present. Transformants occur at a much reduced frequency and harbor either the parent plasmid or a plasmid with deletion at the site of the cleavage. When the linearized plasmid is introduced along with sonicated carrier DNA and a homologous DNA restriction fragment that spans the site of plasmid cleavage, homologous recombination restores the plasmid to its original circular form. Inverted dimer plasmids are not detected. This relationship between homologous recombination and a novel DNA transaction that yields rearrangement could be important to the cell, as the latter could lead to a loss of gene function and lethality.

The formation of an inverted dimer plasmid on transformation with linear molecules is formally analogous to the fusion of the daughters of a broken chromosome at their broken ends. In the latter case, this leads to the formation of a dicentric chromosome, which could break at anaphase. Hence the process is cyclic. Similarly, when our linear molecules are modified by the addition of a cloned yeast centromere, dicentric inverted dimers are not obtained. Instead, we obtain monocentric plasmids with partial duplication and deletion that apparently derive from a process of fusion, bridge-breakage, and fusion. This is not surprising, since it is known that dicentric plasmids undergo breakage in yeast (Mann and Davis 1983). However, any apparent similarity of this process to that which occurs with a broken chromosome in maize must be tempered by the special nature of the transformation process. Most significantly, inverted dimers are rare when sonicated carrier DNA is not present during the transformation. This requirement is not understood, but it is a condition that may not be met in a yeast cell harboring a broken chromosome. It is possible that carrier DNA induces a repair process that results in fusion. On the other hand, a property of the transformation process that results in an inhibition of fusion may be overcome by the presence of carrier DNA. Most inverted dimers are apparently formed from an interaction between two input linear molecules. We cannot rule out the possibility that a minor fraction derive from a single molecule. Thus, the fusion of two input molecules is a much more efficient process than a replicative process that could occur with single linear molecule. For a similar fusion process to occur with a broken yeast chromosome, replication would be required. We do not know if a broken yeast chromosome can replicate. Evidence consistent with the presence of a breakage-fusion-bridge process in yeast has been obtained through the formation of dicentric chromosomes via meiotic recombination (Haber et al. 1984). Spores from these meioses sometimes give rise to a clone that is mixed for markers of the chromosome that could have been dicentric. A process of fusion-bridge-breakage could account for the formation of some of these mixed clones. However, the dicentric chromosomes apparently often survive meiotic disjunction and break in the spore's first mitotic anaphase or possibly in a later generation. Thus, the interpretation of the origin of these mixed clones is uncertain. Some aspects of the fusion process are especially intriguing.(ABSTRACT TRUNCATED AT 400 WORDS)

Chi recombinational hotspots are sites around which the rate of Rec-promoted recombination in bacteriophage lambda is elevated. Examination of a derivative of lambda into which the plasmid pBR322 was inserted reveals that pBR322 lacks Chi sites. Using this lambda-pBR322 hybrid, we obtained mutations creating Chi sites at three widely separated loci within pBR322. Nucleotide sequence analysis reveals that the mutations are single base-pair changes creating the octamer 5' GCTGGTGG 3'. This sequence is present at three previously analyzed Chi sites in lambda, and all analyzed mutations creating or inactivating these Chi sites occur within this octamer. We conclude that Chi is 5' GCTGGTGG 3', or its complement, or both.