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We have developed an effective method to delete or invert a chromosomal segment and to create reciprocal recombination between two nonhomologous chromosomes in Saccharomyces cerevisiae, using the site-specific recombination system of pSR1, a circular cryptic DNA plasmid resembling 2,um DNA of S. cerevisiae but originating from another yeast, Zygosaccharomyces rouxii. A 2.1-kilobase-pair DNA fragment bearing the specific recombination site on the inverted repeats of pSR1 was inserted at target sites on a single or two different chromosomes of S. cerevisiae by using integrative vectors. The cells were then transformed with a plasmid bearing the R gene of pSR1, which encodes the site-specific recombination enzyme and is placed downstream of the GAL] promoter. When the transformants were cultivated in galactose medium, the recombination enzyme produced by expression of the R gene created the modified chromosome(s) by recombination between two specific recombination sites inserted on the chromosome(s).Although recombinant DNA technology is effective for creating novel strains in various organisms, the DNA that can be manipulated in a vector is limited to a few genes or a DNA fragment less than 100 kilobase pairs (kb) long, and the vector is, in general, unstable. To overcome these difficulties, the artificial chromosome vector YAC, which is able to clone an exogenous DNA fragment of several hundred kilobase pairs and is maintained stably, has been developed in Saccharomyces cerevisiae (6). Another possible procedure for integrating large exogenous DNA efficiently, and also to delete, invert, and translocate a chromosomal segment at a predetermined location on a chromosome by using the cre-lox site-specific recombination system of bacteriophage P1, has been suggested by Sauer (27). We have succeeded in effecting such chromosomal modifications in S. cerevisiae by using a site-specific recombination system of plasmid pSR1, which was isolated from a strain of Zygosaccharomyces rouxii.Plasmid pSR1 (1, 35) is a 6,251-base-pair (bp) circular DNA molecule having an architecture similar to that of 2,um DNA (3), the plasmid widely distributed in S. cerevisiae strains. The DNA sequence of the pSRl molecule has no similarities with that of 2,um DNA (1) or genomic DNA of S. cerevisiae (our unpublished results). The pSR1 molecule has a pair of inverted repeats, each composed of a 959-bp sequence, which divide the plasmid molecule into two unique regions of 2,654 and 1,679 bp (Fig. la). pSRl can replicate in S. cerevisiae as well as in its native host and exists in two isomeric forms generated by intramolecular recombination at the inverted repeats. The intramolecular recombination is initiated at a restricted region, at most a 58-bp sequence, in the inverted repeats and is catalyzed by the R protein encoded by the plasmid molecule (19).The site-specific recombination system of pSRl has distinct advantages in modification of S. cerevisiae chromo-* Corresponding author. somes because it operates efficiently in S. cerevisiae and is i...

The nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. An open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a GUG codon as methionine. It was shown by NH2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. Thus the NH2-terminal portion of the gene encodes 34 amino acid residues as a signal peptide. The amino acid sequence deduced from the alpha-amylase gene was fairly homologous (61%) with that of another thermostable amylase from Bacillus amyloliquefaciens.

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The PHO80/TUP7 locus in Saccharomyces cerevisiae is reported to be located on the right arm of chromosome XV close to its centromere. In the present study, the locus has been reassigned to the left arm of the same chromosome by reciprocal recombination between chromosomes V and XV at URA3 (on chromosome V) and PHO80/TUP7 loci by using the site-specific recombination system of the yeast plasmid pSR1.