The nucleotide sequence of two Zygosaccharomyces plasmids, pSB2 (5,415 base pairs), isolated from Zygosaccharomyces bailii, and pSMl (5,416 base pairs), isolated from Zygosaccharomycesfermentati Naganishi, was determined. The predicted amino acid sequences of open reading frames among six yeast plasmids that resemble 2,um DNA indicated regional sequence similarities among FLP proteins. Greater similarities were seen among Zygosaccharomyces plasmids (pSB2, pSB3, pSRl, and pSM1) than other combinations. A putative recognition site for the FLP enzyme of a Zygosaccharomyces plasmid also showed some conservation, especially in the 4 nucleotides flanking the central spacer region. From comparative studies of the sequences of putative genes of each plasmid, we propose an apparent phylogenetic relationship among yeast plasmids resembling 2,Lm DNA. Among the Zygosaccharomyces plasmids, pSB2 and pSRl are most closely related, since not only were the FLP enzymes of these two plasmids most closely related, but also the amino acid sequence of the putative P gene of pSRl showed clear homology with that of open reading frame B of pSB2.Since the discovery of 2,um DNA in Saccharomyces species (21; see reference 6 for a review), several yeast plasmids resembling the 2Rm plasmid were isolated and characterized. We call these plasmids 2,um DNA-like plasmids. For unknown reasons, 2ptm DNA-like plasmids were found frequently in Zygosaccharomyces species. We have found five plasmids in Zygosaccharomyces yeasts: pSB1 and pSB2 from Z. bailii (23), pSR1 and pSB3 from Z. rouxii (23,24), and pSM1 from Z. fermentati (unpublished data). Recently, another 2,um DNA-like plasmid, pKD1, was discovered in Kluyveromyces drosophilarum (8). Of these, the nucleotide sequences of 2,um DNA (13), pSR1 (2), pSB3 (25), and pKD1 (8) have been published. These plasmids, along with 2,um DNA from Saccharomyces cerevisiae, have several structural features in common: (i) they are closed circular DNA plasmids whose size is ca. 6 kilobases (kb), (ii) they have a pair of inverted repeats (IRs) consisting of several hundred bases, (iii) they are cryptic and exist in high copy number, and (iv) intramolecular recombination results in two isomeric forms. In spite of resemblances in their global structure, homology at the nucleotide sequence level is poor (23).Plasmid-encoded functions have been most extensively studied in 2,um DNA. The prominent features of genes on 2,um DNA are briefly summarized as follows. It has one replication origin, which is activated once in the cell cycle by the same replication mechanism as that for replication of chromosomes (16,28). It has four open reading frames (ORFs) or genes, A, B, C, and D. The A gene (or FLP gene) encodes a recombination enzyme which functions as a site-specific recombinase (7). The B and C genes produce trans-acting factors that are needed for the stable maintenance of this plasmid. These two genes, along with the cis-acting locus STB, participate in partitioning of plasmid molecules during cell division (14,15 Our...

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Genomic DNAs isolated from 420 yeast strains stocked in the Department of Fermentation Technology, Hiroshima University (HUT) were screened for the presence of a plasmid sequence both as plasmid or in the chromosome. Five DNA samples gave rise to a positive hybridization signal when 32P-labelled Zygosaccharomyces plasmid pSR1 was used as a probe. Two among these contain hybridizing sequences as plasmids while the other three apparently were chromosomal. Two chromosomal DNA segments of HUT 7195 (Zygosaccharomyces spp.) which hybridized with pSR1 probe were cloned and sequenced. Both DNAs hybridized with a plasmid sequence covering the P gene of pSR1. One of the two segments contains a large open reading frame which can encode 410 amino acid residues. The deduced amino acid sequence is closely related with that of the P gene of pSR1. The present finding suggests that there was an interchange(s) of a gene between yeast plasmid(s) and chromosomes.

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Weevaluated the expression of three yeast genes in Zygosaccharomyces rouxii using lacZ as a reporter gene; they were the gIyceraldehyde-3-phosphate dehydrogenase gene of Z. rouxii (GA P-Zr), that of S. cerevisiae (GAP-Sc) and the PHO5gene of S. cerevisiae. The GAP-Scpromoter showed high expression in the Z. rouxii host as well as in the S. cerevisiae host. The GAP-Zrpromoter showed a comparable level of expression as the GAP-Scboth in the S. cerevisiae host and the Z. rouxii host. The transcription initiation site of the GAP-Zr promoter in the heterologous host seems to be the same as that in the homologous host. The PHO5promoter showed constitutive expression, as efficient as in the case of the GAP-Zrpromoter, in the Z. rouxii host. AHthe promoters mentioned above can be used as sources of a high expression vector in the Z. rouxii host.