Properties of a Saccharomyces cerevisiae mtDNA segment conferring high-frequency yeast transformation.

Abstract

The bakers' yeast Saccharomyces cerevsiae is a facultative anaerobe, tolerant to mutations in its mitochondrial genome. Individual cytoplasmic petite mutants retain genetic information derived from any portion of the parental mtDNA, prompting questions concerning distribution of the DNA replication origin(s) on the yeast mitochondrial genome. The experiments described in this paper were designed to test the possibility of using high-frequency yeast transformation as a selection for yeast mtDNA sequences conferring autonomously replicating function. A complete petite mitochondrial genome was inserted into the yeast vector YIp5, and the hybrid plasmid (YRMpl) was used to transform yeast. YRMpl promoted high-frequency transformation ofboth wild-type yeast cells and petite mutant hosts lacking mtDNA and was maintained in each of these strains as a highcopy-number extrachromosomal element. The stability and copynumber properties of YRMpl are similar to those of YRp12, a recombinant plasmid containing a yeast chromosomal autonomously replicating sequence. sequence (ars) are able to transform yeast at high frequency, a readily selectable property (10). This rationale has been used to isolate putative DNA replication origins from yeast chromosomes (11,12) and from a wide variety of heterologous eukaryotic genomes (13).The experiments described here were undertaken to isolate and map yeast mtDNA sequences that are ars elements as assayed by high-frequency yeast transformation. Since many individual petite mtDNAs exist in vivo as amplified tandemly repeated segments (2), it was anticipated that the repetitive nature of these mitochondrial genomes might provide an enriched source ofDNA replication origins. We have shown that a cloned intact petite genome is capable of conferring ars characteristics to a nonreplicating vector and that ars properties of this cloned segment, such as mitotic stability and copy number, are similar to those of the chromosomal DNA sequence TRPl-arsl (7,8,10 Ag of DNA) as a result of stable integration into host cell chromosomal DNA. A few yeast sequences transform at high frequency (500-20,000 transformants per ,g of DNA) and are maintained within the cell as extrachromosomal elements. These properties result from the presence on the transforming plasmid of a DNA sequence conferring autonomously replicating functions (7-9). Hybrid molecules that combine a low-frequency transforming plasmid with an autonomously replicating MATERIALS AND METHODS Strains and Culture Conditions. The p-petite mutant A17-10 was isolated after ethidium bromide mutagenesis of S. cerevisiae strain A10 (a his ade p+). JHC8-24C (a ura3-52 his3-11 his3-15 leu2-3 leu2-112 inol -13 ino4-8 p+) and an ethidium bromide-induced p0 derivative served as hosts in yeast transformation experiments. Bacterial cloning was carried out in a thymine-requiring derivative ofEscherichia coli HB101 (14). Yeast culture media were used as described (15) Rapid Yeast DNA Preparation. High-molecular-weight total cellular DNA was prepared fr...

Upstream region of the SUC2 gene confers regulated expression to a heterologous gene in Saccharomyces cerevisiae.

“…Nonetheless, a much more dramatic, 300-fold derepression occurred in cells carrying pLS7. The 10-fold-higher derepressed activity observed in strains carrying the multicopy episome pLS7 as compared with strains carrying a single integrated copy of pLS11 is consistent with the difference in copy number (16).…”

Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element.

“…They transformed at high frequencies and were unstable, with the notable exception of pFL20 (see below). Their copy number varied between 15 and 80 as compared with 10 to 200 for different ars plasmids in S. cerevisiae (19,39). In both yeasts only a fraction of cells grown under selective conditions contain an ars plasmid (19,34,39).…”

Surrogate origins of replication in the mitochondrial genomes of ori-zero petite mutants of yeast.

“…Another general conclusion is that the present work provides precise examples of how physiological replication origins can be replaced by sequences which, though much less efficient, are still capable of insuring replication. It is very likely that sequences of the oril type also account for the replication as an extrachromosomal element in yeast of a hybrid plasmid containing a petite repeat unit devoid of any ori sequences (Hyman et al, 1982), but derived from a region rich in sequences of the orn family (de Zamaroczy et al, in preparation). As for the role of oril sequences as surrogate origins of replication, it should be stressed (Bernardi, 1982a) that the expansion (by replication slippage and unequal crossing-overs) of the duplicated and translocated ori sequences to form the intergenic sequences results in partial copies of ori sequence segments, still capable of interacting with the replication complex, being spread all over the genome.…”