Do yeast require an origin of replication and if so, what is the yeast origin of replication in BG1805?

Yes, yeast plasmids do require a sequence that promotes replication. In BG1805, this function is supplied by sequences from the yeast 2-micron plasmid. The 2-micron plasmid is an endogenous plasmid that is stably maintained in many yeast strains at high copy number (>60 copies per cell). Replication of 2-micron DNA and stability of the plasmid requires 2 cis-acting sequences (neither of which is very large), and several trans-acting factors (Rep1 and Rep2 proteins and others). See summary of REP1 found at SGD below. Sequences from endogenous 2-micron have been incorporated into yeast plasmids used for cloning. Based on available literature in this area, the 2-micron sequences have both a replication origin and a partitioning element (~62 base pairs). Thus 2-micron based yeast plasmids are stable and high copy number unlike ARS based plasmids (ARS = chromosomal origins of replication). It is likely that replication of these 2-micron based plasmids depends upon trans-factors supplied by the endogenous copy. BG1805 plasmid is supposed to contain sequences homologous to the yeast 2-micron plasmid. Furthermore, it had been found that around base pair 6700 to ~ 8,000 contains a region that is an exact match to the 2-micron DNA. (Sequencing was performed on BG1766, the one step precursor to BG1805). Summary paragraph for the REP1 protein in found on the SGD website (http://www.yeastgenome.org/): The 2-micron plasmid is a relatively small (6318 bp) nuclear multicopy extrachromosomal element found in most common strains of Saccharomyces cerevisiae ( 4). Its presence confers no obvious advantage to its host, nor does it appear to impose any disadvantage at its steady-state copy number of 40-60 molecules ( 5). The plasmid contains 4 protein-coding loci ( FLP1, REP1, REP2, RAF1) and 4 cis-acting loci (an origin of replication, a partitioning locus called STB, and 2 Flp Recombination Targets or FRTs) ( 6). The 2-micron plasmid propagates itself with chromosome-like stability through the combined action of a plasmid amplification system and a plasmid partitioning system. The amplification system compensates for any copy number decreases caused by missegregation events, and consists of Flp1p along with a pair of 599-bp FRT sites present in the plasmid genome in a head-to-head orientation ( 7). The partitioning system ensures roughly equal distribution of replicated plasmids to daughter cells by overcoming the normal segregation bias that favors the mother cell over daughters in plasmid retention ( 8, 7). This system comprises Rep1p, Rep2p, and the partitioning locus STB which consists of two subloci, one containing approximately six iterations of a consensus 65-bp repeat, and another which is involved in maintaining the active configuration of STB and contains the termination site for two transcripts directed toward the plasmid replication origin ( 7). Multiple copies of the 2-micron plasmid exist as a tight-knit cluster within the nucleus that stays together throughout the cell cycle ( 9), and this plasmid cluster is the segregation entity, effectively reducing copy number to one ( 9, 10). For more information, go to the SGD site (http://www.yeastgenome.org/). Type REP1 into the blank box at the top of the SGD website to get to the REP1 gene page. The 2mm-Plasmid-Encoded Rep1 and Rep2 Proteins Interact with Each Other and Colocalize to the Saccharomyces cerevisiae Nucleus, JOURNAL OF BACTERIOLOGY, 0021-9193/97/$04.0010 Dec. 1997, p. 7497–7506. Ahn et. al.

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