The TRP1 RI circle (TRP1, ARS1, YARp1) is a plasmid which was constructed by self-ligation of a 1.45 kb EcoRI fragment from chromosome number IV of the yeast Saccharomyces cerevisiae. It contains a sequence called ARS (for Autonomously Replicating Sequence) and the TRP1 gene which codes for the enzyme N-(5'-phosphoribosyl)-anthranilate isomerase required in the third step of the biochemical synthesis of tryptophan. The ARS1 sequence confers to this plasmid the ability to be replicated independently of the chromosomal DNA. The TRP1 RI circle exists in a high copy number (100-200 molecules/cell) as compared to the endogenous 2-micron plasmid in yeast (30-60 molecules/cell). It has been suggested that the high copy number of the TRP1 RI circle mat be attributed to the fact that the TRP1 gene incorporated into the circle, doesn't include its natural strong promoter but a weak minor one. To test the validity of this hypothesis two different versions of the plasmid were constructed: one including the full promoter and the other with both promoters. Yeast cells were transformed with both constructs and levels of expression of the two versions of the gene as well as copy number of the two constructs were studied. Results of this analyis indicated that levels of expression are higher for the new version of the gene. Southern blot analysis showed that the copy number of the construct including the full promter is reduced to about half as compared with the partial promoter version. This data indicates that the increase in transcription levels of the full TRP1 gene is a direct consequence of the presence of its complete promoter and this effect is acompanied by a three-fold reduction in copy number. The proximity of the TRP1 gene to the ARS1 region in these constructs is having an effect on both replication and transcription of the plasmids.
The life cycle of Saccharomyces cerevisiae is very interesting and its cell cycle is similar to that of all eukaryotic cells. The DNA replication process of this cell, although not completely unraveled, is known in some detail.
About the method used by yeast to regulate gene expression, the most well known is that mechanism which regulates expresion of the galactose genes cluster.
The maps of each of the constructs used in the project presented here are available. You can see either the TRP1 RI circle or pNEW which is the FullTRP1 Circle.
Two models are proposed in this project to explain the outcome of the experimental analyses presented here: model 1 and model 2. Any suggestions or comment? Please, e-mail me. Thanks!