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Guy's Research: Gluten Digestion Enzyme Plasmid


I am currently viewing Yeast integrative plasmid,(YIp), whhich are yeast vectors that utilize integration into the yeast chromosome for survival and are used when studying a DNA gene.

Vectors derived from the 2mm plasmid are called yeast episomal plasmids (YEps). Some YEps contain the entire 2mm plasmid, others include just the 2mm origin of replication. An example of the latter type is YEp13 (Figure 7.3). YEp13 illustrates several general features of yeast cloning vectors. First, it is a shuttle vector. As well as the 2mm origin of replication and the selectable LEU2 gene, YEp13 also includes the entire pBR322 sequence, and can therefore replicate and be selected for in both yeast and E. coli. There are several lines of reasoning behind the use of shuttle vectors. One is that it may be difficult to recover the recombinant DNA molecule from a transformed yeast colony. This is not such a problem with YEps, which are present in yeast cells primarily as plasmids, but with other yeast vectors, which may integrate into one of the yeast chromosomes (p. 135), purification may be impossible.This is a disadvantage because in many cloning experiments purification of recombinant DNA is essential in order for the correct construct to be identified by, for example, DNA sequencing.

The standard procedure when cloning in yeast is therefore to perform the initial cloning experiment with E. coli, and to select recombinants in this organism. Recombinant plasmids can then be purified, characterized, and the correct molecule introduced into yeast (Figure 7.4). (1)

(1) Yeast integrative plasmids (YIps) are basically bacterial plasmids carrying a yeast gene. An example is YIp5, which is pBR322 with an inserted URA3 gene (Figure 7.6(a)). This gene codes for orotidine-5¢-phosphate decarboxylase (an enzyme that catalyses one of the steps in the biosynthesis pathway for pyrimidine nucleotides) and is used as a selectable marker in exactly the same way as LEU2. A YIp cannot replicate as a plasmid as it does not contain any parts of the 2mm plasmid, and instead depends for its survival on integration into yeast chromosomal DNA. Integration occurs just as described for a YEp (Figure 7.5).

(2) Yeast replicative plasmids (YRps) are able to multiply as independent plasmids because they carry a chromosomal DNA sequence that includes an origin of replication. Replication origins are known to be located very close to several yeast genes, including one or two which can be used as selectable markers. YRp7 (Figure 7.6(b)) is an example of a replicative plasmid. It is made up of pBR322 plus the yeast gene TRP1. This gene, which is involved in tryptophan biosynthesis, is located adjacent to a chromosomal origin of replication.The yeast DNA fragment present in YRp7 contains both TRP1 and the origin. (1)

1. "Chapter 7 Cloning Vectors." Gene Cloning. N.p.: n.p., n.d. 135-37. Http:// 22 Aug. 2005. Web.