Yeast cloning vectors - Discover Biotech

In this article, I briefly describe yeast cloning vectors.

Table of Contents

Cloning vectors

A small piece of DNA into which a foreign DNA fragment is inserted for cloning purposes is known as a cloning vector. The cloning vector may be the plasmid from a bacterium, a higher organism’s cell, or DNA taken from a virus. The vector contains restriction sites that help in the convenient insertion and removal of a DNA fragment. The cloning vector and the foreign DNA are treated with a restriction enzyme that creates the same overhang, then ligating the fragments together. Genetically engineered plasmids and bacteriophages (such as phage λ) are perhaps most commonly used. Other types of cloning vectors are also used, which include bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).

Yeast plasmids

Yeast are eukaryotes and possess complex internal structures similar to plants and animals. Yeasts can post-translationally modify proteins, unlike bacteria. However, while working with them they share the same technical advantages like rapid growth, ease of replica plating, a well-defined genetic system, and a highly versatile DNA transformation system as prokaryotes. In biotechnology experiments, the yeast Saccharomyces cerevisiae is one of the most important organisms. Yeasts possess a stable diploid and a haploid state, making them suitable for genetic analysis. They also possess an efficient mechanism of homologous recombination, which helps in simple gene replacement. The discovery of a plasmid in most strains of S. cerevisiae stimulated the development of cloning vectors for yeast. Vectors derived from the 2µm plasmid are yeast episomal plasmids or YEps. The plasmid is called a 2µm circle and is one of only a minimal number of plasmids found in eukaryotic cells.

Yeast episomal plasmid

Vectors derived from the 2-micron circle (a natural yeast plasmid) are called yeast episomal plasmids or YEps. These plasmids are similar to bacterial plasmids. YEps may contain the entire 2-micron plasmid or include just the 2-micron origin of replication. YEp13 is a shuttle vector and illustrates several general features of yeast cloning vectors. It contains the 2-micron origin of replication and the selectable LEU2 gene, along with the entire pBR322 sequence (figure 1), and can therefore replicate, and be selected for in both yeast and E. coli.

The cloning procedure in yeast starts with the cloning experiment with E.coli with the selection of recombinants (figure 2). The standard cloning procedure in yeast (figure 2) is to perform the initial cloning experiment with E.coli and to select recombinants. Recombinant plasmids can then be purified, and characterized, and the appropriate molecule is introduced into yeast. YEps may integrate into a yeast chromosome by homologous recombination with the defective genomic copy of the selection gene.

**Figure 2: Cloning with a yeast episomal plasmid**

Yeast integrating plasmid (YIp)

This plasmid lacks an origin of replication and is inserted into the host chromosome through homologous recombination. YIps rely on integration into the host chromosome for survival and replication and are usually used when studying the functionality of a solo gene or a toxic gene.

Yeast replicating plasmid (YRp)

These vectors can replicate independently of the yeast chromosome. However, they tend to be unstable and may be lost during budding. They contain an Autonomously Replicating Sequence (ARS) derived from the yeast chromosome.

Yeast centromere plasmid (YCp)

These are low-copy vectors and involve part of an ARS along with part of a centromere sequence (CEN). These vectors replicate as small independent chromosomes and are thus typically found as a single copy. CEN vectors are stable without integration.

Yeast artificial chromosome (YAC)

For cloning huge pieces of DNA, artificial chromosomes are used. A YAC was first described in 1983 by Murray and Szostak. It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences named autonomous replicating sequences needed for replication and preservation in yeast cells (figure 3).

A YAC is constructed using an initial circular plasmid. Restriction enzymes typically break the circular plasmid into two linear plasmids. DNA ligase is then used to ligate a sequence or gene of interest between the two linear molecules, forming a single large linear piece of DNA. The construction of the YAC clone is similar to that for cosmids. YAC vectors can accommodate genomic DNA fragments of more than 1 Mb and hence can be used to clone entire human genes, such as the cystic fibrosis gene, which is 250 kb in length.

pYAC3 vector includes the yeast sequences TEL, CEN4, ARS, TRP1, URA3, and SUP4. TEL represents a segment of the telomeric DNA sequence extending by the telomerase enzyme inside the yeast cell. CEN4 is the centromere sequence for chromosome 4 of S. cerevisiae. The ARS (autonomously replicating sequence) functions as a yeast origin of replication. To ensure the survival of only properly reconstituted YACs, TRP1 and URA3 are the yeast-selectable markers present at each end. SUP4 is a gene, which is insertionally inactivated in recombinants (figure 3). It is the basis of a red-white color test, which is analogous to blue-white screening in E.coli.

Cloning with pYAC3 vector

The procedure of cloning with pYAC3 begins with the digestion of the vector with a combination of BamHI and SnaBI, which cuts the molecule into three fragments. The BamHI fragment is removed, leaving two arms, each bounded by one TEL sequence and one SnaBI site. The DNA to be inserted is ligated between the two arms producing the artificial chromosome (figure 4). The restriction enzyme SnaBI is a blunt-end cutter. Thus, the inserted DNA must have blunt ends. Then, through the method of protoplast transformation, the artificial chromosome is introduced into S. cerevisiae. The yeast strain used is a double auxotrophic mutant, trp1^–and ura3^–, which will be converted to trp1⁺and ura3⁺by the two markers on the artificial chromosome.

Selection in S.cerevisiae

The selectable markers in Saccharomyces cerevisiae do not normally confer resistance to toxic substances, as in E.coli plasmids. However, they enable the growth of yeast on selective media lacking specific nutrients. Only cells containing a correctly constructed artificial chromosome will be able to grow on a minimal medium. Then, transformants are selected. Cells transformed with an incorrect artificial chromosome will fail to grow on a minimal medium as one of the markers will be absent.

Conclusion

Yeast are eukaryotes and possess complex internal structures similar to plants and animals. The discovery of a plasmid in most strains of S. cerevisiae stimulated the development of cloning vectors for yeast.

Yeast episomal plasmids are similar to bacterial plasmids. YEp13 is a shuttle vector and illustrates several general features of yeast cloning vectors. Yeast integrating plasmid lacks an origin of replication and is inserted into the host chromosome through homologous recombination. The yeast-replicating plasmid can replicate independently of the yeast chromosome. Yeast centromere plasmid vectors replicate as small independent chromosomes and are thus typically found as a single copy.

A yeast artificial chromosome (YAC) is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences named autonomous replicating sequences needed for replication and preservation in yeast cells. Artificial chromosomes are used for cloning large pieces of DNA.