The URA3 and LYS2 yeast genes have an additional advantage because both positive and negative selections are possible, as discussed below (Section 10.1, URA3 and LYS2).

Although there are numerous kinds of yeast shuttle vectors, those used currently can be broadly classified in either of following three types as summarized in Table 9.1: integrative vectors, YIp; autonomously replicating high copy-number vectors, YEp; or autonomously replicating low copy-number vectors, YCp. Another type of vector, YACs, for cloning large fragments are discussed in Section 13.2 (Yeast Artificial Chromosomes).

The YpI integrative vectors do not replicate autonomously, but integrate into the genome at low frequencies by homologous recombination. Integration of circular plasmid DNA by homologous recombination leads to a copy of the vector sequence flanked by two direct copies of the yeast sequence as illustrated in the top of Figure 5. The site of integration can be targeted by cutting the yeast segment in the YIp plasmid with a restriction endonuclease and transforming the yeast strain with the linearized plasmid. The linear ends are recombinogenic and direct integration to the site in the genome that is homologous to these ends. In addition, linearization increases the efficiency of integrative transformation from 10- to 50-fold.

The YIp vectors typically integrate as a single copy. However multiple integration do occur at low frequencies, a property that can be used to construct stable strains overexpressing specific genes. YIp plasmids with two yeast segments, such as YFG1 and URA3 marker, have the potential to integrate at either of the genomic loci, whereas vectors containing repetitive DNA sequences, such as Ty elements or rDNA, can integrate at any of the multiple sites within genome. Strains constructed with YIp plasmids should be examined by PCR analysis, or other methods, to confirm the site of integration.

Strains transformed with YIp plasmids are extremely stable, even in the absence of selective pressure. However, plasmid loss can occur at approximately 10-3 to 10-4 frequencies by homologous recombination between tandemly repeated DNA, leading to looping out of the vector sequence and one copy of the duplicated sequence as illustrated in Figure 9.1 and discussed below in Section 11.2 (Two-Step Gene Replacement).

 

Figure 9.1. Two-step gene replacement. The wild-type chromosomal YFG1⁺ allele can be replaced by the mutant yfg1-1 allele from a YIp integrating plasmid. The plasmid is first integrated in the chromosome corresponding to the site on the plasmid that was cleaved by a restriction endonuclease. Strains that have excised the URA3 marker in vivo by homologous recombination are selected on FOA medium. Either the original YFG1⁺ allele, or the yfg1-1 allele remains in the chromosome, depending on the site of the cross-over.

Most YEp plasmids are relatively unstable, being lost in approximately 10-2 or more cells after each generation. Even under conditions of selective growth, only 60% to 95% of the cells retain the YEp plasmid.

The copy number of most YEp plasmids ranges from 10-40 per cell of cir⁺ hosts. However, the plasmids are not equally distributed among the cells, and there is a high variance in the copy number per cell in populations.

Several systems have been developed for producing very high copy-numbers of YEp plasmids per cell, including the use of the partially defective mutation leu2-d, whose expression is several orders of magnitude less than the wild-type LEU2⁺ allele. The copy number per cell of such YEp leu2-d vectors range from 200-300, and the high copy-number persists for many generations after growth in leucine-containing media without selective pressure. The YEp leu2-d vectors are useful in large-scale cultures with complete media where plasmid selection is not possible. The most common use for YEp plasmid vectors is to overproduce gene products in yeast.

The YCp yeast centromere plasmid vectors are autonomously replicating vectors containing centromere sequences, CEN, and autonomously replicating sequences, ARS. The YCp vectors are typically present at very low copy numbers, from 1 to 3 per cell, and possibly more, and are lost in approximately 10^-2 cells per generation without selective pressure. In many instances, the YCp vectors segregate to two of the four ascospore from an ascus, indicating that they mimic the behavior of chromosomes during meiosis, as well as during mitosis. The ARS sequences are believed to correspond to the natural replication origins of yeast chromosomes, and all of them contain a specific consensus sequence. The CEN function is dependent on three conserved domains, designated I, II, and III; all three of these elements are required for mitotic stabilization of YCp vectors. YRp vectors, containing ARS but lacking functional CEN elements, transform yeast at high frequencies, but are lost at too high a frequency, over 10% per generation, making them undesirable for general vectors.

The stability and low copy-number of YCp vectors make them the ideal choice for cloning vectors, for construction of yeast genomic DNA libraries, and for investigating the function of genes altered in vivo. ARS1, which is in close proximity to TRP1, is the most commonly used ARS element for YCp vectors, although others have been used. CEN3, CEN4 and CEN11 are commonly used centromeres that can be conveniently manipulated. For example, the vector YCp50 contains CEN4 and ARS1.