Most of studies on gene cloning have been carried out in bacteria, E. Coli. So, vectors are based on plasmid or virus that can survive in E. Coli.
Eukaryotic cloning vectors are used when cloning is performed for correction of introns of eukaryotic mRNAs, to control or improve synthesis of an important metabolic product (e.g. a hormone, such as insulin), or to protect from hazardous effects or change the properties of the organism. Introduction of herbicide resistance into a crop plant is an example of such change. It is often convenient to use eukaryotic hosts for the study of expression and regulation of eukaryotic genes.
Cloning vectors for yeast and other fungi
Saccharomyces cerevisiae (Yeast) is one of the most important organisms in the field of biotechnology as well as pharmaceutical research. It is a single-cell eukaryotic microbe, which can be cultured as well as manipulated using standard methodology. In 1976, Struhl et al reported that a fragment of yeast DNA when cloned with E.coli, restored histidine dependent growth in strains carrying the hisB mutation.
Most strain of yeast contain 2μm plasmid (6 kb in size) that contains protein encoding REP1 and REP2 genes and exists in the yeast cell in between 70 and 200 copy number. Most plasmids used for yeast studies are shuttle vectors, containing sequences permitting them to be selected and propagated in E.coli, thus allowing convenient amplification and subsequent alteration in vitro. This DNA provides a model for the study of eukaryotic DNA replication as it is small and is found in abundance.
Most common yeast vectors originated from pBR322 that contain the following:
- An origin of replication (ori)
- Selectable antibiotic markers
- High copy number maintenance in E.Coli.
- β-lactamase gene
- AmpR or/and TetR.
Most commonly used yeast markers include, URA3, HIS3, LEU2, TRP1 and LYS2, which complement specific auxotrophic mutations in yeast, such as ura3-52, his3-D1, leu2-D1, trp1-D1 and lys2-201. URA3, HIS3, LEU2 and TRP1 yeast markers can complement specific E. coli auxotrophic mutations
Yeast vector system developed shows mostly the following common features:
- They are mostly derived from bacterial plasmids and retain ability to replicate and remain as selection markers suitable to use in bacterial system. They also act as shuttle vectors.
- Selection markers that are generally nutritional markers.
Yeast shuttle vectors, which are used currently can be broadly classified in either of following types:
- Integrative vectors (YIp)
- Yeast episomal plasmids or autonomously replicating high copy-number vectors (YEp)
- Yeast replicating plasmid (YRp)
- Autonomously replicating low copy-number vectors (YCp)
- Yeast linear plasmid (YLp)
Yeast integrating plasmid (YIp)
These vectors are unable to replicate autonomously within a yeast cell so, they integrate into genome at low frequencies by homologous recombination. Site of integration can be targeted by cutting the yeast segment with a restriction endonuclease and transforming the yeast strain with linearized plasmid. Linearization increases the efficiency of integrative transformation (as integration is essential for successful transformation ) from 10- to 50-fold. The frequency of yield is very slow i.e. one transformant per μg of DNA. YIp plasmids with two yeast segments, such as YFG1 and URA3 marker, have 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 transformed with YIp plasmids are extremely stable.
Yeast episomal plasmid (YEp)
Yeast episomal plasmid vectors replicate autonomously due to the presence of a segment of the yeast 2μm plasmid that serves as an origin of replication (ori). The ori is responsible for the high copy-number and high frequency of transformation of YEp vectors. YEp vectors contain either a full copy of the 2μm plasmid, or, a region that encompasses the ori and the REP3 gene. Most YEp plasmids are relatively unstable. 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.
Yeast replicating plasmid (YRp)
Replacement of 2μm by autonomously replicating sequences, ARS (100 bp sequence) gives rise to YRp. These vectors are based on chromosomal elements. These vectors undergo autonomous replication of plasmids in yeast by virtue of the presence of a functional origin of DNA replication. These were first discovered when a region closely linked to TRp1 gene allowing high frequency transformation and propagation without need for integration was observed.
Yeast centromeric plasmid (YCp)
The YCp are autonomously replicating vectors containing centromere sequences, CEN, and autonomously replicating sequences, ARS. These vectors are also based on chromosomal elements as YRp. 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, so they are useful in situations that require low copy number. The stability and low copy-number of YCp vectors make them the ideal choice for cloning vectors, for investigating the function of genes altered in vivo and for construction of yeast genomic DNA libraries.
Yeast linear plasmid (YLp)
YLp is artificial chromosomal plasmid containing telomeric sequences for maintaining a linear state. Telomeric sequences are added to the needs of a cut ARS-based plasmid. It was originally constructed using telomeres from ciliate protozoan Tetrahymena.
Three factors that decide which type of yeast vector is most suitable for a cloning experiment is as follows:
- Transformation frequency: It a measure of the number of transformants that can be obtained per microgram of plasmid DNA. A high transformation frequency is necessary if many recombinants are needed, or if the amount of starting DNA is very small. YEps have high transformation frequency, providing between 10,000 and 100,000 transformed cells per μg, whereas YIp yields less than 1000 transformants per μg,
- High copy numbers: YEps and YRps have high copy numbers, 20–50 and 5–100 per cell, respectively. In contrast, a YIp is usually present at just one copy per cell.
- Stability: YIps produce very stable recombinants, as loss of YIp that got integrated into a chromosome occurs at only very low frequency. Whereas, YRp recombinants are extremely unstable.