Classes Of Saccharomyces Cloning Plasmid Vectors

Saccharomyces plasmids were developed from Escherichia coli plasmid vectors. The basic E. coli vector is small [2-4 kilobasepairs (kbp) of DNA] and includes genes needed for plasmid replication, an origin of replication (ORI) derived from an E. coli plasmid, and a selectable marker gene such as AMPr (for ampicillin resistance) to be used to identify E. coli transformants containing the plasmid. The E. coli ORI allows the plasmid to replicate independent of the E. coli chromosome as an extrachromosomal element or plasmid. As such it is easy to purify in large amounts. Additionally, one or more restriction sites will be present for cloning foreign DNA sequences.

E. coli plasmids are the foundation for the construction of the Saccharomyces yeast cloning vectors. Saccharomyces sequences were added to the E. coli vectors to create what are referred to as E. eo/;/yeast shuttle vectors, meaning that these plasmid vectors are able to establish themselves in either organism. First, a marker gene capable of being selected in a yeast host strain was included in order to be able to select yeast transformants. Good antifungal agents, comparable to the ampicillin and tetracycline used in E. coli, were not initially available. Therefore, nutritional genes encoding enzymes in biosynthetic pathways were the first to be used as selectable marker genes in Saccharomyces. More recently antifungal agents like kanamycin (also called G418 or neomycin) and hygromycin have come into use.

URA3, LEU2, TRP1, and HIS3 are the genes most commonly used as selectable marker genes for Saccharomyces transformation. The Saccharomyces strains used as hosts for plasmid vectors carrying these nutritional marker genes must contain recessive mutant alleles of these genes in order to be an appropriate host. Suitable mutant alleles of URA3, LEU2, TRP1, HI S3 and other genes are available. Strains like YPH500 have been specially constructed to carry several of these mutant genes. It is important to keep in mind that transformation is rare, about one in 1000 cells or less, and not so different from the rate of mutation. To facilitate selection of transformants as opposed to the back mutations to wild-type, the mutant alleles of these genes do not revert at any appreciable rate because they are deletions, multiple point mutations, or transposon insertion mutations.

A typical Saccharomyces transformation is carried out as follows. An appropriate host/vector pair is selected. For example, a host strain carrying the ura3-52 allele is unable to grow on a minimal medium that lacks uracil because it is unable to synthesize uracil, which is essential for various cellular processes including RNA synthesis. If a plasmid carrying the wild-type dominant URA3 gene is introduced into this host strain by transformation, then the transformant will be able to grow on a minimal medium lacking uracil. The plasmid DNA is transformed into the host cells by any one of a number of methods including chemical treatments, electro-poration, or pellet guns. The DNA treated cells are plated on a solid synthetic medium lacking uracil. Only those individuals that have acquired a stable copy of URA3 by transformation with the plasmid vector will be able to form colonies. Of course this must be confirmed by appropriate tests.

The fate of the plasmid after entering a Saccharomyces cell depends on the particular Saccharomyces sequences it contains. If a Saccharomyces origin of replication is included, then the plasmid will replicate as an extrachromosomal element. Its copy number, the average number of plasmids per cell, is determined in part by the class of Saccharomyces ORI and whether or not a Saccharomyces centromere is also included in the plasmid. If the plasmid vector lacks a Saccharomyces replication origin, then the plasmid must integrate at a chromosomal site (usually by homologous recombination between vector sequences and the chromosome) to produce a stable transformant. If the plasmid vector integrates, then it will replicate as part of the chromosome.

Yip PLASMID

A Yip plasmid consists of the basic E. coli vector described above plus a Saccharomyces selectable marker gene, but does not contain a Saccharomyces origin of replication. Therefore, Yip plasmids must integrate into a chromosome in order to be replicated at each cell division. If integration does not occur, the transforming DNA will be lost due to degradation or dilution by cell division.

Integration occurs by means of a single crossover (recombination) event between the plasmid DNA and the chromosome. This is illustrated below in Figure 1.2. The crossover occurs only between homologous DNA sequences and is carried out by the generalized recombination enzymes. After the integration event, the plasmid sequences are part of the chromosome, are replicated when the chromosome is replicated, and are passed into both the mother and daughter cells during cell division, as are all the other sequences of the chromosome. Figure 1.2 shows a recombination event occurring between a yeast sequence carried by this vector and a homologous chromosomal sequence. This recombination event might also have occurred between the URA3 sequence on the plasmid and the mutant ura3-52 gene in the host since sequences are still present at this site. To prevent this, one could use ura3 deletion mutation.

URA3

LEU2

LEU2

1

leu2-6

J

LEU2

URA3

Ieu2~6

Figure 1.2 Targeted integration

Figure 1.2 Targeted integration

The integration of a YIp plasmid can be targeted to a specific chromosomal site (shown in Figure 1.2). Integration requires recombination between homologous sequences on the plasmid and chromosome. For supercoiled plasmid DNA, this recombination occurs in about one in 10000-100000 transformed cells. But, if prior to transformation the plasmid is digested with a restriction enzyme that cuts at a site within the homologous sequence creating a highly recombinogenic double-strand break, then the frequency of recombination will increase about 1000-fold. Therefore, as is shown in Figure 1.2, if a particular YIp plasmid carrying two Saccharo-myces sequences, for example URA3 and LEU2, is digested at a site in the LEJJ2 gene and transformed into a ura3 leu2 host strain, then it will integrate 1000 times more often at the leu2 locus than at the ura3 locus. If transformant strains are crossed to another strain of opposite mating type with the ura3 leu2 genotype and sporulated, then all of the tetrads will be PD with two uracil" leucine" spores and two uracil+ leucine+ spores. That is, the URA3 LEU2 alleles will segregate together because they are both linked to the site of plasmid integration.

YRp PLASMID

YRp plasmids are constructed from the basic YIp vector by the addition of a Saccharomyces origin of replication derived from a chromosomal sequences. These yeast ORI sequences are commonly called ARS sequences for autonomously replicating sequence. Chromosomal replication initiates at these sites and, on average, they are found every 40 kilobasepairs of DNA in Saccharomyces. YRp plasmids can be integrated but normally they are not and are able to replicate as independent extrachromosomal plasmids. Depending on the particular ARS element, they are present in 5-10 copies per cell on average.

YRp plasmids are unstable because there is no mechanism to move the plasmid copies into the bud (like a spindle) and they often get left behind in the mother nucleus. Because of this, the growth of cells transformed with YRp plasmids in nonselective media (that is, media containing the nutrient synthesized by the selection marker carried on the plasmid) leads to the spontaneous loss of the pi^smid. To ensure that the plasmid is maintained by most of the cells in a culture, transformants must be grown in a selection medium lacking the nutrient at all times so that only cells inheriting the plasmid will be capable of growing and dividing.

YEp PLASMID

A YEp plasmid contains an origin of replication derived from the naturally occurring Saccharomyces plasmid called the 2/i circle in the basic Yip vector. The Saccharomyces 2/j circle is a very abundant plasmid found in many natural strains and most laboratory strains. The 2/i circle ORI is a very active origin and YEp plasmids normally replicate as high-copy independent extrachromosomal plasmids present in 25-50 copies per cell on average. YEp plasmids are also unstable and transformants must be grown under selection to maintain the plasmid.

YCp PLASMID

A YCp plasmid contains a centromere sequence, CEN, derived from one of the 16 Saccharomyces chromosomes added to a YRp vector. These plasmids are treated like mini chromosomes by the dividing Saccharomyces cell. YCp plasmids attach to spindle fibers during division and are very efficiently transmitted to both mother and daughter cells in mitosis and meiosis, although not as efficiently as the normal chromosomes. Therefore, YCp plasmids are very stable plasmids that replicate and segregate along with the remainder of the chromosomes. As a result, YCp plasmids are low-copy independent extrachromosomal plasmids present in 1-2 copies per cell on average and are lost from transformant cells at a very low rate even in the absence of nutritional selection.

YAC PLASMID

YAC vectors are designed to carry large chromosomal fragments of DNA and have been very useful in cloning fragments for various genome sequencing projects and for positional cloning studies. YAC stands for yeast artificial chromosome. The major difference between YAC vectors and YCp vectors is the inclusion of two copies of a sequence derived from Saccharomyces telomere DNA consisting of many repeats of the short nucleotide sequence 5'C2_3A(CA)|_3 on one strand with the complementary GT-rich sequence repeated on the other strand. In the circular YAC vector plasmid, the two copies are separated by a stuffer fragment that is cut out using restriction enzymes prior to transformation into the host Saccharomyces strain. Once in the host cell, endogenous telomerase enzyme will elaborate a full telomere at each end of the linearized YAC vector DNA. Natural Saccharomyces chromosomes range from 230 to 1700 kbp but these have several origins of replications each. Inserts up to 1400 kbp can be accommodated in certain YAC vectors.

LIBRARIES

Saccharomyces plasmid libraries can be constructed from any of these types of vector. The choice depends on whether a stable or unstable Saccharomyces transformant is desired and whether one or many copies per cell are needed.

Was this article helpful?

0 0

Responses

  • michael griffin
    What class is saccharomyces?
    7 years ago

Post a comment