Plasmid replication depends on host cell polymerases
Plasmids are found naturally in many microorganisms. Plasmids can be transferred between species by transformation or conjugation, but they generally have a restricted host range. When you think of plasmids, you probably also think of bacteria, but plasmids are not restricted to bacteria. In fact, most S. cerevisiae strains carry a large plasmid known as the 2 micron or 2 μm plasmid. Multiple copies of the 2 μm plasmid are usually present in the nucleus of a yeast cell, and the plasmid number is stable through many rounds of cell division.
Although plasmids replicate independently of the chromosomal DNA, they rely on host enzymes to catalyze their replication. Host DNA polymerases bind to an origin of replication (ori) sequence in the plasmid. Plasmids that replicate in bacteria have ori sequences that bind bacterial DNA polymerase, while plasmids that replicate in yeast have distinct ori sequences
that bind yeast DNA polymerase. The plasmids that we are using are sometimes referred to as “shuttle vectors,” because they are able to replicate in more than one kind of cell. Our plasmids contain the ori of plasmid, pBR322, which is replicated in E. coli to a copy number of 30-40. The plasmids also contain the ori of the S. cerevisiae 2 μm plasmid described above. In this class, we will propagate the shuttle vectors in bacteria, because bacteria grow more rapidly than yeast and because the yield of plasmid from bacteria is higher than the yield from yeast. We will harvest the plasmids from bacteria and then use them to transform yeast cells.
Laboratory plasmids carry selectable markers
Plasmids place a toll on the host cell’s metabolism, and they would normally be lost from their host cells if they did not confer some selective advantage to the host. The plasmids used in molecular biology therefore carry genes for selectable markers, which allow transformed cells to grow under non-premissive conditions, conditions where untransformed cells are unable to grow. Our plasmids contain the b-lactamase (ampR) gene, which allows E. coli to grow in the presence
of ampicillin, an antibiotic that interferes with bacterial cell wall synthesis. The plasmids also contain the S. cerevisiae URA3 gene, which allows ura3 mutants like BY4742, the parent strain of our mutants, to grow in the absence of uracil only after they are transformed with plasmids (Chapter 12).
Promoters control transcription of coding sequences for fusion proteins
The plasmids that we will use this semester contain MET genes that have been cloned into plasmids directly downstream of the promoter sequence for the yeast GAL1 gene (Johnston, 1987). Transcription from the GAL1 promoter is normally controlled by regulatory proteins that sense glucose and galactose levels in yeast (Chapter 13). In the plasmids, the GAL1 promoter has been placed at the 5’-ends of protein coding sequences for S. cerevisiae Met proteins, their
S. pombe orthologs or bacterial LacZ. The presence of the GAL1 promoter will allow you to manipulate expression of the Met proteins or LacZ in transformed yeast cells.
The figure below shows a map of the pYES2.1 plasmid. ORFs for the S. cerevisiae and S. pombe proteins were individually cloned into the pYES2.1 plasmid by students in an advanced laboratory class and by the BIOL2040 staff. In all cases, the ORFs were cloned downstream of theGAL1 promoter (element 5 in the diagram).
The proteins expressed from pYES2.1 plasmids are fusion proteins that are ~5 k Da longer than the natural coding sequences. During the cloning processes used to construct the overexpression plasmids, researchers deleted the natural stop codons of the ORFs so that transcription would continue into plasmid-encoded sequences. The pYES sequence adds C-terminal tags that can be used for procedures such as western blots (Chapter 15) or protein purification. These tags will be discussed in greater detail in Chapter 13.