Vectors and Selection Markers

Over the past 30 years, a wide range of selection markers have become available for P pastoris. Recessive markers, which require a mutation in the host genome that is complemented by the marker, mostly derive from amino acid or nucleotide synthesis pathways, as traditionally applied for other yeasts or bacteria as well (Table 19.3). The disadvantage of these auxotrophic markers is the possibility 

Biosynthetic marker Gene Useful property Referenc s 

ADEl Argininosuccinate lyase Pink color of adel strains [75. 76] 

URA3 Orotidine 5 -phosphate decarboxylase 5-FOA selection for Ura strains [75] 

---

As host cells that have been successfully transformed proliferate, they rapidly amplify the recombinant DNA. For example, under favorable conditions of nutrient availability and temperature, a single recombinant plasmid introduced into an E. coli cell can be replicated a billion times in about 11 hours. However, transformed and untransformed cells usually have identical appearances. (See Figure 18C for an example of an exception to this mle.) Consequently, researchers often design cloning protocols utilizing vectors with selectable marker... 

As vectors do not replicate in the CF system, plasmid compatibility and selection markers are not important. However, the supplied plasmid and the linear DNA templates must be of high quality. 

DNA construct will often contain an effect gene and a selectable marker gene (such as antibiotic or herbicide resistance), both of which are bracketed by promoter and terminator sequences. A plasmid vector carries this cassette of genetic information into the plant genome by one of the above methods. 

---