Experimental Techniques

Outlined below are a few laboratory techniques whose discovery was facilitated by the sequencing of the yeast genome.

Yeast Artificial Chromosomes (YACs)  

        One of the goals of molecular genetics is to obtain physical data about the genomic organization of DNA.  In order to characterize genomes requires the cloning of large chromosomal fragments.  YAC is a vector system that allows large segments (between 200 kb and 800 kb) of DNA to be cloned.  These cloning systems are based on yeast linear plasmids containing three important DNA elements required for mitotic stability: telomere (TEL), origin of replication (ARS) and centromere (CEN).  Using YAC vectors, recombinational cloning can be achieved.

Figure of a yeast artificial chromosome cloning system. Reproduced with permission from Fred Sherman. http://dbb.urmc.rochester.edu/labs/sherman_f/yeast/14.html

Genetic Transformation

Due to the fact that yeast can transform plasmids, using S. cerevisiae facilitates the introduction of a specific marker into its genome that is associtated with a novel characteristic or phenotype of interest.  This process, called genetic transformation, plays a critical role in the manipulation and studying the behaviour of genes.  S. cerevisiae cells are converted into transformable spheroplasts, yeast cells with a deficient cell wall, with treatment by lithium salts and the use of electroporation.  The following figure, reproduced with permission from Guo et la, 1999, diagrams the use of plasmids to transform markers into the yeast genome and how the process depends on homologous recombination.                      

Sequence Alignment

Due to the fact that the entire genome sequence is known, any gene of interest can be compared to all yeast genes using online tools such as BLAST or FASTA.  Similarly, one can find similar sequences to a particular yeast gene.   During the comparison, the homology between sequences is calculated using statistical methods by the computer program and can serve as an indicator of evolutionary relatedness between the two genes. Sequence alignment serves as an indicator of the genetic relatedness between the organisms.  

Homologous Recombination

The basis for the construction of genetic maps, which displays the determined traits of a chromosome, is homologous recombination.  As a consequence of knowing the genome sequence, researchers can genetically engineer recombinations S. cerevisiae alleles.  This process requires several steps including the introduction of strand nicks into each strand followed by strand exchange and sealing of the nicks.

Yeast two-hybrid Systems 

This is a powerful experimental tool which can be used to determine if certain proteins can interact with each other and how strong that interaction is.  Fusion proteins, which have a protein of interest fused to either the activation or DNA binding domain of a transcriptional enhancer of a reporter gene,  are expressed by transforming genetically engineer plamids into yeast cells.  If two proteins interact, the activation domain and DNA binding domain of the transcription factor will interact and produced an increase in the level of reporter gene transcribed.  Below is a figure reproduced with permission of the author, Dr. F. Sherman that illustrates two protein interacting to bring both domains of Gal4 together to enhance trancription of the lacZ reporter gene..