Yeast Artificial Chromosomes (YACs)
Bacterial Artificial Chromosomes (BACs)
Library Screening and Gene Sequencing
Yeast Artificial Chromosomes (YACs)
One goal of molecular genetics is to obtain physical data about the genomic organization of long stretches of DNA. Traditionally, this data has been obtained by a technique called chromosome walking. Walking is performed by subcloning the end of a lamda or cosmid clone and screening a library for other clones that contain similar sequence information. If this new clone overlaps a portion of the original clone, then the length of the DNA of interest is extended by the length of DNA in the second clone that is not found in the original clone. By performing these steps successive times, a long distance map can be obtained. This technique though has difficulties. First, each step is technically slow. Second, if you use lambda or cosmid clones, you might only extend the region of interest by 5-10 kb in each step of the walk. Finally, if any of the clones that are obtained contain repeated sequences, the subclone could lead you to another region of the genome that is not contiguous with the region of interest.Yeast artificial chromosomes can alleviate some of these problems because of the large (100-1000kb) amount of DNA that can be cloned. First of all, YACs cannot speed up each step of the walk because the subcloning and screening steps can only be performed so quickly. But they can solve the other two problems. Because they carry large amounts of DNA, each step can easily extend the region of interest by 50-100 kb and up to as much as 500 kb. Thus a long distance map of the region can be obtained in several steps. Secondly, although repetitive regions may be 10-20 kb in length they are rarely, longer than 50 kb. Thus a YAC with 100kb will contain some region that is single copy which can be used for further steps in the walk.
Features of YACs
- Large DNA (>100 kb) is ligated between two arms. Each arm ends with a yeasttelomere so that the product can be stabilized in the yeast cell. Interestingly, larger YACs are more stable than shorter ones, which favors cloning of large stretches of DNA.
- One arm contains an autonomous replication sequence (ARS), a centromere (CEN) and aselectable marker (trp1). The other arm contains a second selectable marker (ura3).
- Insertion of DNA into the cloning site inactivates a mutant expressed in the vector DNA and red yeast colonies appear.
- Transformants are identified as those red colonies which grow in a yeast cell that is mutant for trp1 and ura3. This ensures that the cell has received an artificial chromosome with both telomeres (because of complementation of the two mutants) and the artificial chromosome contains insert DNA (because the cell is red).