Doaa.M.Reda YAC

Yeast artificial chromosome “YAC”: 

By Doaa M.Reda Hala Bhig Yeast artificial chromosome “YAC” Under supervision of Dr. Hala Eissa

DNA Cloning : 

DNA Cloning DNA cloning is a technique that allows the wholesale production of a specific DNA sequence. DNA containing a gene of interest is inserted into a self replicating element .

PowerPoint Presentation: 

A genomic library is a population of hosts, each of which carries a DNA molecule that was inserted into a cloning vector , such that the collection of cloned DNA molecules represents the entire genome of the source organism. This term also represents the collection of all of the vector molecules , each carrying a piece of the chromosomal DNA of the organism. Genomic library

Types of cloning vector: 

Types of cloning vector Plasmid Cosmid Phagmid Bacteriophage Bacterial artificial chromosome “BAC” Yeast artificial chromosome “YAC”

Yeast Artificial Chromosome “YAC”: 

Yeast Artificial Chromosome “YAC ” A vector used to clone DNA fragments larger than 100 kb and up to 3000 kb . Or A linear molecules which behave like a yeast chromosome.

YAC construction : 

YAC construction YAC is consist of several important regions ans they are : TEL : The telomere which is located at each chromosome end, protects the linear DNA from degradation by nucleases. CEN : The centromere which is the attachment site for mitotic spindle fibers, "pulls" one copy of each duplicated chromosome into each new daughter cell. ORI : Replication origin sequences which are specific DNA sequences that allow the DNA replication machinery to assemble on the DNA it can also called autonomously replicating sequence (ARS) trp1 ura3

PowerPoint Presentation: 

trp1 ura3 Yeast selectable marker A and B : selectable markers that allow the easy isolation of yeast cells that have taken up the artificial chromosome. Bacterial selectable marker : such as Ampicillin resistance marker Recognition site : for the two restriction enzymes EcoRI and BamHI . Also it contains few other specific sequences

Cloning process using YAC: 

Cloning process using YAC F irst the YAC vectors are linearized by restriction digestion. S econd the recombinant DNA is then transformed into the protoplast of the yeast cells “ a double auxotrophic mutant, ura3 ­ and trp1 - , yeast strain is used ” T hird transformants are selected on the minimal medium in which uracil and tryptophan remains absent.

Advantages of using of YACs : 

Advantages of using of YACs Maximum length of DNA that can be cloned into vectors “ 1000 Kb ” and 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 a selectable 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 .

Disadvantages of using of YACs: 

YAC s are not used as extensively anymore because of some problems : YAC clones can contain non-contiguous segments of the genome. This means that 2 or more DNA fragments from separate parts of the genome can be integrated into an individual YAC (because they are able to support rather large inserts). YAC s are unstable and frequently lose parts of the DNA during propagation. Disadvantages of using of YACs

Conclusion : 

Conclusion Artificial chromosomes are useful for genome sequencing , for functional characterization of entire genomic regions and for the transduction of large DNA segments into human and nonhuman mammalian cells but with a limitation usage .