ARTIFICIAL CHROMOSOMES IN BACTERIA AND YEAST YEAST ARTIFICIAL

ARTIFICIAL CHROMOSOMES IN BACTERIA AND YEAST

YEAST ARTIFICIAL CHROMOSOMES • First described by Murray and Szostak. • YAC is a human engineered DNA molecule used to clone DNA sequences in yeast cells. • p. YAC 3 and p. YAC 4 are different types of YAC plasmids. • In this method, segments of an organism’s DNA are inserted into YACs, which are taken by yeast cells. • As the yeast cells grow and divide, they amplify the YAC DNA, which is isolated and used.

COMPONENTS OF YAC is an artificially constructed chromosome that contains a • Centromere; Yeast centromere that ensure chromosome partitioning between two daughter cells and a selective marker gene. • Telomere; Yeast telomere stabilize chromosome ends. • ARS element; Autonomous replicating sequence elements are required for replication and preservation of YAC in yeast cells.

• Selectable marker genes, such as antibiotic resistance and a visible marker, are utilized to select transformed yeast cells. • Yeast selectable marker such as URA 3 and TRP 1 and various bacterial selectable markers. • YACs can carry up to 1000 kbp of inserted DNA sequence.

CONSRTUCTION OF YACS Construction of YAC occurs in three steps; ü Plasmid DNA purification. ü Treatment with restriction enzymes. ü Ligation and yeast transformation. § A YAC is built using an initial circular DNA plasmid, which is typically cut into a linear DNA molecule using restriction enzymes. § DNA ligase is then used to ligate a DNA sequence or gene of interest into the linearized DNA. § Which forms a single large, circular piece of DNA.

CONSTRUCTION OF YAC

APPLICATIONS OF YAC • YACs can be used in many organisms for cloning. • Another application of YAC is the study of regulation of gene expression. • Application of YAC includes generating whole DNA libraries of the genome of higher organisms. • Also to identify essential mammalian chromosomal sequences necessary for future construction of Specialized mammalian artificial chromosomes (MACs).

BACTERIAL ARTIFICIAL CHROMOSOME • A BACTERIAL ARTIFICIAL CHROMOSOME (BAC) IS A DNA CONSTRUCT, BASED ON FUNCTIONAL FERTILITY PLASMID. • USED FOR TRANSFORMING AND CLONING IN BACTERIA. • BACTERIAL ARTIFICIAL CHROMOSOME’S USUAL INSERT SIZE IS 150 -350 KBP.

• BAC are used to sequence the genome of organisms in Genome projects. E. g Human genome Project. • Short piece of organism’s DNA is amplified as an insert in BACs. • Sequenced parts are rearranged in Silico (is expression meaning’performed in computer).

Common Gene components • Rep E ; for plasmid replication and regulation of copy number. • par. A and par B ; for partitioning F plasmid DNA to daughter cells during division. • Selectable marker ; for antibiotic resistence some BACs also have lac. Z at cloning site.

• T 7 and Sp 6 ; phage promotes for transcription of inserted genes. • Ori S ; the origin of replication.

Contribution to models of diseases Inherited disease • • BACs are utilized in modeling genetic diseases. BACs have been used to study some neurological diseases such as Alzheimer’s disease or • In case of ANEUPLOIDY associated with Down syndrome. • They have also been used to study Oncogenes associated with cancer.

• BACs can also be utilized to detect genes or large sequences of interest. • They are preferred because they accommodate much larger sequences without risk. Infectious disease • Genomes of several large DNA viruses and RNA viruses have been cloned as BACs. • These constructs are referred to as ‘infectious clones’.