Fundamentals of Biotechnology

Lecture #07

Bacterial Artificial Chromosomes• Many vectors which are popularly used for DNA cloning in

bacterial cells contain high to medium copy number replicons.

• disadvantage is that such vectors often show structural instability of inserts, resulting in deletion or rearrangement of portions of the cloned DNA.

• Common in the case of DNA inserts of eukaryotic origin where repetitive sequences occur frequently and, as a result, it is difficult to clone and maintain intact large DNA in bacterial cells.

Continue!!• BAC: A high-capacity cloning vector based on the F

plasmid of Escherichia coli

• is a DNA construct, based on a functional fertility plasmid (or F-plasmid).

• The bacterial artificial chromosome's usual insert size is 150-350 kb, but can be greater than 700 kb.

• BACs are often used to sequence the genome of organisms in genome projects.

Common gene components• oriS, repE - F for plasmid replication and regulation of

copy number.

• parA and parB for partitioning F plasmid DNA to daughter cells during division and ensures stable maintenance of the BAC. (at 1–2 per E. coli cell).

• A selectable markers for antibiotic resistance, some

BACs also have lacZ at the cloning site for blue/white selection.

• T7 & Sp6 phage promoters for transcription of inserted genes.

Yeast Artificial Chromosomes

• YAC: is a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb).

• It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin.

• The most popularly used system for cloning very large DNA fragments.

• Can clone >2,000 kb (2 Mb).

Continue!!!• Cloning in yeast cells offers, in principle, some advantages

over cloning in bacterial cells.

• Certain eukaryotic sequences, notably those with repeated sequence organizations,

• Difficult, or impossible, to propagate in bacterial cells which do not have such types of DNA organization,

• but would be anticipated to be tolerated in yeast cells which are eukaryotic cells.

• However, the main advantage offered by YACs has been the ability to clone very large DNA fragments.

Essential functional components • Centromeres, required for disjunction of sister

chromatids in mitosis and of homologous chromosomes at the first meiotic division.

• Telomeres, required for complete replication of linear molecules and for protection of the ends of the chromosome from nuclease attack.

• Autonomous replicating sequence (ARS) elements, required for autonomous replication of the chromosomal DNA. They are thought to act as specific replication origins.

It also contains few other specific sequences like:

Selectable markers (A & B):that allow the easy isolation of yeast cells that have taken up the artificial chromosome.

Recognition site for the two restriction enzymes EcoRI and BamHI.

Cloning human genomic DNA into a YAC

Genomic DNA is partially digested by the restriction enzyme EcoRI. Very large DNA fragments are obtained.

The YAC is digested by the two restriction enzymes EcoRI and BamHI.

Those two elements recombine at the EcoRI sites and are covalently linked by the DNA ligase.

A recombinant YAC vector, a yeast artificial chromosome with genomic DNA inserted, is produced. This vector can be used to infect yeast cells and generated an unlimited number of copies.

• The resulting construct cannot be transfected directly into yeast cells.

• Instead, yeast cells have to be treated in such a way as to remove the external cell walls.

• The resulting yeast spheroplasts can accept exogenous fragments but are osmotically unstable and need to be embedded in agar.

• The overall transformation efficiency is very low and the yield of cloned DNA is low (about one copy per cell).