They involve engineered chromosomes derived from different organisms. Examples of artificial chromosomes include YACs, BACs, human artificial chromosomes, and P1-derived artificial chromosomes. The yeast artificial chromosomes (also known as YACs) include chromosomes derived from the yeast with the help of genetic engineering tools and techniques. The genetic engineers use the inserted sequences for cloning. Human Genome Project worked with YACs. The only disadvantage of YACs involves a stability issue. The basic components of the yeast artificial chromosomes include the autonomously replicating sequences (ARS), the centromeric regions, the telomeres, and selectable markers for antibiotic resistance. These properties help in transforming the yeast cells.
Construction of yeast artificial chromosomes:
For initiating the construction of YACs, the first requirement involves the circular DNA plasmid. It gets cut into a linear DNA molecule using the process of restriction digestion. This process requires the action of the restriction enzymes. The gene of interest or the DNA molecule then gets ligated into the linear DNA using the enzyme known as DNA ligase. Thus, it forms a circular DNA with the gene of interest. Next, the selectable marker gets ligated into the plasmid vector. The plasmid is an extrachromosomal material present in the bacterial cells. The plasmid vectors play a crucial role in recombinant DNA technology. Ligating the selectable marker into it helps in differentiating the colonies expressing it. Hence, it becomes easy to amplify the YAC vector and screen the clones. Other selectable marker genes include TRP1 and URA3. The cloning site suitable for inserting the foreign DNA is known as the SUP4 gene. The gene causes an accumulation of red pigment.
Thus, it compensates for the related mutation. The transformed ones form colorless colonies. The wild-type ones form red colonies. Cloning a foreign DNA into the transformed ones results in insertional inactivation of the gene. Thus, it forms red colonies again. Ligating the Centromeric sequences helps in stabilizing the mitosis. The YACs also involves ligation of the autonomously replicating sequences. It also accompanies the ligation of origin of replication. It plays a crucial role in replication and mitosis. It helps the plasmid to replicate independently of the nuclear replication machinery. The ligation of the artificial telomeric sequences plays a crucial role in converting the circular plasmid to a linear one. Finally, the genetic engineers insert the sequence to be amplified. The final step involves the transformation of yeast colonies.
Image: Yeast Artificial Chromosomes (pYAC4)
Thus, a YAC consists of the following:
· CEN region (the Centromeric region)
· ARS (autonomously replicating region)
· TEL (two telomeres)
· TRP1 gene
· SUP4 gene
· URA3 gene
YAC vectors accommodate DNA segments with several base pairs. Thus, they help in cloning large segments. The instability of the YAC vectors occurs due to the frequent rearrangements in the genomic segments. Thus, it becomes very difficult to assemble the genome sequence using these artificial chromosomes.
Mega-YACs possess a capacity of occupying large segments with the insert size of 1400 kilobase pairs. Thus, they help in preparing the human genome libraries.
Bacterial artificial chromosomes:
The abbreviation for the bacterial artificial chromosomes is known as BACs. It involves a DNA construct based on the bacterial plasmids. The bacteria possess fertility plasmid known as the F-plasmid. It helps in the process of conjugation or a unidirectional gene transfer. Genetic engineers use F-plasmid for transformation and cloning experiments. Their insert size involves 100-350 kbp. The F-plasmid consists of partition genes. These genes promote even distribution of the plasmids. Human Genome Project highly utilized BACs. They involve high accuracy and stability. They show an ability to replace the whole genome shotgun approach. These chromosomes become easy to handle. The bacterial cells undergo a process of conjugation involving the transfer of the F-plasmid from the donor to the recipient cell. Construction of the BACs requires F-plasmid like a raw material. BACs get purified as plasmid DNA. The components of BACs include the origin of replication, par A and par B components, antibiotic resistance genes, and the phage promoters. The origin of replication or the oriS (also known as rep E-F) plays a crucial role in initiating the replication. It regulates the copy number of the plasmids. The maintenance of BACs requires components such as the parA and parB. These components help in stabilizing the bacterial artificial chromosomes. They play an important role in the cell division process and help in the formation of daughter cells. The antibiotic gene help in identifying the recombinants. They act as selectable markers. The phage promoters also serve in constructing used the BACs. Examples include T1 and Sp6 promoters. They help in studying diseases.
BAC libraries get constructed based on the collection of BAC clones. The BAC clones storage occurs at a regulated temperature. BACs help us in studying large genes. Hence, these artificial chromosomes worked well in the Human Genome Project. BAC clones also help us in studying several genes at once. They help in developing vaccines.
Human artificial chromosomes:
These chromosomes are also known as microchromosomes. They act as new chromosomes. They hold large amounts of DNA. Most of them get constructed using natural human chromosomes as the starting materials. The HACs get classified as artificial chromosomes. They get inserted into the cell as an extra chromosome or the 47th chromosome. The cytogeneticists working with HACs suggest that these artificial chromosomes help in gene therapy strategies. They also help to study the cell and the genetic material functioning. Preparation of HACs also involves synthetic DNA and Yeast artificial chromosomes. These elements help in studying the centromere or construction of gene carriers.
P-1 Derived artificial chromosomes (PACs):
We obtain this type of artificial chromosome from the P1 bacteriophage. It carries large amounts of DNA. The P1 bacteriophage exists in the prophage state. Hence, it follows the lysogenic pathway. However, the PAC acts as a plasmid by not integrating into the bacterial DNA. Its genome size is larger when compared with that of the lambda phage. It consists of two regions for replication. These regions include the region involved in the lytic DNA replication. The other region maintains the plasmid during the non-lytic growth.
References:
[1] Genomes, T.A. Brown
[2] Molecular Biology and Genomics, Page 121-130, Cornel Mulhardt
[3] An Introduction to Genetic Engineering, Desmond S. T. Nicholl
[4] Molecular Biology, Page 225-226, David P. Clark, Nanette J. Pazdernik, Michelle R. McGehee
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