Artificial Chromosomes

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

      Copyrights, 2019 All Rights Reserved

Cloning vectors

Image 1: Cloning vectors

The concept of cloning vectors is similar to the example described below. Imagine you want to reach a destination far away from your house. So, you use a vehicle to reach that place, isn’t it? The vehicle carries you to the desired destination. In the molecular world, the extrachromosomal DNA known as the plasmids act as vehicles in carrying the fragment of interest to the desired location. Thus, in DNA cloning, a plasmid or phage chromosome acts as a vector (vehicle) to carry the cloned DNA segment. There are many types of vectors such as plasmids, cosmids, phagemids, shuttle vectors, expression vectors, and artificial chromosomes.

1.   Plasmid vectors:

The extrachromosomal elements present in the bacterial cells replicate autonomously or independently. Plasmids have a circular DNA with double strands. It consists of sequences capable of independent function. Naturally prevalent plasmids serve as the main source of plasmid vectors. They are engineered to get the desired features. Plasmid extraction is easily possible with the E. coli bacterial cells. These plasmids pass from one cell to another cell. Replication of plasmids occurs during each cell division. We obtain plasmids through disruption of the bacterial cells followed by centrifugation. Next step involves treatment with the restriction enzymes. Cleavage of a plasmid produces staggered ends. The foreign DNA gets incorporated in the vacant site. Note the two important points about the plasmids. Firstly, the plasmids possess a limited number of restriction sites. Secondly, the plasmids carry genes for resistance to antibiotics. Care must be taken to avoid incorporation of any harmful gene into it. 

Image 2: Plasmid Vector

The features of E. coli plasmids are as follows:

·        Origin of replication: The site for starting or initiating the replication is known as the origin of replication. It permits an independent replication of the plasmid.

·        Selectable marker: It is usually dominant. A selectable marker gets readily detected. Thus, it exhibits a trait suitable for artificial selection. One more type of marker is known as a screenable marker. It allows the researchers to distinguish between the required cells and the unwanted cells. An antibiotic resistance gene acts as a selectable marker. Examples include ampicillin or a tetracycline resistance gene. The positive and negative markers are the two types of markers. The markers conferring a selective advantage to the host are known as positive markers. The markers eliminating the growth of the markers are known as negative markers.

·        Restriction enzyme cleavage sites: The restriction sites are present just once in a vector. The DNA fragments get inserted into the vector at specific sites. An appropriate restriction enzyme recognizing this sequence gets selected for cleaving that particular site. Later on, the fragment of interest gets inserted into that site. Some of the plasmids have multiple cloning sites. Plasmids act as disease models. Protein productions and gene therapy also involve the use of plasmid vectors.

Example of a plasmid vector: pUC 19 vector

pUC 19 or “puck” 19 vector is a 2686 base pair vector with multiple cloning sites. They are unique restriction sites clustered in one region. A high copy number of the plasmids and amp^R selectable marker make it a good choice for cloning experiments. The polylinker has restriction sites for various restriction enzymes such as Eco RI, Bam HI, HindIII, PstI, SacI, Sma I and many other enzymes. The polylinker is within the beta-galactosidase gene (lac z+). The pUC 19 vector gets cleaved at its polylinker site leaving a vacant space. Next, a piece of DNA to be cloned gets separated through electrophoresis and obtained for further procedures. The piece of DNA, the vector, and the ligating enzymes are mixed. The DNA gets inserted into the plasmid and the gaps sealed by DNA ligase. The resulting plasmid gets transformed into E. coli. It involves incubation of the recombinant plasmids with E. coli cells and treating chemically or by electroporation. Next step includes plating of the transformed cells on a suitable medium to observe the colonies. Blue-white screening distinguishes recombinant DNA clones from the plasmids. Episomes are the plasmids capable of integrating into the chromosome.

2.   Phagemids or Phasmid:

It is a cloning vector with two properties such as bacteriophage and plasmid properties. They are DNA based vectors. These phasmids exhibit a property of getting packed into phage heads.

3.   Fosmids:

They resemble cosmids and involve bacterial F plasmids. E. coli serves as a source of fosmid vectors and contains only one fosmid.

4.   Shuttle vectors:

The vectors introduced into two or more organisms are known as shuttle vectors. For example, some vectors get transformed into E. coli and also get transformed into the yeast. These plasmids propagate in the prokaryotes and eukaryotes. Example, an Adenovirus shuttle vector propagates into both prokaryotes and mammals. Multiple copies of a gene get synthesized. A shuttle vector consists of two origins of replication specific to each host. Shuttle vectors are also known as bifunctional vectors. A yeast shuttle vector consists of bacterial origin of replication, yeast origin of replication, yeast selectable marker, bacterial selectable markers, and an origin of transfer.

5.   Cosmids:

Cosmids are also known as hybrid plasmids. They consist of a lambda phage cos sequence. The genetic engineering technique uses cosmids as cloning vectors. They have a suitable origin of replication and an antibiotic resistance gene. These plasmids get packaged into phage capsids thereby allowing transduction. The cos sequences present in a cosmid are just a few hundred base pairs in length. The availability of the cosmids as the shuttle or mammalian cosmids depends on the need of the experiment. A typical feature of the cosmid involves the formation of colonies instead of forming plaques.

6.   Expression vectors:

A plasmid or a virus behaves like an expression vector. However, an expression vector designing must boost the expression of the desired gene. The gene of interest gets inserted into the expression vector. The gene contains a protein-coding region. The expression of the desired gene produces a protein product.

Image 3: The Expression vector

7.   Plant vectors:

The plant vectors involve Ti-plasmid vectors. The Ti-plasmids belong to a soil bacterium known as Agrobacterium tumefaciens. It consists of T-DNA capable of integrating into a plant genome. Integration of plasmid into the plant genome occurs when the bacterium infects the plant and causes crown gall disease. An example of plant cloning vector involves the pBIN vector. It consists of a lac z’ gene, a right T-DNA boundary, a left T-DNA boundary, an origin of replication, and kanamycin resistance gene.

8.   BAC’s and YAC’s:

The bacteria exchange their plasmids through a process known as conjugation. The donor bacterial cell transfers its F plasmid through a cellular bridge. BACs or bacterial artificial chromosome construction involves F-plasmids. The human genome project utilized bacterial artificial chromosomes for various projects.

·        Components of BACs:

·        oriS, rep E-F: It is the origin of replication. It regulates the copy number of the plasmids.

·        The parA and par B components help in the partition of the cells into daughter cells during cell division.

·        Antibiotic resistance gene acts as a selectable marker.

·        Phage promoters induce transcription of an inserted gene such as T1 and Sp6 promoters.

Various disease studies involve BACs as models. A BAC library is a collection of BAC clones stored in regulated temperatures. Several genes and large gene studies involve BACs. They are useful in the development of vaccines.

Yeast artificial chromosomes or YACs help in cloning large DNA thereby guiding in an accurate construction of a physical map. They involve genetically engineered yeast DNA.

References:

[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2]Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh

[3] Gene Cloning and Manipulation, Christopher Howe
[4] Genetic Engineering, Verma P.S. & Agarwal V.K.

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