Artificial

chromosomes

Presented by:

1. Sadaf Raza (1515054064)

2. Ananya Srivastava (1515054007)
3. Saumya Gupta (1515054067)

Batch: 2015-2019
Introduction
• Artificial chromosomes are DNA molecules or fragments assembled in
vitro from defined constituents, which guarantee stable maintenance of
large DNA fragments, with the properties of natural chromosomes.

• Artificial chromosomes are useful for genome sequencing

programmes, for functional characterization of entire genomic regions
and for the transduction of large DNA segments into human and non
human mammalian cells.

• Types: BAC, YAC, PAC, HAC

Yeast artificial chromosomes (YACs)
• The YAC was devised and first reported in 1987 by David Burke.

• Yeast artificial chromosomes (YACs) are genetically engineered

chromosomes derived from the DNA of the yeast, Saccharomyces
cerevisiae.

• Yeast artificial chromosome (YAC) vectors allow the cloning, within

yeast cells, of fragments of foreign genomic DNA that can approach
500kbp in size.
Construction of YAC

• 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, forming a single large, circular piece of
DNA.
Fig : Yeast Artifical Chromosome
Applications of YAC
• YACs can be utilized to clone and assemble the entire genomes of an
organism.
• It can be used to find genes, segments of DNA that code for a specific
protein or phenotype.
Limitations of using YAC vectors
• Very large DNA molecules are very fragile and breakage, leading to
problem of rearrangement.
• Difficult to separate YAC from the other host chromosomes because of
their similar size.
• Yield of DNA is not high when the YAC is isolated from yeast cells.
• Clones tend to be unstable, with their foreign DNA inserts often being
deleted.
Bacterial artificial chromosome
• A bacterial artificial chromosome (BAC) is a DNA construct, based on
a functional fertility plasmid (or cloning in F-plasmid), used for
transforming in bacteria, usually E. Coli.
• They are capable of carrying approximately 300 kbp of inserted DNA
sequence.
• Ex: pBAC108L pBeloBAC11
Fig. Genetic map of a Bacterial Artificial Chromosome
(BAC) The genes oriS, repE, sopB, sopA are involved in
plasmid replication.
Common gene Components of BAC

• Ori s:origin of replication from Ecoli F-plasmid.

• Rep E: encode a rep protein.
• parA,parB,parC: partitioning of F plasmid during cell division.
• CMr : chloramphenical resitance.
• LacZ: β galactosidase gene.
Applications of BAC
Contribution to models of disease: Inherited disease
BACs are now being utilized in modeling diseases, often alongside
transgenic mice.

BACs have been used to study neurological diseases such as

Alzheimer's disease or as in the case of aneuploidy associated with
Down syndrome. There have also been instances when they have been
used to study specific oncogenes associated with cancers.
Applications of BAC [contd.]
Contribution to models of disease: Infectious disease
The genomes of several large DNA viruses and RNA viruses have been
cloned as BACs.
These constructs are referred to as "infectious clones".
The infectious property of these BACs has made the study of many
viruses such as the Herpesviruses, Poxviruses and Coronaviruses more
accessible.
Applications of BAC [contd.]

Sequencing:
BACs are often used to sequence the genome of in genome projects, for
example the Human Genome Project. A short piece of the organism's
DNA is amplified as an insert in BACs, and then sequenced. Finally, the
sequenced parts are rearranged in silico, resulting in the genomic
sequence of the organism.
P1 derived artificial chromosome
• P1 was developed as a cloning vector by Nat Sternberg and colleagues
in the 1990s.

• P1 derived artificial chromosome are DNA construct that are derived

from DNA of p1 bacteriophage they can carry large amount(about
100-300 kbs ) of other sequence for a variety of bioengineering
purpose.
Human artificial chromosome
• A human artificial microchromosome (HAC) can act as a new
chromosome in a population of human cells.
• That is, instead of 46 chromosomes, the cell could have 47 with the
47th being very small, roughly 6-10 megabases (Mb) in size for
natural chromosomes, and instead able of 50-250 Mb to carry new
genes introduced by human researchers.
• Ideally, researchers could integrate different genes that perform a
variety of functions, including disease defense.
Construction methods HAC
• There are currently two accepted models for the creation of human
artificial chromosome vectors.

1. Top Down Approach

2. Bottom Up Approach
Top Down Approach
• Where you take a natural human chromosome and strip it all of its
genes so there is kind of this skeleton of the chromosome so we add
our own artificial genes into produce the proteins that we desire.
• The chromosomes endogenous can then be microcell-mediated
transferred into other cell lines by chromosome transfer (MMCT).
Bottom Up Approach

• Where we build the artificial chromosome piece by piece & this is

called the de novo artificial human chromosome, superior approach,
since it gives a full control over what we put in the genes, also full
control over it's shape and the structure of the chromosome.
Production

First create the centromere and then, create origins of replication

(initiation genes) by using 2 arms containing a telomere & origin of
replication of BAC & add this into a human cell where the two arms are
attached and we produce the functional HAC.
To introduce HAC, we use a technique micro cell mediated
chromosome transfer uses a donor cell with HAC in it, & it fuses into
the new human cell, the recipient cell, it does this by exposing the donor
cell with the hac to a chemical called colcemid, which causes the donor
cell to micro nucleate each nucleus containing one of the hac
chromosomes, micro cells are fused to the recipient cell using a different
exposure to chemicals eg PEG, this causes the fusion to the recipient
cell causing a new human cell with the successful functional HAC.
Applications of HAC
• HACs are useful in expression studies as gene transfer vectors, as a
tool for elucidating human chromosome function, and as a method for
actively annotating the human genome.
• HACs have been used to create transgenic animals for use as animal
models of human disease and for production of therapeutic products.
• HAC can carry genes to be introduced into the cells in gene therapy.
Thank you.