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1. ( Genetic engineering of plant and microbes )
‘’ – ARTIFICIAL CHROMOSOME- ’’

2. SYNOPSIS :-
 Introduction of artificial chromosome
 Types of artificial chromosome
 Bacterial artificial chromosome-
• Introduction of BAC
• Component of BAC
• Cloning method of BAC
• Application of BAC
 Yeast artificial chromosome-
• Introduction of YAC
• Component of YAC
• Cloning method of YAC
• Application of YAC
 Human artificial chromosome
 PI-derived artificial chromosome
 Conclusion
 Reference

3. Artificial chromosomes are synthetically designed DNA molecules of known
structure, which are assembled in vitro (in the laboratory) from specific DNA
sequences that acts like a natural chromosome.
 Artificial chromosomes are circular or linear vectors that are stably maintained in,
usually, 1-2 copies per cell.
They are huge in size in comparison to other vectors but can clone very large
segments of chromosomes (even an entire chromosome)
INTRODUCTION
 Types of artificial chromosomes
1. Bacterial artificial chromosome (BAC)
2. Yeast artificial chromosome (YAC)
3. Human artificial chromosome (HAC)
4. PI-derived artificial chromosome (PAC)

4. 1. bacterial Artificial Chromosome (bac)
 First developed by Hiroaki Shizuya in 1992.
 A bacterial artificial chromosome are cloning vectors based on the extra-chromosomal
plasmid of E.coli, called F factor or fertility factor.
 These vector enable the construction of artificial chromosome, which can be cloned in
E.coli.
 This vector is useful for cloning DNA fragment up to 350kb.
 It can be handled like regular bacterial plasmid vector, and is very useful for
sequencing large stretches of chromosomal DNA.

5.  Components of bacterial artificial chromosome (BAC)-
1. RepE- for plasmid replication and
regulation of copy number.
2. ParaA & ParaB & ParaC – for
partitioning F plasmid DNA to
daughter cells during division and
ensure stable maintenance of the BAC.
3. Selectable marker- for antibiotic
resistance; some BAC also have lacZ at
the cloning site for blue/white
selection.
4. Ori- origin of replication.

6.  DNA fragments of inserted are isolated and cleaved using
restriction enzymes.
 The BAC is digested by restriction enzymes around the
cloning site (Hind-III & Bam-HI).
 Recombinant DNA is formed ( F plasmid and target DNA)
using DNA ligase.
 New recombinant DNA is inserted into compliant cells and
plated.
 As bacterial cell grow and divide they also amplify the BAC
DNA which can be isolate.
 CmR and LacZ distinguish between successful transmission
of target gene into bacterium.
Cloning experiment using a BAC vector:

7.  Applications of Bacterial Artificial Chromosome (BAC)-
 BACs are often used to sequence the genome of organisms in genome
projects.
 A short piece of the organism DNA is amplified as an insert in BAC and
then sequenced.
 Now being use for modelling genetic diseases.
 The infectious property of these BACs has made study of many viruses
such as herpes virus an pox virus more easy.

8. 2. Yeast Artificial Chromosome (yac)
 First described in 1983 by Murray and Szostak.
 Yeast artificial chromosome is a human engineered DNA molecule used to clone DNA
sequence in yeast cells.
 YAC are shuttle vector capable of replicating and being selected in common bacterial
host such as E.coli as well as yeast.
 Yeast artificial chromosome (YAC) Is a vector used to clone DNA fragments larger
than 100 kb and up to 3000 kb. YACs are useful for the physical mapping of complex
genomes and for the cloning of large genes.

9. 1. The Telomere (TEL): The telomere which is located at
each chromosome end, protects the linear DNA from
degradation by Nucleases.
2. 2. The Centromere(CEN): The centromere which is the
attachment site for mitotic spindle fibers, “pulls” one
each duplicated chromosome into each new daughter
3. Origin of Replication(OR): Replication origin
sequences which are specific DNA sequences that allow
DNA replication machinery to assemble on the DNA
at the replication forks.
4. A and B: Selectable markers that allow easy isolation
of yeast cells that have taken up artificial chromosome.
5. Recognition Site: Recognition Site for two restriction
enzymes EcoRI and Bam Hl.
 Components of yeast artificial chromosome (YAC)-

10. Cloning experiment using a
YAC vector:
1. Large DNA fragments are
carrying out restriction digestion
EcoRI.
2. 2. The YAC is digested by two
restriction enzymes EcoRI and
3. 3. Those two elements recombine
EcoRI sites and are covalently
DNA ligase.
4. 4. A recombinant YAC vector, a
artificial chromosome with
DNA inserted, is produced. This
can be used to infect yeast cells
generate an un limited number of

11.  Applications of Yeast Artificial Chromosome (YAC)-
 YAC vectors can be used to clone genes that are too larged to be cloned as
a single fragment in an E.coli vector, e.g.- Human cystic fibrosis gene
(250kb).
 YAC vector opened the way to studies of the functions and modes of
expression of gene that has previously be intractable to analysis by RDT
technique.
 Production of gene libraries.
 YACs have been of immense value in providing long pieces of cloned DNA
for use in large scale DNA sequencing project.

12. 3. Human Artificial Chromosomes (HAC):
 A human artificial chromosome (HAC) is a mini-chromosome that is constructed artificially in
human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very
small, roughly 6-10 mega-bases in size, and able to carry new genes introduced by human
researchers.
 Using its own self-replicating and segregating systems, a HAC can behave as a stable chromosome
that is independent of the chromosomes of host cells.
4. Pl-Derived Artificial Chromosome (PAC):
These are DNA constructs which are derived from the DNA of PI bacteriophage. They can carry
large amounts (about 100-300 kilo-bases) of other sequences for a variety of bio-engineering
purposes. It is one type of vector used to clone DNA fragments (100- to 300-kb insert size; average,
150 kb) in E.coli cells.

13. CONCLUSION:-
REFERENCE :-
Artificial chromosome are laboratory construct that contain DNA sequence and
that perform the critical functions of natural chromosomes. They are used to
introduce and control new DNA in a cell, to study how chromosome function, and
to map genes in genomes.
Willard, H. F. – “ Genomics and Gene Therapy: Artificial chromosomes
coming to life.”
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