4) Yeast Artificial chromosomes

Yeast artificial chromosomes ( YAC ) such small plasmids that grown in bacteria
which is Escherichia Coli and introduced into yeast cells for example Saccharyomyces
cerevesiae . Yeast artificial chromosomes is a miniature version of a eukaryote
chromosomes . It contain an origin of replication , selectable markers , two telomers and ,
a centromere that allows for replication of the Yeast artificial chromosomes and
segregation into daughter cells during the process of cell division . Next , the foreign
DNA fragments are cloned into a restriction site in the center of the Yeast artificial
chromosomes . YACs are particularly useful for cloning large fragments of DNA from
200kb to almost 1 millions bases ( megabase = mb ) in term of size . Yeast artificial
chromosomes also played an important role in the cloning efforts of the Human Genome
Projects which are similar to Bacterial artificial chromosomes ( BACs ) .




Basically , for cloning human genomic DNA into a Yeast artificial chromosomes , the
Genome DNA is partially will be digested by the restricted enzyme called EcoRI . EcoRI is
the enzyme that isolated from strains of bacteria Escherichia Coli . and used for restriction
modification system . The result obtained will be large DNA fragments . Next , The Yeats
artificial chromosome ( YACs) is digested by the two enzymes EcoRI and BamHI . BamHI is
a type II restriction endonuclease, having the capacity for recognizing short sequences (6
b.p.) of DNA and specifically cleaving them at a target site. BamHI is derived from Bacillus
amyloliquefaciens . 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.

Yeast artificial chromosomes can be considered as a functional artificial chromosome
since it includes three specific DNA sequences that enable it to propagate from one cell to its
offspring:

i. TEL: The telomere which is located at each chromosome end, protects the linear
DNA from degradation by nucleases.
ii. CEN: The centromere which is the attachment site for mitotic spindle fibers, "pulls"
one copy of each duplicated chromosome into each new daughter cell.
iii. ORI: Replication origin sequences which are specific DNA sequences that allow
the
DNA replication machinery to assemble on the DNA and move at the replication
forks.

As a conclusion , Yeast artificial chromosomes is used for to clone and map
chromosomal segments and genomic clones to localize genes and approach genetic
disease .
5) Human Artificial Chromosomes

A human artificial chromosome (HAC) can be defined as a mini-chromosome
that is constructed artificially in human cells. Its self-replicating and segregating systems
by its own , a HAC can behave as a stable chromosome that is independent from the
chromosomes of host cells.

In human artificial chromosome ( HAC ) , there are elements that important to
know in order to understand Human artificial chromosome . The 3 important elements are
replication region , centromere and telomere . The replication region is the place when
the duplication of DNA begins . Next , centromere function as in proper chromosome
segregation during the cell division process . Last element is telemore which acts to
protect the end linear chromosomes .


Human artificial chromosomes (HACs) represent another extrachromosomal
gene delivery and gene expression vector system (711). Although this technology is less
advanced than virus derived vectors, HACs have several potential advantages over
currently used episomal viral vectors for gene therapy applications . These are
multimerized in human cells, forming HACs with sizes of approximately 1 to 3 Mb (7, 8,
10, 19) .


6) Ti Vectors

Ti vectors are naturally occurring plasmids which size around 200kb that isolated
from the bacterium Agrobacterium tumefaciens , which is a soilborne plant pathogen that
cause a condition in the plants called crown gall disease . For example , when
Agrobacterium tumefaciens enters host plants , a piece of DNA from the tumor inducing
plasmid ( Ti plasmid ) inserts into the host chromosome . T-DNA encodes for the
synthesis of a hormone called auxin , which weakens the host cell wall . Auxin acts as a
cardinal role in coordination of many growth and behavioral processes in the plant's life
cycle and are essential for plant body development . Infected plants cells divide and
enlarge to form a tumor gall . If we remove the auxin and other detrimental genes , the
resulting vector could be used to deliver gens into plan cells . The size of Ti vector
around 250kb . Its usually used for the creation of transgenic plants.