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15-03-2011, 09:28 PM
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Human Artificial Chromosome
Human Artificial Chromosome
Shreya Sara Ittycheria & Sherin J.S.
(S6, Biotechnology and Biochemical Engineering, Mohandas College of Engineering and
Technology, Anad, Nedumangad, Trivandrum – 695544)


.pdf  Human Artificial Chromosome.pdf (Size: 246.98 KB / Downloads: 117)

Abstract
The human artificial chromosome (HAC) is a microchromose that can act as a new chromosome
in a population of human cells. Yeast artificial chromosome and bacterial artificial chromosome
were created for human artificial chromosome which first appeared in 1997. They are useful in
expression studies as gene transfer vectors and are a tool for elucidating human chromosome
function. Grown in HT1080 cells, they are mitotically and cytogenetically stable for up to six
months. The use of a similar strategy in human cells to produce human artificial chromosomes
(HACs) might be expected to provide an important tool for the manipulation of large DNA
sequences in human cells. However, of the three required chromosomal elements, only telomeres
have been well defined in human cells to date. It has been demonstrated that telomeric DNA,
consisting of tandem repeats of the sequence T2AG3, can seed the formation of new telomeres
when reintroduced into human cells (4–6). And recently, two telomeric binding proteins, TRF1
and TRF2, have been described (7–9). The second required element, a human centromere, is
thought to consist mainly of repeated DNA, specifically the alpha satellite DNA family, which is
found at all normal human.The production of HACs from cloned DNA sources should help to
define the elements necessary for human chromosomal function and to provide an important
vector suitable for the manipulation of large DNA sequences in human cells.
Key Words : Artificial chromosome, michromosome, gene transfer vector

ntroduction
A human artificial chromosome (HAC) is a
microchromose that 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 in size and are able to carry new
genes introduced by human researchers. Yeast
artificial chromosome and bacterial artificial
chromosome were created for human artificial
chromosome which first appeared in 1997. They
are useful in expression studies as gene transfer
vectors and are a tool for elucidating human
chromosome function. Grown in HT1080 cells,
they are mitotically and cytogenetically stable
for up to six months.
History
John J. Harrington, Gil Van Bokkelen, Robert
W. Mays, Karen Gustashaw & Huntington F.
Willard of Case Western Reserve University
School of Medicine published the first report of
human artificial chromosomes in 1997. They
were first synthesized by combining portions of
alpha satellite DNA with telomeric DNA and
genomic DNA into linear microchromosomes.
Construction using Yeast Artificial
Chromosome
A human artificial chromosome (HAC) vector
was constructed from a 1-Mb yeast artificial
chromosome (YAC) that was selected based on
its size from among several YACs identified by
screening a randomly chosen subset of the
Centre d’Étude du Polymorphisme Humain
(CEPH) (Paris) YAC library with a degenerate
alpha satellite probe. This YAC, which also
included non-alpha satellite DNA, was modified
to contain human telomeric DNA and a putative
origin of replication from the human β-globin
locus. The resultant HAC vector was introduced
into human cells by lipid-mediated DNA
transfection, and HACs were identified that
bound the active kinetochore protein CENP-E
and were mitotically stable in the absence of
selection for at least 100 generations.
Microdissected HACs used as fluorescence in
situ hybridization probes localized to the HAC
itself and not to the arms of any endogenous
human chromosomes, suggesting that the HAC
was not formed by telomere fragmentation. Our
ability to manipulate the HAC vector by
recombinant genetic methods should allow us to
further define the elements necessary for
mammalian chromosome function.
As the time rapidly approaches when the
complete sequence of a human chromosome will
be known, it is striking how little is known about
how human chromosomes function. In contrast,
the necessary elements for chromosomal
function in yeast have been defined for several
years. Three important elements appear to be
required for the mitotic stability of linear
chromosomes: centromeres, telomeres, and
origins of replication. The ascertainment of these
elements in Saccharomyces cerevisiae provided
the basis for the construction of yeast artificial
chromosomes (YACs), which have proven to be
important tools both for the study of yeast
chromosomal function and as large capacity
cloning vectors.
The use of a similar strategy in human cells to
produce human artificial chromosomes (HACs)
might be expected to provide an important tool
for the manipulation of large DNA sequences in
human cells. However, of the three required
chromosomal elements, only telomeres have
been well defined in human cells to date. It has
been demonstrated that telomeric DNA,
consisting of tandem repeats of the sequence
T2AG3, can seed the formation of new telomeres
when reintroduced into human cells. And
recently, two telomeric binding proteins, TRF1
and TRF2, have been described. The second
required element, a human centromere, is
thought to consist mainly of repeated DNA,
specifically the alpha satellite DNA family,
which is found at all normal human centromeres.
However, normal human centromeres are large
in size and complex in organization, and
sequences lacking alpha satellite repeats also
have been shown to be capable of human
centromere function. As for the third required
element, the study of origins of DNA replication
also has led to conflicting reports, with no
apparent consensus sequence having yet been
determined for the initiation of DNA synthesis in
humancells.
The production of HACs from cloned DNA
sources should help to define the elements
necessary for human chromosomal function and
to provide an important vector suitable for the
manipulation of large DNA sequences in human
cells. Two approaches to generate chromosomes
with the “bottom up” strategy in human cells
from human elements have recently been
described. Harrington et al. (17) synthesized
arrays of alpha satellite DNA, which were
combined in vitro with telomeres and fragmented
genomic DNA, and transfected into HT1080
cells. The undefined genomic DNA component
appeared to play an important role in the ability
to form HACs, leaving unanswered questions as
to what sequences, other than telomeres and
alpha satellite DNA, were necessary for
chromosome formation. Ikeno et al. (18) used
two 100-kb YACs containing alpha satellite
DNA from human chromosome 21 propagated in
a recombination deficient strain, which
necessitated transient expression of a
recombination protein (Rad52) to modify the
YAC with telomere sequences and selectable
markers (19). Only one of the two YACs was
able to form HACs in HT1080 cells, suggesting
that not all alpha satellite sequences may be able
to form centromeres. Here we report
construction of functional HACs from a YAC
that was propagated in a recombination-
proficient yeast strain and was chosen solely for
its size (1 Mb) and the presence of alpha satellite
DNA. This YAC contains both alpha satellite
and non-alpha satellite DNA and was modified
to include a putative human origin of replication
and human telomeric DNA. The function and
stability ofHACs generated from this 1-Mb YAC in
humancell line are described.
Advances in HAC Technology
Human artificial chromosome (HAC) technology
has developed rapidly over the past four years.
Recent reports show that HACs are useful gene
transfer vectors in expression studies and
important tools for determining human
chromosome function. HACs have been used to
complement gene deficiencies in human cultured
cells by transfer of large genomic loci also
containing the regulatory elements for
appropriate expression. And, they now offer the
possibility to express large human transgenes in
animals, especially in mouse models of human
geneticdiseases
.Conclusion
Artificial chromosomes (ACs) are highly
promising vectors for use in gene therapy
applications. They are able to maintain
expression of genomic-sized exogenous
transgenes within target cells, without
integrating into the host genome. Although
these vectors have huge potential and
benefits when compared against normal
expression constructs, they are highly
complex, technically challenging to
construct and diffcult to deliver to target
cells.
Reference
1. Nature Genetics 15, 345 - 355 (1997)
Harrington and Bokkelen et al.)
2. Grimes et al. Genome Biology 2004 5:R89)
3. Formation of de novo centromeres and
construction of first-generation human artificial
microchromosomes in Nature Genetics15, 345 -
355 (1997) Harrington and Bokkelen et al. )
4.Advances in human artificial chromosome
technology-Larin Z, Mejía JE.)
5.Murray A W , Szostak J W (1983) Nature
(London) 305:189–193, pmid:6350893.
6.Murray A W ,Szostak J W (1985) Annu Rev
Cell Biol 1:289–315, pmid:3916318.
7.Burke D T , Carle G F , Olson M V -(1987)
Science 236:806–812, pmid:3033825.
8.Farr C ,Fantes J ,Goodfellow P ,Cooke H
(1991) Proc Natl Acad Sci USA 88:7006–7010,
pmid:1871116.
9. nett M A ,Buckle V J ,Evans E P ,Porter A C
G ,Rout D ,Smith A G ,Brown W R A (1993)
Nucleic Acids Res 21:27–36, pmid:8441617.
10.Hanish J P ,Yanowitz J L ,De Lange T (1994)
Proc Natl Acad Sci USA 91:8861–8865,
pmid:8090736.
11. Bilaud T ,Brun C ,Ancelin K ,Koering C E
,Laroche T , Gilson E (1997) Nat Genet 17:236–
239, pmid:9326951.
12. Chong L ,van Steensel B ,Broccoli D
,Erdjument H ,Hanish J ,Tempst P ,de Lange T
(1995) Science 270:1663–1667, pmid:7502076.
13. van Steensel B ,Smogorzewska A ,de Lange
T (1998) Cell 92:401–413, pmid:9476899.
14. Lee C ,Wevrick R ,Fisher R B ,Ferguson-
Smith M A ,Lin C C (1997) Hum Genet
100:291–304, pmid:9272147.
14. Manuelidas L -(1978) Chromosoma 66:23–
32, pmid:639625.
15. Willard H F- (1985) Am J Hum Genet
37:524–532, pmid:2988334.
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