Human Artificial Chromsomes

dellaire dellaire at odyssee.net
Tue Apr 15 16:33:43 EST 1997


Torsten,

I just got a copy of the Nature Genetics Article by Harrington et al.
(1997) Vol 15 pg 345

"Formation of de novo centromeres and construction of first-generation
human artificial minichromosomes"

In the article they used a very simple approach of randomly joining
centromeric repeats to identify what consitutes a centromere, they ende=
d up
with
workable minichromosomes.

The approach involved cloning long sythetic arrays (concatamers) of
alpha stellite DNA (based on Chrm 17 centromeric repeats) with telomeri=
c
DNA and genomic DNA to generate mammalian artificial chromosomes (MAC's=
) in
HT1080 human cells.  These MAC`s are about 1 Mb and are stable for 6
months+. They [authors] basically let nature select what combination of
chromosomal components that is required for maintenance of a chromosome
(with proper replication and segregation at mitosis). They selected for
these chromosomes using  G418 resistance, as the recombinant MAC's cont=
ain
a betageo gene (neo/betagal fusion (P. Soriano's I presume)). =20

You could have found Nature Genetics On-line from this list of on-line
journals from Experimental Medicine at McGill University

http://www.medcor.mcgill.ca/EXPMED/DOCS/on-linej.html

Or could have gone there directly at:

http://genetics.nature.com/

The abstract can be found here:

http://genetics.nature.com/1997-April/formationofidenovoicentromeres

Or if you don`t have a WWW browser here is the abstract:


++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Formation of de novo centromeres and construction of first-generation
human artificial microchromosomes

John J. Harrington1,2*, Gil Van Bokkelen1,2*, Robert W. Mays1,2, Karen
Gustashaw1 & Huntington F. Willard1

1. Department of Genetics and Center for Human Genetics, Case Western
Reserve University School of Medicine and University Hospitals of
Cleveland,Cleveland, Ohio 44106, USA.=20
2. Athersys, Inc., Cleveland, Ohio 44106, USA. *J.J.H. & G.V.B.
contributed equally to this work.=20
Correspondence should
be addressed to: H.F.W. e-mail: HFW at po.CWRU.edu

     We have combined long synthetic arrays of alpha satellite DNA with
telomeric DNA and genomic DNA to generate artificial chromosomes in
human
HT1080 cells. The resulting linear microchromosomes contain exogenous
alpha satellite DNA, are mitotically and cytogenetically stable in the
absence of selection for up to six months in culture, bind centromere
protein specific for active centromeres, and are estimated to be 6=9610
megabases in size, approximately one-fifth to one- tenth the size of
endogenous human chromosomes. We conclude that this strategy results in
the formation of de novocentromere activity and that the
microchromosomes so generated contain all of the sequence elements
required for stable mitotic chromosome segregation and maintenance. Thi=
s
first-generation system for the construction of human artificial
chromosomes should be suitable for dissecting the sequence requirements
of human centromeres, as well as developing constructs useful for
therapeutic applications.=20
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++


Regards,


Graham Dellaire
McGill Experimental Medicine
dellaire at odyssee.net
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