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Abstract

To construct a mammalian artificial chromosome (MAC), telomere repeats and selectable markers were introduced into a 100 kb yeast artificial chromosome (YAC) containing human centromeric DNA. This YAC, which has a regular repeat structure of alpha-satellite DNA and centromere protein B (CENP-B) boxes, efficiently formed MACs that segregated accurately and bound CENP-B, CENP-C, and CENP-E. The MACs appear to be about 1-5 Mb in size and contain YAC multimers. Structural analyses suggest that the MACs have not acquired host sequences and were formed by a de novo mechanism. The accurate segregation of the MACs suggests they have potential as vectors for introducing genes into mammals.

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A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite

H Masumoto, H. Masukata, T Okazaki … (1989)

We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170- bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere.

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

J J Harrington, G Van Bokkelen, R W Mays … (1997)

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 proteins specific for active centromeres, and are estimated to be 6-10 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 novo centromere activity and that the microchromosomes so generated contain all of the sequence elements required for stable mitotic chromosome segregation and maintenance. This 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.

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Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors

D Burke, G Carle, Gregory Olson (1987)

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PubMed ID:: 9592390

DOI:: 10.1038/nbt0598-431

ScienceOpen disciplines: Chemistry

Keywords: Animals,Autoantigens,Cell Line,Cell Line, Transformed,Centromere,genetics,Centromere Protein B,Chromosomal Proteins, Non-Histone,chemistry,Chromosomes,Chromosomes, Artificial, Yeast,Chromosomes, Human, Pair 21,Cloning, Molecular,DNA, Satellite,DNA-Binding Proteins,Genetic Vectors,Humans,In Situ Hybridization, Fluorescence,Kinetochores,Polymerase Chain Reaction,Telomere,Transfection

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ScienceOpen disciplines: Chemistry

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Construction of YAC-based mammalian artificial chromosomes.

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Most cited references 42

A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite

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

Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors

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