Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Human Artificial Episomal Chromosomes (HAECs) for Cloning, Mapping and Functional Testing of Large Genetic Units in Human Cells

Tian-Qiang Sun[1], Michael Grosz[3], Zachary Kelleher[2] & Jean-Michel H. Vos[1-3]
[1]Department of Biochemistry and Biophysics; [2]Curriculum in Genetics and Molecular Biology; [3]UNC Lineberger Comprehensive Cancer Center, CB#7295, 349 LCCC, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

Of approximately 100,000 human genes, only a few thousands have been cloned or mapped so far. For other chromosomal regions, such as those involved in DNA replication, chromatin packaging and chromosome segregation, much less is known. The construction of detailed physical maps is only the first step for the localization, identification and functional determination of genetic units in human cells. To study the function and regulation of human genes and other critical genomic regions which span hundreds of kilobase pairs of DNA, one has to be able to clone an entire functional unit as a single DNA fragment and transfer it into human cells. Current large-insert heterologous cloning systems, such as YACs, BACs and PACs, do not appear suitable for transferring and functional analysis of genetic units in human cells.

We have developed a human artificial episomal chromosome (HAECs) system, based on the latent replication origin of the large herpes Epstein-Barr virus (EBV) for the propagation and stable maintenance of DNA as circular minichromosomes in human cells [1]. Individual HAECs carried human genomic inserts ranging from 60 to 330 kb and appeared genetically stable. A HAEC library of 1,500 independent clones carrying random human genomic fragments with average sizes of 150-200 kb was established. The multicopy circular HAEC DNA was selectively recovered and used for restriction mapping, PCR amplification and fluorescent in situ hybridization. This autologous HAEC system, with human DNA segments directly cloned in human cells, provides an important tool for the mapping, sequencing and, most importantly, the functional study of large mammalian DNA regions [2]. Current efforts are focused on a) the packaging of the HAEC-based library as infectious EBV for shuttling large human genomic inserts in human cells [4], and b) the construction of human chromosome-specific HAEC libraries to complement the bacterial- and yeast-based mapping efforts.

[1] Sun T-Q and Vos J-MH (1994) "Human Artificial Episomal Chromosomes for Cloning Large DNA in Human Cells" Nature Genetics, in press.
[2] Vos J-MH (1994) "Herpesviruses as Genetic Vectors" in "Viruses for Human Gene Therapy" ed. J-MH Vos, Carolina Academic Press, Durham, NC, USA, pp. 109-140.
[3] Sun T-Q and Vos J-MH (1994) "Cloning Large DNA in Human Cells with the HAEC system" Methods in Molecular Genetics, ed. K. Adolph, Academic Press, San Diego, CA, Vol. 3, in press.
[4]Sun T-Q and Vos J-MH (1992) Packaging of 150-200 kb DNA as infectious Epstein-Barr virus, Int. J. Genome Res. 1: 45-57.