DEVELOPEMENT OF A STABLE GENETIC VECTOR FOR THE ANIMALS AND THE YEASTS.
An attempt has been made to construct linear vectors and artificial chromosomes which replicate extrachromosomally in a variety of cells and organisms. For this purpose biologically relevant sequences, like replication origins, telomeres and eventually centromeres, from various sources are assembled and tested in different cells and organisms. A circular plasmid containing the entire BPV-1 genome which replicates extrachromosomally in Xenopus embryos has been constructed. This plasmid is now being used to identify genes relevant during early embryogenesis. Efforts are also being made to construct a linear version of this plasmid which will function in Xenopus.

Research has been carried out to isolate and study replication origins and telomeres and to assemble the different elements into linear vectors, in order to improve gene transfer in eukaryotes. The replication origins studied came from viruses (bovine papilloma virus (BPV); simian virus 40 (SV40) and African swine fever virus (ASFV)) and chromosomes from animal cells. Plasmids containing the BPV origin of replication function could be injected into Xenopus embryos. Much is known about the structure of telomeres from lower eucaryotes and these sequences were used for the assembly experiments. In addition, telomeric segments were isolated and analyzed from human cells and ASFV. The role of simple repeating deoxyribonucleic acid (DNA) sequences on integration was determined and a putative signal for integration identified. Several linear vectors were assembled and their stability determined. Extracts from cells infected with ASFV enabled the in vitro replication of DNA. Human telomeric sequences were cloned in Escherichia coli and in yeast, and theviral telomeres of ASFV were isolated. A putative signal for integration (d(CG)) was identified. A linear vector containing the BVP origin of replication and Tetrahymena telomeres was shown to be stable in Xenopus oocytes.
GOOD, AUTONOMOUSLY REPLICATING VECTORS FOR ANIMAL OR PLANT CELLS DO NOT EXIST, AND MOST ATTEMPTS TO INTRODUCE GENES INTO THESE CELLS HAVE RELIED ON THE MATERIAL BECOMING INTEGRATED INTO ONE OF THE CHROMOSOMES OF THE HOST CELL.
ATTEMPTS TO MAKE USE OF ARTIFICIALLY CONSTRUCTED CIRCULAR PLASMIDS HAVE BEEN TOTALLY UNSUCCESSFUL. IT MAY THUS VERY WELL BE THAT THE MOST APPROPRIATE VECTOR WILL NOT BE CIRCULAR, BUT WILL HAVE A LINEAR STRUCTURE. THIS PROPOSITION IS REINFORCED IF ONE CONSIDERS THAT THE CHROMOSOMES OF HIGHER ORGANISMS ARE ALMOST INVARIABLY LINEAR AND THAT CIRCULAR PLASMIDE ARE NOT NORMALLY FOUND IN THE CELLS OF THESE ORGANISMS.
IDEALLY SUCH A VECTOR SHOULD CONTAIN A CENTROMERE FUNCTION TO ENSURE ITS STABILITY AT LOW COPY NUMBER.

IT IS THE AIM OF THIS PROPOSAL TO CONSTRUCT LINEAR VECTORS, AND EVENTUALLY ARTIFICIAL LINEAR CHROMOSOMES, FOR THE PURPOSE OF IMPROVING GENE TRANSFER AND GENE CLONING IN EUKARYOTIC CELLS. THE VECTORS WILL BE CONSTRUCTED USING COMPONENTS DERIVED FROM NATURALLY OCCURING LINEAR PLASMIDS, CHROMOSOMES, AND ANIMAL VIRUSES. THE VECTORS WILL BE DESIGNED FOR USE IN MAMMALIAN HOST CELLS, BUT THERE IS A VERY GOOD CHANCE THAT VECTORS USEFUL FOR YEASTS WILL BE PRODUCED AS FALLOUT OF THE MAIN PROJECT.
APART FROM THE PRODUCTION OF POTENTIALLY USEFUL EUKARYOTIC VECTORS, FURTHER UNDERSTANDING OF REPLICATION AS WELL AS TELOMERE AND CENTRIMERE FUNCTIONS WILL NO DOUBT BE GAINED. THE POTENTIAL FOR NATURALLY OCCURING LINEAR PLASMIDS AND THE ABILITY OF THEIR ENDS TO FUNCTION AS TELOMERES WILL BE TESTED IN THE CELLS OF HIGHER ORGANISMS. VARIOUS ELEMENTS IDENTIFIED AS BEING POTENTIAL COMPONENTS OF AN ARTIFICIAL MINICHROMOSOME, BUT DERIVED FROM DIVERSE SOURCES, WILL BE ASSEMBLED AND THEIR FUNCTIONS TESTED AFTER INTRODUCTION INTO THE CELLS OF YEAST AND HIGHER ORGANISMS.