A basic element employed in recombinant DNA technology is the plasmid, which is extrachromosomal, double-stranded DNA found in some microorganisms. Where plasmids have been found to naturally occur in microorganisms, they are often found to occur in multiple copies per cell. Most reported naturally occurring plasmids are of covalently closed circular DNA; however, linear plasmids have been found to exist in organisms such as Kluyveromyces lactis and Pichia Acaciae.
The utility of isolated linear plasmids has traditionally revolved around their use as hybrid or shuttle vectors. These plasmids can be specifically cleaved by restriction endonucleases or other restriction enzymes, each of which recognizes a specific, unique site on the plasmid DNA. Thereafter, homologous genes as well as heterologous genes, i.e., genes derived from organisms other than the host, or gene fragments, may be inserted into the plasmid by endwise joining of the cleaved plasmid and the desired genetic material at the cleavage site or at reconstructed ends adjacent to the cleavage site. The resulting recombined DNA material can be referred to as a hybrid or shuttle vector which is then able to be expressed in a host microorganism.
In addition, linear plasmids are sources of autonomous replication sequences (ARS) which are required to produce the plasmid in daughter cells. Thus, linear plasmids are convenient vehicles for the modification of host cells. However, linear plasmids found in other microorganisms have demonstrated utility which extends beyond that of use as sources of ARS and subsequent shuttle vectors. For example, a linear DNA can serve as a linear DNA vector and was found to be stably maintained in cytoplasm. These newly developed vectors are extremely stable and have high copy numbers.
Also linear plasmids of have been found to code for a secreted killer toxin which is capable of killing a wide variety of sensitive yeast strains while immunizing the producing organism from the toxin. These linear plasmids are believed to be the source of a secretion signal which is useful in the secreted production of heterologous proteins. A killer system in which toxin production and immunity are both associated with linear double-stranded DNA was found to exist in Pichia acacial. The toxins produced might prove useful in the biotyping of pathogenic fungi as has been demonstrated for other Pichia toxins.
The uses of linear plasmids are continuously being explored. In addition to having already demonstrated their utility as shuttle vectors and sources of ARS, they are potentially useful as recombinant linear plasmids which are highly stable and maintain heterologous DNA at a high copy number; sources of secretion signals; sources of promoters for the expression of proteins; as well as utilities yet to be discovered with continued experimentation. An ongoing interest in linear plasmids and their use in recombinant DNA technology exists; therefore, providing additional linear plasmids is a significant contribution to the art.
USPTO Backgrounds
USPTO Backgrounds