Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1992-06-25
1995-02-14
Nucker, Christine M.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
43525411, 4353201, 4351723, 435 9142, C12N 115, C12N 1580, C12P 2100
Patent
active
053895266
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates generally to the fields of molecular biology and the production of recombinant proteins by the biotechnology industry. More particularly, the present invention relates to novel strains of the genus Dictyostelium, recombinant plasmid vectors for use with strains of the genus Dictyostelium, and polypeptides which facilitate the extrachromosomal replication of such plasmids in strains of the genus Dictyostelium. Such extrachromosomally replicating plasmids, constructed with the art disclosed in this invention, are suitable for carrying a wide variety of genes and promoter sequences for the controlled production of recombinant proteins by the biotechnology industry.
BACKGROUND ART
As is well known in the art, genetic information is encoded on double stranded DNA molecules according to the sequence of four nucleotides containing different bases, adenine (A), thymine (T), cytosine (C) and guanine (G). Blocks of DNA sequences flanking genes often control gene activity by binding regulatory proteins and acting as recognition signals for enzymes of the cells biosynthetic machinery. Thus each cell contains a web of regulatory molecules which, by binding to specific DNA sequences, control gene activity. Other DNA sequences have crucial functions related to the control of DNA synthesis and partitioning of DNA into separate cells during cell division. These functions must be present on every DNA molecule in every cell or the DNA will be lost within a few cell generations.
Plasmids are usually circular DNA molecules possessing DNA sequences allowing them to replicate independently from chromosomal DNA. The DNA sequence block where the replication of plasmid DNA is initiated is commonly called the "origin of replication" and the ability to replicate independently from chromosomal DNA is referred to as "extrachromosomal" replication.
Molecular biologists have developed techniques for cutting DNA molecules into fragments using sequence specific restriction enzymes, purifying the fragments and rejoining them in a different order. If one of the fragments of DNA used contains an origin of replication from an E. coli plasmid, the DNA can be inserted (transformed) into E. coli where it will replicate as a plasmid and can be produced in relatively large quantities. These techniques mean that genes from one organism, for example a human gene, can be flanked by regulatory DNA sequences from another organism, for example the bacterium E. coli, causing the human gene to be active in E. coli under entirely different regulatory controls. If the plasmid in question is constructed to include a second origin of replication allowing replication in a separate host cell, for example a mouse cell line, the gene can easily be transferred to the second host cell. Such a plasmid containing origins of replication for more than one host is commonly called a "shuttle vector". Plasmids are usually constructed to contain selectable markers, which are usually genes that confer antibiotic resistance or a metabolic advantage on the host cell to allow cells containing the plasmid to be distinguished from cells that have not received any plasmid during the transformation. Selectable marker genes must be flanked by appropriate DNA sequences to permit gene activity in the required host cell. It is possible to insert a plasmid into a host cell where it will be unable to replicate and so the only cells that survive the selection procedure will be those with the plasmid inserted into the host's chromosomal DNA. Such a plasmid without an appropriate origin of replication is called an "integrating plasmid".
A cell produces polypeptides and proteins by initially making a messenger RNA copy of the gene, a process called transcription which is under the control of the flanking DNA sequences as summarised above. The cellular biosynthetic machinery then reads (translates) the RNA sequence in three nucleotide groups called codons which specify the amino acids to be incorporated into the polypeptide chain. The g
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Chang Andy C. M.
Slade Martin B.
Williams Keith L.
Mosher Mary E.
Nucker Christine M.
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