EP0048470A2
Expired / Lapsed
EP
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Eucaryotically active recombinant DNA
Assignee: University of Southern California
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Filing: 1981-09-18
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Granted: —
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Expires: 2001-09-18
Abstract
Abstract Eucaryotic DNA replication origin sequences are used as cloning vectors in placing DNA sequences into eucaryotic cells. By a "shotgun" procedure, random DNA segments, connected to eucaryotic replication origin sequences, are transfected into transformed eucaryotic cells which are then selected for cells that have reverted to normal cell phenotype. From the reverted cells, plasmid DNA molecules are isolated and when attached to procaryotic plasmids are amplified in bacteria. The eucaryotic replication origin sequences are obtained by isolating scrambled and palindromic (i.e. inverted repeat) eucaryotic DNA segments, joining the palindromic segments to procaryotic plasmids, transfecting the set of recombinant DNA molecules into eucaryotic cells and selecting for surviving, replicating, supercoiled recombinant molecules. These molecules are replicated in bacteria and then isolated as supercoiled plasmids. Random DNA segments are preferably obtained from the genome of eucaryotic cells of the same phenotype as the phenotypically transformed cells and are scrambled by dividing and randomly joining the segments. Sets of these scrambled segments are attached to eucaryotic replicating origin sequences and used as reverter plasmids in transformed cells. Efficiency in changing cellular phenotypes is enhanced by incorporating additional palindromic eucaryotic DNA sequences, each at least 25 base pairs long. By appropriate survival selection techniques, reverter plasmids are obtained from tumorigenic cells and from virally infected cells. Reverter plasmids are potentially obtainable from other phenotypically altered eucaryotic cells, for example, cells containing DNA sequences which code for auto-immune disease, and cells manifesting the effects of aging.
Inventors
- 👤 Robert Frank Baker
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 18:53
CPC Classifications
C; C12; C12N; C12N15/00
Claims Analysis
Total Claims: 28
Claim 1
Independent Claim
86 words
A method for preparing eucaryotically active recombinant DNA, comprising: preparing DNA molecules containing eucaryotic replication origin sequences; joining first heterogenous DNA segments to said eucaryotic replication origin segments to form recombinant molecules; inserting said recombinant molecules into phenotypically transformed eucaryotic cells whereby to replicate therein; treating said recombinant molecule-containing cells to suppress said transformed cells whereby to select, as survivors, cells that have reverted to normal cell phenotype; and isolating and replicating reverter molecules from said survivor cells whereby to constitute said eucaryotically active recombinant DNA.
Claim 2
Independent Claim
86 words
A method for preparing eucaryotically active recombinant DNA, comprising: preparing DNA molecules containing eucaryotic replication origin sequences; joining first heterogenous DNA segments to said eucaryotic replication origin segments to form recombinant molecules; inserting said recombinant molecules into phenotypically transformed eucaryotic cells whereby to replicate therein; treating said recombinant molecule-containing cells to suppress said transformed cells whereby to select, as survivors, cells that have reverted to normal cell phenotype; and isolating and replicating reverter molecules from said survivor cells whereby to constitute said eucaryotically active recombinant DNA.
Claim 3
Dependent Claim
93 words
The method of claim 1 in which said eucaryotic replication origin segments are jointed to procaryotic plasmids, said procaryotic plasmids having genes encoding resistance to a predetermined drug, and in which the step of isolating and replicating reverter plasmids comprises; isolating supercoiled reverter plasmids from said survivor cells; inserting said reverter plasmids into procaryotic cells; replicating said inserted reverter plasmids; selecting said reverter plasmid-containing procaryotic cells in accordance with their resistance to said drug; amplifying said reverter plasmids in said selected procaryotic cells; and isolating supercoiled reverter plasmids from said selected procaryotic cells.
Claim 4
Dependent Claim
50 words
The method of claim 1 or 2 in which said eucaryotic replication origin sequences are chosen to impart relaxed replication properties to said recombinant molecules, and said DNA molecules containing eucaryotic replication origins are obtained by selection from first substantially palindromic eucaryotic DNA segments joined with second heterogenous DNA segments.
Claim 5
Independent Claim
46 words
A method for preparing eucaryotically active DNA molecules serving as procaryotic cloning vectors, comprising joining first substantially palindromic eucaryotic DNA segments to procaryotic plasmids and selecting from the resultant plasmids those plasmids containing eucaryotic replication origins serving to impart relaxed eucaryotic replication properties to said plasmids.
Claim 6
Independent Claim
46 words
A method for preparing eucaryotically active DNA molecules serving as procaryotic cloning vectors, comprising joining first substantially palindromic eucaryotic DNA segments to procaryotic plasmids and selecting from the resultant plasmids those plasmids containing eucaryotic replication origins serving to impart relaxed eucaryotic replication properties to said plasmids.
Claim 7
Dependent Claim
122 words
The method of claim 1 or 4 in which said procaryotic plasmids have genes encoding resistance to a predetermined drug, and wherein the step of selecting plasmids containing eucaryotic replication origins comprises: inserting said resultant plasmids into eucaryotic cells; replicating said plasmid-containing eucaryotic cells; isolating supercoiled plasmids from said replicated eucaryotic cells; inserting said supercoiled plasmids into procaryotic cells; replicating the resultant procaryotic cells; selecting said resultant procaryotic cells; selecting said resultant procaryotic cells in accordance with their resistance to said drug; isolating from said selected procaryotic cells, supercoiled plasmids containing eucaryotic replication origin sequences; and selecting those supercoiled plasmids, isolated from said replicated eucaryotic cells, that have shown substantial replication whereby to constitute the plasmids that are inserted into procaryotic cells.
Claim 8
Independent Claim
43 words
A method for preparing eucaryotically active DNA molecules, comprising joining first substantially palindromic eucaryotic DNA segments to heterogenous eucaryotic DNA segments and selecting from the resultant molecules those molecules containing eucaryotic replication origins serving to impart relaxed eucaryotic replication properties to said plasmids.
Claim 9
Independent Claim
43 words
A method for preparing eucaryotically active DNA molecules, comprising joining first substantially palindromic eucaryotic DNA segments to heterogenous eucaryotic DNA segments and selecting from the resultant molecules those molecules containing eucaryotic replication origins serving to impart relaxed eucaryotic replication properties to said plasmids.
Claim 10
Independent Claim
27 words
A method for preparing eucaryotically active DNA molecules having enhanced recombinant properties, comprising joining to said molecules substantially palindromic DNA sequences at least 25 base pairs long.
Claim 11
Independent Claim
27 words
A method for preparing eucaryotically active DNA molecules having enhanced recombinant properties, comprising joining to said molecules substantially palindromic DNA sequences at least 25 base pairs long.
Claim 12
Dependent Claim
12 words
The method of claim 7 in which said palindromic sequences are eucaryotic.
Claim 13
Independent Claim
39 words
A method for preparing scrambled DNA segments, comprising: dividing DNA into first segments at first DNA sequence locations; joining said first segments; and dividing said joined segments into other segments at DNA sequence locations different from said first locations.
Claim 14
Independent Claim
39 words
A method for preparing scrambled DNA segments, comprising: dividing DNA into first segments at first DNA sequence locations; joining said first segments; and dividing said joined segments into other segments at DNA sequence locations different from said first locations.
Claim 15
Dependent Claim
16 words
The method of claim 9 including the step of joining said scrambled segments to procaryotic plasmids.
Claim 16
Dependent Claim
56 words
The method of claim 9 or 10 in which said step of dividing DNA into first segments is conducted in a solution of said DNA and including the step of halting the division of said DNA in successive increments of said solution over a period of time whereby to obtain a size distribution of DNA segments.
Claim 17
Dependent Claim
34 words
The method of claim 11 in which said solution contains an endonuclease that is sequence non-specific and said halting step comprises treating said successive increments to terminate the DNA cutting ability of said endonuclease.
Claim 18
Dependent Claim
67 words
The method of claim 12 in which said endonuclease is a DNase requiring a metal ion for enzyme activation, said DNA solution containing said metal salt, and in which said successive increments are obtained by continuous transfer of said solution, over said period of time, to a vessel containing a reagent comprising a chelating agent for said metal ion serving to terminate the cutting.effectiveness of said endonuclease.
Claim 19
Dependent Claim
23 words
Eucaryotically active DNA, comprising: a circular molecule of DNA containing a substantially palindromic eucaryotic DNA segment serving as a eucaryotic replication origin sequence.
Claim 20
Dependent Claim
23 words
Eucaryotically active DNA, comprising: a circular molecule of DNA containing a substantially palindromic eucaryotic DNA segment serving as a eucaryotic replication origin sequence.
Claim 21
Dependent Claim
34 words
The DNA of claim 14 containing a substantially palindromic DNA segment at least 25 base pairs long and different in sequence from said eucaryotic replication origin, whereby said DNA molecule has enhanced recombinant properties.
Claim 22
Dependent Claim
44 words
A procaryotic plasmid containing a recombinant DNA segment obtained by the steps comprising: dividing DNA into first segments at first DNA sequence locations; joining said first segments; and dividing said joined segments into other segments at DNA sequence locations different from said first locations.
Claim 23
Dependent Claim
44 words
A procaryotic plasmid containing a recombinant DNA segment obtained by the steps comprising: dividing DNA into first segments at first DNA sequence locations; joining said first segments; and dividing said joined segments into other segments at DNA sequence locations different from said first locations.
Claim 24
Dependent Claim
23 words
DNA having enhanced recombinant properties, comprising a circular molecule of DNA containing a substantially palindromic DNA segment at least 25 base pairs long.
Claim 25
Dependent Claim
23 words
DNA having enhanced recombinant properties, comprising a circular molecule of DNA containing a substantially palindromic DNA segment at least 25 base pairs long.
Claim 26
Dependent Claim
12 words
The DNA of claim 17 in which said palindromic sequence is eucaryotic.
Claim 27
Independent Claim
30 words
A method for causing a phenotypic change in eucaryotic cells, comprising inserting into said eucaryotic cells recombinant DNA prepared in accordance with the method of any one of claims 1-13.
Claim 28
Independent Claim
25 words
A method for causing a phenotypic change in eucaryotic cells, comprising inserting into said eucaryotic cells the recombinant DNA of any one of claims 14-18.
References Cited (Backward Citations)
No citation records indexed.
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