US4761367A
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Vectors suitable for detection of eukaryotic DNA regulatory sequences
Assignee: University of North Carolina at Chapel Hill
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Filing: 1984-11-07
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Granted: 1988-08-02
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Expires: 2004-11-07
Abstract
Abstract The present invention relates to vectors useful for identifying eukaryotic regulatory sequences and to a method for identifying these sequences. The vectors comprise a first nucleotide sequence which allows for replication in a eukaryotic host, a second nucleotide sequence which codes for a first product that controls replication at said first sequence, and a third nucleotide sequence which codes for a second product that is detectable. The method comprises inserting putative eukaryotic regulatory sequences at a position on the 5'-side of the second sequence and measuring the production of the second product.
Inventors
- 👤 Marshall H. Edgell
- 👤 Steven K. Nordeen
- 👤 Clyde A. Hutchinson, III
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 16:49
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/67; C12N15/69; C12N15/79; C12N15/85; C12Q; C12Q1/00; C12Q1/68; C12Q1/6897
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
247 words
A method for identifying eukaryotic regulatory sequences which comprises: (a) inserting putative eukaryotic regulatory sequences into a vector which comprises: (i) a first nucleotide sequence which provides for replication of the vector in a eukaryotic host and is the SV40 origin of replication; (ii) a second nucleotide sequence which codes for a first product which controls replication at said first sequence, said second sequence lacks the native promoter and the expression of which is not under the control of said promoter and said first product is SV40 large T-antigen; (iii) a cloning site on the 5'-side of said second sequence suitable for the insertion of a putative eukaryotic regulatory sequence; and (iv) a third nucleotide sequence which codes for a second product which is different from said first product and is detectable in a positive assay, said positive assay detecting a product directly as a result of said product's presence and not indirectly as a result of a growth requirement or survivability, whereby the insertion of a eukaryotic regulatory sequence results in the amplification of the vector; (b) transforming a first eukaroytic host with the vector of step (a); (c) transforming a second eukaryotic host with said vector lacking the putative eukaryotic regulatory sequences; and (d) measuring the amount of said second product produced in said first and second hosts and comparing the amounts of said second product produced, whereby an increase in the amount in said first host indicates that said sequence is a regulatory sequence.
Claim 2
Independent Claim
247 words
A method for identifying eukaryotic regulatory sequences which comprises: (a) inserting putative eukaryotic regulatory sequences into a vector which comprises: (i) a first nucleotide sequence which provides for replication of the vector in a eukaryotic host and is the SV40 origin of replication; (ii) a second nucleotide sequence which codes for a first product which controls replication at said first sequence, said second sequence lacks the native promoter and the expression of which is not under the control of said promoter and said first product is SV40 large T-antigen; (iii) a cloning site on the 5'-side of said second sequence suitable for the insertion of a putative eukaryotic regulatory sequence; and (iv) a third nucleotide sequence which codes for a second product which is different from said first product and is detectable in a positive assay, said positive assay detecting a product directly as a result of said product's presence and not indirectly as a result of a growth requirement or survivability, whereby the insertion of a eukaryotic regulatory sequence results in the amplification of the vector; (b) transforming a first eukaroytic host with the vector of step (a); (c) transforming a second eukaryotic host with said vector lacking the putative eukaryotic regulatory sequences; and (d) measuring the amount of said second product produced in said first and second hosts and comparing the amounts of said second product produced, whereby an increase in the amount in said first host indicates that said sequence is a regulatory sequence.
Claim 3
Dependent Claim
36 words
The method of claim 1 wherein said second product is selected from the group consisting of an antigen, an immunoglobulin, an enzyme, a transport protein, a structural protein and a protein expressed at the cell surface.
Claim 4
Dependent Claim
12 words
The method of claim 2 wherein said second product is hemagglutinin antigen.
Claim 5
Dependent Claim
35 words
The method of claim 1 wherein said vector further comprises a fourth nucleotide sequence containing a weak promoter to insure low basal transcription of said first product inserted on the 5'-side of said second sequence.
Claim 6
Dependent Claim
23 words
The method of claim 4 wherein said second product is hemagglutinin antigen and said weak promoter is the mouse mammary tumor virus promoter.
Claim 7
Dependent Claim
167 words
A vector suitable for detecting eukaryotic regulatory sequences which comprises: (a) a first nucleotide sequence which provides for replication of the vector in a eukaryotic host and is the SV40 origin of replication; (b) a second nucleotide sequence which codes for a first product which controls replication at said first sequence, said second sequence lacks the native promoter and the expression of which is not under the control of said promoter and said first product is SV40 large T-antigen; (c) a cloning site on the 5'-side of said second sequence suitable for the insertion of a putative eukaryotic regulatory sequence; and (d) a third nucleotide sequence which codes for a second product which is different from said first product and is detectable in a positive assay, said positive assay detecting a product directly as a result of said product's presence and not indirectly as a result of a growth requirement or survivability, whereby the insertion of a eukaryotic regulatory sequence results in the amplification of the vector.
Claim 8
Dependent Claim
167 words
A vector suitable for detecting eukaryotic regulatory sequences which comprises: (a) a first nucleotide sequence which provides for replication of the vector in a eukaryotic host and is the SV40 origin of replication; (b) a second nucleotide sequence which codes for a first product which controls replication at said first sequence, said second sequence lacks the native promoter and the expression of which is not under the control of said promoter and said first product is SV40 large T-antigen; (c) a cloning site on the 5'-side of said second sequence suitable for the insertion of a putative eukaryotic regulatory sequence; and (d) a third nucleotide sequence which codes for a second product which is different from said first product and is detectable in a positive assay, said positive assay detecting a product directly as a result of said product's presence and not indirectly as a result of a growth requirement or survivability, whereby the insertion of a eukaryotic regulatory sequence results in the amplification of the vector.
Claim 9
Dependent Claim
11 words
A eukaryotic host cell transformed by the vector of claim 6.
Claim 10
Dependent Claim
36 words
The vector of claim 6 wherein said second product is selected from the group consisting of an antigen, an immunoglobulin, an enzyme, a transport protein, a structural protein and a protein expressed at the cell surface.
Claim 11
Dependent Claim
33 words
The vector of claim 6 which further comprises a fourth nucleotide sequence containing a weak promoter to insure low basal transcription of said first product inserted on the 5'-side of said second sequence.
Claim 12
Dependent Claim
11 words
A eukaryotic host cell transformed by the vector of claim 9.
Claim 13
Dependent Claim
197 words
A eukaryotic vector suitable for detecting eukaryotic regulatory sequences which comprises: (a) a first nucleotide sequence which provides for replication of the vector in a eukaryotic host and is the SV40 origin of replication; (b) a second nucleotide sequence which codes for a first product which controls replication at said first sequence, said second sequence lacks the native promoter and the expression of which is not under the control of said promoter and said first product is SV40 large T-antigen; (c) a cloning site on the 5'-side of said second sequence suitable for the insertion of a putative eukaryotic regulatory sequence, and (d) a third nucleotide sequence which codes for a second product which is different from said first product and is detectable in a positive assay, said positive assay detecting a product directly as a result of said product's presence and not indirectly as a result of a growth requirement or survivability, whereby the insertion of a eukaryotic regulatory sequence results in the amplification of the vector, said second product selected from the group consisting of an antigen, an immunoglobulin, an enzyme, a transport protein, a structural protein and a protein expressed at the cell surface.
Claim 14
Dependent Claim
197 words
A eukaryotic vector suitable for detecting eukaryotic regulatory sequences which comprises: (a) a first nucleotide sequence which provides for replication of the vector in a eukaryotic host and is the SV40 origin of replication; (b) a second nucleotide sequence which codes for a first product which controls replication at said first sequence, said second sequence lacks the native promoter and the expression of which is not under the control of said promoter and said first product is SV40 large T-antigen; (c) a cloning site on the 5'-side of said second sequence suitable for the insertion of a putative eukaryotic regulatory sequence, and (d) a third nucleotide sequence which codes for a second product which is different from said first product and is detectable in a positive assay, said positive assay detecting a product directly as a result of said product's presence and not indirectly as a result of a growth requirement or survivability, whereby the insertion of a eukaryotic regulatory sequence results in the amplification of the vector, said second product selected from the group consisting of an antigen, an immunoglobulin, an enzyme, a transport protein, a structural protein and a protein expressed at the cell surface.
Claim 15
Dependent Claim
11 words
A eukaryotic host cell transformed by the vector of claim 11.
Claim 16
Dependent Claim
12 words
The vector of claim 10 wherein said second product is hemagglutinin antigen.
Claim 17
Dependent Claim
8 words
The vector of claim 13 identified as pEN-1.
Claim 18
Dependent Claim
11 words
A eukaryotic host cell transformed by the vector of claim 13.
Claim 19
Dependent Claim
33 words
The vector of claim 11 which further comprises a fourth nucleotide sequence containing a weak promoter to insure low basal transcription of said first product inserted on the 5'-side of said second sequence.
Claim 20
Dependent Claim
23 words
The vector of claim 11 wherein said second product is hemagglutinin antigen and said weak promoter is the mouse mammary tumor virus promoter.
Claim 21
Dependent Claim
11 words
A eukaryotic host cell transformed by the vector of claim 16.
Claim 22
Dependent Claim
8 words
The vector of claim 17 identified as pEN-2.
Claim 23
Dependent Claim
11 words
A eukaryotic host cell transformed by the vector of claim 17.
References Cited (Backward Citations)
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1991-08-02 | 1992-02-01 | 1992-08-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-08-02 | 1996-02-01 | 1996-08-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-08-02 | 2000-02-01 | 2000-08-02 | $7700.00 | Window Closed / Lapsed |