US4762786A
Expired / Lapsed
US
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Vectors and conditions which allow genetic transformation of cephalosporium
Assignee: Eli Lilly and Co
•
Filing: 1984-09-27
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Granted: 1988-08-09
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Expires: 2004-09-27
Abstract
Abstract A method for transforming Cephalosporium and other lower eukaryotes is disclosed. The method involves inserting a recombinant DNA cloning vector comprising a Saccharomyces cerevisiae transcriptional and translational activating sequence positioned for expression of hygromycin phosphotransferase into a host cell and then growing the host cell under selective conditions. The vectors optionally further comprise Cephalosporium ribosomal DNA and also sequences that allow for replication and selection in E. coli and Streptomyces.
Inventors
- 👤 Jerry L. Chapman, Jr.
- 👤 Thomas D. Ingolia
- 👤 Kevin R. Kaster
- 👤 Stephen W. Queener
- 👤 Paul L. Skatrud
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 15:38
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/10; C12N9/12; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/80; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/886; Y10S435/911; Y10S435/925; Y10S435/926
Claims Analysis
Total Claims: 48
Claim 1
Independent Claim
95 words
A method for transforming a Cephalosporium host cell which comprises: (1) introducing a recombinant DNA cloning vector into a Cephalosporium host cell, said vector comprising (a) a Saccharomyces cerevisiae transcriptional and translational activating sequence positioned for expression of a sequence that codes for hygromycin B phosphotransferase and (b) a sequence that codes for hygromycin B phosphotransferase, subject to the limitation that the sequence of (a) is positioned for the expression of the sequence of (b), and (2) growing said host cell under selective conditions suitable for maintaining DNA comprising said vector in said host cell.
Claim 2
Independent Claim
95 words
A method for transforming a Cephalosporium host cell which comprises: (1) introducing a recombinant DNA cloning vector into a Cephalosporium host cell, said vector comprising (a) a Saccharomyces cerevisiae transcriptional and translational activating sequence positioned for expression of a sequence that codes for hygromycin B phosphotransferase and (b) a sequence that codes for hygromycin B phosphotransferase, subject to the limitation that the sequence of (a) is positioned for the expression of the sequence of (b), and (2) growing said host cell under selective conditions suitable for maintaining DNA comprising said vector in said host cell.
Claim 3
Dependent Claim
14 words
The method of claim 1 wherein the recombinant DNA cloning vector is a plasmid.
Claim 4
Dependent Claim
16 words
The method of claim 2 wherein the plasmid integrates into the genome of said host cell.
Claim 5
Dependent Claim
28 words
The method of claim 2 wherein the plasmid further comprises an E. coli origin of replication and a sequence that codes for a selectable phenotype in E. coli.
Claim 6
Dependent Claim
15 words
The method of claim 2 wherein the plasmid further comprises a Cephalosporium ribosomal DNA sequence.
Claim 7
Dependent Claim
30 words
The method of claim 2 wherein the plasmid further comprises a transcriptional and translational activating sequence positioned for expression of a sequence that codes for a functional polypeptide in Cephalosporium.
Claim 8
Dependent Claim
15 words
The method of claim 4 wherein the plasmid further comprises a Cephalosporium ribosomal DNA sequence.
Claim 9
Dependent Claim
26 words
The method of claim 4 wherein the plasmid further comprises a Streptomyces origin of replication and a sequence that codes for a selectable phenotype in Streptomyces.
Claim 10
Dependent Claim
15 words
The method of claim 8 wherein the plasmid further comprises a Cephalosporium ribosomal DNA sequence.
Claim 11
Dependent Claim
30 words
The method of claim 9 wherein the plasmid further comprises a transcriptional and translational activating sequence positioned for expression of a sequence that codes for a functional polypeptide in Cephalosporium.
Claim 12
Dependent Claim
24 words
The method of claim 2 wherein the Saccharomyces cerevisiae transcriptional and translational activating sequence is the phosphoglycerate kinase gene transcriptional and translational activating sequence.
Claim 13
Dependent Claim
127 words
The method of claim 2 wherein the sequence that codes for hygromycin B phosphotransferase is ##STR8## ##STR9## ##STR10## ##STR11## ##STR12## ##STR13## ##STR14## ##STR15## ##STR16## ##STR17## wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytidyl, T is thymidyl, R and R 2 are deoxyribonucleotide triplets that independently encode lysine, R 1 and R 3 are deoxyribonucleotide triplets wherein the nitrogenous bases are complementary to the respective and corresponding bases of R and R 2 , m and n=0 or 1, subject to the limitation that when n=0, then m=0 and when m=1, then n=1, R 4 is a deoxyribonucleotide triplet that encodes a translational stop codon and R 5 is a deoxyribonucleotide triplet wherein the nitrogenous bases are complementary to the corresponding bases of R 4 .
Claim 14
Dependent Claim
24 words
The method of claim 12 wherein the Saccharomyces cerevisiae transcriptional and translational activating sequence is the phosphoglycerate kinase gene transcriptional and translational activating sequence.
Claim 15
Dependent Claim
28 words
The method of claim 13 wherein the plasmid further comprises an E. coli origin of replication and a sequence that codes for a selectable phenotype in E. coli.
Claim 16
Dependent Claim
15 words
The method of claim 13 wherein the plasmid further comprises a Cephalosporium ribosomal DNA sequence.
Claim 17
Dependent Claim
26 words
The method of claim 13 wherein the plasmid further comprises a Streptomyces origin of replication and a sequence that codes for a selectable phenotype in Streptomyces.
Claim 18
Dependent Claim
15 words
The method of claim 16 wherein the plasmid further comprises a Cephalosporium ribosomal DNA sequence.
Claim 19
Dependent Claim
30 words
The method of claim 17 wherein the plasmid further comprises a transcriptional and translational activating sequence positioned for expression of a sequence that codes for a functional polypeptide in Cephalosporium.
Claim 20
Dependent Claim
19 words
The method of claim 1 wherein said host cells are grown on a growth medium containing antibiotic hygromycin B.
Claim 21
Dependent Claim
23 words
The method of claim 19 wherein the concentration of hygromycin B is within the range of about 8 μg/ml to about 5 mg/ml.
Claim 22
Dependent Claim
13 words
The method of claim 20 wherein the hygromycin B concentration is 100 μg/ml.
Claim 23
Dependent Claim
14 words
The method of claim 19 wherein the recombinant DNA cloning vector is plasmid pIT221.
Claim 24
Dependent Claim
14 words
The method of claim 19 wherein the recombinant DNA cloning vector is plasmid pPS6.
Claim 25
Dependent Claim
14 words
The method of claim 19 wherein the recombinant DNA cloning vector is plasmid pETS702.
Claim 26
Dependent Claim
14 words
The method of claim 19 wherein the recombinant DNA cloning vector is plasmid pPLS221.
Claim 27
Dependent Claim
13 words
The method of claim 1 wherein said Cephalosporium host cell is C. acremonium.
Claim 28
Dependent Claim
12 words
The method of claim 1 wherein the host cell is Cephalosporium acremonium/pIT221.
Claim 29
Dependent Claim
12 words
The method of claim 19 wherein the host cell is Cephalosporium acremonium/pIT221.
Claim 30
Dependent Claim
12 words
The method of claim 1 wherein the host cell is Cephalosporium acremonium/pPS6.
Claim 31
Dependent Claim
9 words
A recombinant DNA cloning vector which is plasmid pPS5.
Claim 32
Dependent Claim
9 words
A recombinant DNA cloning vector which is plasmid pPS5.
Claim 33
Dependent Claim
17 words
A recombinant DNA cloning vector which is selected from the group consisting of plasmids pIT221 and pPS6.
Claim 34
Dependent Claim
17 words
A recombinant DNA cloning vector which is selected from the group consisting of plasmids pIT221 and pPS6.
Claim 35
Dependent Claim
16 words
A recombinant DNA cloning vector selected from the group consisting of plasmids pIT220, pETS702 and pPLS221.
Claim 36
Dependent Claim
16 words
A recombinant DNA cloning vector selected from the group consisting of plasmids pIT220, pETS702 and pPLS221.
Claim 37
Dependent Claim
12 words
A transformed Cephalosporium host cell employed in the method of claim 1.
Claim 38
Dependent Claim
10 words
The host cell of claim 33 which is Cephalosporium acremonium.
Claim 39
Dependent Claim
10 words
The host cell of claim 34 which is Cephalosporium acremonium/pPS5.
Claim 40
Dependent Claim
19 words
The host cell of claim 34 which is selected from the group consisting of Cephalosporium acremonium/pIT221 and Cephalosporium acremonium/pPS6.
Claim 41
Dependent Claim
36 words
A host cell selected from the group consisting of Streptomyces lividans/pETS702, Streptomyces lividans/pPLS221, E. coli K12/pETS702, E. coli K12/pPLS221, E. coli K12 RR1/pIT221, E. coli K12 RR1/pPSI, E. coli K-12 RR1/pPS5 and E. coli K12 RR1/pPS6.
Claim 42
Dependent Claim
36 words
A host cell selected from the group consisting of Streptomyces lividans/pETS702, Streptomyces lividans/pPLS221, E. coli K12/pETS702, E. coli K12/pPLS221, E. coli K12 RR1/pIT221, E. coli K12 RR1/pPSI, E. coli K-12 RR1/pPS5 and E. coli K12 RR1/pPS6.
Claim 43
Dependent Claim
15 words
A Streptomyces host cell transformed with a plasmid employed in the method of claim 8.
Claim 44
Dependent Claim
58 words
A method for transforming a lower eukaryotic host cell which comprises introducing a recombinant DNA cloning vector into a lower eukaryotic host cell exclusive of yeast and growing said host cell under selective conditions suitable for maintaining DNA comprising said vector in said host cell wherein said vector is a vector employed in the method of claim 1.
Claim 45
Dependent Claim
11 words
The method of claim 39 wherein the host cell is Penicillium.
Claim 46
Dependent Claim
20 words
The method of claim 40 wherein the Penicillium is selected from the group consisting of P. chrysogenum and P. notatum.
Claim 47
Dependent Claim
19 words
The method of claim 39 wherein the host cell is selected from the group consisting of Aspergillus and Paecilomyces.
Claim 48
Independent Claim
25 words
The method of claim I wherein the host cell is selected from the group consisting of Cephalosporium salmyosynnematum, C. chrysogenum, C. purpurascens and C. curtipis.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-08-09 | 1992-02-08 | 1992-08-09 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-08-09 | 1996-02-08 | 1996-08-09 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-08-09 | 2000-02-08 | 2000-08-09 | $7700.00 | Window Closed / Lapsed |