Patent US5436141A successfully refreshed from Google Patents.
US5436141A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method for synthesizing stable single-stranded CDNA in eukaryotes by means of a bacterial retron and products
Assignee: Rutgers Health
•
Filing: 1991-08-30
•
Granted: 1995-07-25
•
Expires: 2011-08-30
Abstract
Abstract A method for producing in vivo stable single-stranded DNAs in eucaryotic cells. The DNAs are multicopy single-stranded DNA (msDNA) structures constituted by a RNA and a DNA portion. The group of genes (retrons) producing said coupled RNA and DNA portions of the msDNAs and the gene encoding reverse transcriptase (RT). The transformed eucaryotes harboring these retrons. The new msDNAs which are encoded by the new retrons. The msDNAs can be used as vectors for antisense DNA and for amplification of inserted genes.
Inventors
- 👤 Shohei Miyata
- 👤 Atsushi Ohshima
- 👤 Sumiko Inouye
- 👤 Masayori Inouye
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-08 05:17
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/113; C12N15/10; C12N15/63; C12N15/79; C12N15/80; C12N15/81; C12N9/00; C12N9/10; C12N9/12; C12N9/1241; C12N9/1276; C12P; C12P19/00; C12P19/26; C12P19/28; C12P19/30; C12P19/34; C12N2310/00; C12N2310/10; C12N2310/11; C12N2310/111
Claims Analysis
Total Claims: 52
Claim 1
Independent Claim
60 words
A method for expressing an msDNA in a yeast host cell which comprises transfecting the cell with a DNA expression vector which replicates in the cell, which vector contains a retron encoding the msDNA, which retron contains msr and msd coding regions (msr-msd) for the msDNA and a gene encoding a reverse transcriptase and expressing the msDNA from the retron.
Claim 2
Independent Claim
60 words
A method for expressing an msDNA in a yeast host cell which comprises transfecting the cell with a DNA expression vector which replicates in the cell, which vector contains a retron encoding the msDNA, which retron contains msr and msd coding regions (msr-msd) for the msDNA and a gene encoding a reverse transcriptase and expressing the msDNA from the retron.
Claim 3
Dependent Claim
12 words
The method of claim 1 wherein the expression vector is a plasmid.
Claim 4
Dependent Claim
16 words
The method of claim 1 wherein the reverse transcriptase gene is downstream of the msr-msd region.
Claim 5
Dependent Claim
49 words
The method of claim 3, wherein the expression vector contains a promoter located upstream of msr, wherein a non-coding DNA fragment of the 5' end upstream of msr, which fragment starts from the promoter and ends immediately upstream of msr, and which contains several AUG codons, has been deleted.
Claim 6
Dependent Claim
16 words
The method of claim 1 wherein the reverse transcriptase gene is upstream of the msr-msd region.
Claim 7
Dependent Claim
16 words
The method of claim 5 wherein a foreign DNA fragment is contained in the msd region.
Claim 8
Dependent Claim
17 words
The method of claim 1 wherein the expression vector contains a promoter located upstream of the msr.
Claim 9
Dependent Claim
13 words
The method of claim 7 wherein the promoter is a foreign strong promoter.
Claim 10
Dependent Claim
12 words
The method of claim 1 wherein the retron is in vector YEp521-M1.
Claim 11
Dependent Claim
12 words
The method of claim 1 wherein the retron is in vector YEp521-M3.
Claim 12
Dependent Claim
12 words
The method of claim 1 wherein the retron is in vector YEp521-M4.
Claim 13
Dependent Claim
12 words
The method of claim 1 wherein the retron is in vector YEp521-M5.
Claim 14
Independent Claim
60 words
A method for expressing an msDNA in a saccharomyces host cell which comprises transfecting the cell with a DNA expression vector which replicates in the cell, which vector contains a retron encoding the msDNA, which retron contains msr and msd coding regions (msr-msd) for the msDNA and a gene encoding a reverse transcriptase and expressing the msDNA from the retron.
Claim 15
Independent Claim
60 words
A method for expressing an msDNA in a saccharomyces host cell which comprises transfecting the cell with a DNA expression vector which replicates in the cell, which vector contains a retron encoding the msDNA, which retron contains msr and msd coding regions (msr-msd) for the msDNA and a gene encoding a reverse transcriptase and expressing the msDNA from the retron.
Claim 16
Dependent Claim
13 words
The method of claim 13 wherein the msd region contains a cloning site.
Claim 17
Dependent Claim
100 words
The method of claim 13 wherein the msDNA molecule comprises a branched single-stranded RNA which is covalently linked to a single-stranded DNA by a 2',5'-phosphodiester bond between the 2'-OH group of an internal rG residue and the 5'-phosphate of the DNA molecule, which RNA is non-covalently linked to the DNA by base-pairing between the complementary 3' ends of the RNA and DNA molecules, which RNA-DNA form stable stem-loop secondary structures, which msDNA is encoded by a primary RNA transcript, pre-msDNA which contains an open-reading frame (ORF) downstream of the msr locus, the ORF encoding a polypeptide having reverse transcriptase activity.
Claim 18
Dependent Claim
11 words
The method of claim 13 wherein the msDNA produced is msDNA-Ec67.
Claim 19
Dependent Claim
25 words
The method of claim 13 wherein the msDNA which is expressed is selected from the group consisting of msDNA-Mx162,msDNA-Mx65 msDNA-Ec107, msDNA - Mx86 and Sa163.
Claim 20
Dependent Claim
17 words
The method of claim 13 wherein the gene encoding reverse transciptase located upstream of the msr region.
Claim 21
Dependent Claim
11 words
The method of claim 18 wherein the msDNA produced is msDNA-Ec67.
Claim 22
Dependent Claim
38 words
A mammalian cell transfected with a DNA expression vector for replication, which vector contains a retron for msDNA synthesis, which retron contains msr and msd coding regions (msr-msd) of the msDNA and a gene encoding a reverse transcriptase.
Claim 23
Dependent Claim
38 words
A mammalian cell transfected with a DNA expression vector for replication, which vector contains a retron for msDNA synthesis, which retron contains msr and msd coding regions (msr-msd) of the msDNA and a gene encoding a reverse transcriptase.
Claim 24
Dependent Claim
39 words
A yeast host cell transfected with a DNA expression vector for replication, which vector contains a retron for msDNA synthesis, which retrons contains msr and msd coding regions (msr-msd) of the msDNA and a gene encoding a reverse transcriptase.
Claim 25
Dependent Claim
39 words
A yeast host cell transfected with a DNA expression vector for replication, which vector contains a retron for msDNA synthesis, which retrons contains msr and msd coding regions (msr-msd) of the msDNA and a gene encoding a reverse transcriptase.
Claim 26
Dependent Claim
39 words
A saccharomyces host cell transfected with a DNA expression vector for replication, which vector contains a retron for msDNA synthesis, which retron contains msr and msd coding regions (msr-msd) of the msDNA and a gene encoding a reverse transcriptase.
Claim 27
Dependent Claim
39 words
A saccharomyces host cell transfected with a DNA expression vector for replication, which vector contains a retron for msDNA synthesis, which retron contains msr and msd coding regions (msr-msd) of the msDNA and a gene encoding a reverse transcriptase.
Claim 28
Dependent Claim
18 words
The saccharomyces host cell of claim 22 wherein the reverse transcriptase gene is downstream of the msr-msd region.
Claim 29
Dependent Claim
111 words
The saccharomyces host cell of claim 22, wherein the expression vector contains a promoter located upstream of msr, wherein a non-coding DNA fragment of the 5' end upstream of msr, which fragment starts from the promoter and ends immediately upstream of msr, where the gene encoding the reverse transcriptase is downstream of the msr region, or which fragment starts from the promoter and ends immediately upstream of the gene encoding the reverse transcriptase, where the gene encoding the reverse transcriptase is upstream of the msr region, and which contains several AUG codons, has been deleted but for the initiation codon AUG of the reverse transcriptase gene closest to the 5' end.
Claim 30
Dependent Claim
18 words
The saccharomyces host cell of claim 24 wherein the reverse transcriptase gene is upstream of the msr-msd region.
Claim 31
Dependent Claim
16 words
The saccharomyces host cell of claim 25 wherein the msd region contains a foreign DNA fragment.
Claim 32
Dependent Claim
14 words
The saccharomyces host cell of claim 26 wherein the DNA fragment is 50-bp long.
Claim 33
Dependent Claim
29 words
The saccharomyces host cell of claim 22 wherein the expression vector contains the promoter for the msr-msd region and reverse transcriptase gene, which promoter is located upstream of msr.
Claim 34
Dependent Claim
15 words
The saccharomyces host cell of claim 28 wherein the promoter is a foreign strong promoter.
Claim 35
Dependent Claim
59 words
A DNA expression vector for replication in a yeast host which comprises a retron which encodes a hybrid single- stranded RNA- DNA (msDNA) structure, which retron contains a gene encoding a reverse transcriptase and one coding region which contains two coding sequences, one msr and the other msd for encoding, respectively, the RNA and DNA portions of the msDNA.
Claim 36
Dependent Claim
59 words
A DNA expression vector for replication in a yeast host which comprises a retron which encodes a hybrid single- stranded RNA- DNA (msDNA) structure, which retron contains a gene encoding a reverse transcriptase and one coding region which contains two coding sequences, one msr and the other msd for encoding, respectively, the RNA and DNA portions of the msDNA.
Claim 37
Dependent Claim
57 words
A DNA expression vector for replication in a saccharomyces host which comprises a retron which encodes a hybrid singe-stranded RNA-DNA (msDNA) structure, which retron contains a gene encoding a reverse transcriptase and one coding region which contains two coding sequences, one msr and the other msd for encoding, respectively, the RNA and DNA portions of the msDNA.
Claim 38
Dependent Claim
57 words
A DNA expression vector for replication in a saccharomyces host which comprises a retron which encodes a hybrid singe-stranded RNA-DNA (msDNA) structure, which retron contains a gene encoding a reverse transcriptase and one coding region which contains two coding sequences, one msr and the other msd for encoding, respectively, the RNA and DNA portions of the msDNA.
Claim 39
Dependent Claim
18 words
The DNA expression vector of claim 31 wherein the reverse transcriptase gene is downstream of the msr-msd region.
Claim 40
Dependent Claim
48 words
The vector of claim 32, wherein the expression vector contains a promoter located upstream of msr, wherein a non-coding fragment of the 5' end upstream of msr, which fragment starts from the promoter and ends immediately upstream of msr, and which contains several AUG codons has been deleted.
Claim 41
Dependent Claim
8 words
The vector of claim 32 which is YEp521-M1.
Claim 42
Dependent Claim
16 words
The vector of claim 31 wherein the reverse transcriptase gene is upstream of the msr-msd region.
Claim 43
Dependent Claim
8 words
The vector of claim 35 which is YEp521-M4.
Claim 44
Dependent Claim
25 words
The vector of claim 31 which includes a promoter for the msr-msd region and the reverse transcriptase gene, which promoter is located upstream of msr.
Claim 45
Dependent Claim
13 words
The vector of claim 37 wherein the promoter is a foreign strong promoter.
Claim 46
Dependent Claim
10 words
The vector of claim 38 wherein the promoter is GAL10.
Claim 47
Dependent Claim
8 words
The vector of claim 37 which is YEp521-M3.
Claim 48
Dependent Claim
27 words
The vector of claim 31 in which a foreign DNA fragment is contained in the msd region of the retron which encodes the DNA of the msDNA.
Claim 49
Dependent Claim
11 words
The vector of claim 41 wherein the fragment is 50-bp long.
Claim 50
Dependent Claim
8 words
The vector of claim 41 which is YEp521-M5.
Claim 51
Dependent Claim
18 words
The DNA expression vector of claim 31 wherein the msr or the msd contains a foreign DNA fragment.
Claim 52
Dependent Claim
15 words
The DNA expression vector of claim 44 wherein the msr contains the foreign DNA fragment.
References Cited (Backward Citations)
EP0132309A2
Cited
US5079151A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1998-07-24 | 1999-01-23 | 1999-07-25 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2002-07-24 | 2003-01-23 | 2003-07-25 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2006-07-24 | 2007-01-23 | 2007-07-25 | $7700.00 | Window Closed / Lapsed |