EP0154186A2
Expired / Lapsed
EP
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Isolation of a novel interferon gene and expression thereof
Assignee: Exegenics Inc
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Filing: 1985-02-07
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Granted: —
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Expires: 2005-02-07
Abstract
Abstract A novel mature human alpha-interferon gene is isolated from a human genomic library, said gene is sequenced, Inserted into a recombinant expression vector by recombinant DNA techniques and is thereafter used to transform bacteria which then express the gene as determined by the presence of interferon activity. Novel methods and compositions which allow for the rapid screening and isolation of mammalian genes including low copy genes from genomic libraries employing short probes are also disclosed. The method of the present invention comprises synthesizing at least one DNA probe about 17 nucleotides in length and comprising a DNA sequence homologous to at least one DNA sequence contained within said gene, labeling the DNA probe and thereafter hybridizing the labeled DNA probe to said genomic library employing an improved method for hybridizing labeled DNA probes to recombinant vectors contained within a host cell and affixed to nitrocellulose filters, the improvement comprising filtering the labeled DNA probes prior to its addition to a hybridization solution, adding the labeled DNA probes to the hybridization solution, employing a device to simultaneously rotate and incubate filters during prehybridization and hybridization, and adding 10 percent dextran sulfate to prehybridization and hybridization solutions and thereafter identifying said gene in a manner consistent with the genomic library and label chosen. Two synthetic 17-mer DNA probes with the base sequence of
Inventors
- 👤 Arthur Peter Bollon
- 👤 Motohiro Fuke
- 👤 Richard Michael Torczynski
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 22:41
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/555; C07K14/56; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6832; C12Q1/6876
Claims Analysis
Total Claims: 61
Claim 1
Independent Claim
98 words
A method for identifying a gene, gene fragment or allele contained within a genomic or cDNA library, said method comprising: (a) synthesizing at least one unique sequence DNA probe comprising a DNA sequence homologous to at least one DNA sequence contained within said gene, gene fragment or allele thereof and of an effective size to stably hybridize to said gene, gene fragment or allele, (b) labeling said DNA probe, (c) hybridizing said labeled DNA probe to said genomic or cDNA library and (d) thereafter identifying said gene or gene fragment in a manner consistent with the label chosen.
Claim 2
Independent Claim
98 words
A method for identifying a gene, gene fragment or allele contained within a genomic or cDNA library, said method comprising: (a) synthesizing at least one unique sequence DNA probe comprising a DNA sequence homologous to at least one DNA sequence contained within said gene, gene fragment or allele thereof and of an effective size to stably hybridize to said gene, gene fragment or allele, (b) labeling said DNA probe, (c) hybridizing said labeled DNA probe to said genomic or cDNA library and (d) thereafter identifying said gene or gene fragment in a manner consistent with the label chosen.
Claim 3
Dependent Claim
13 words
The method of Claim 1, wherein said DNA probe is labeled by radiolabeling.
Claim 4
Independent Claim
17 words
The method of Claims 1 or 2, wherein said genomic or cDNA library comprises human chromosomal DNA.
Claim 5
Independent Claim
21 words
The method of Claims 1 to 3, wherein said genomic or cDNA library is contained within a bacteriophage or plasmid vector.
Claim 6
Independent Claim
18 words
The method of Claims 1 to 4, wherein said gene, gene fragment or allele codes for an interferon.
Claim 7
Independent Claim
19 words
The method of Claims 1 to 5, wherein said gene, gene fragment or allele codes for an human interferon.
Claim 8
Dependent Claim
17 words
The method of Claim 6, wherein said gene, gene fragment or allele codes for a human alpha-interferon.
Claim 9
Dependent Claim
14 words
The method of Claim 1 wherein said DNA probe is enzymatically or chemically synthesized.
Claim 10
Dependent Claim
16 words
The DNA probe of Claim 1, wherein said DNA probe is about seventeen nucleotides in length.
Claim 11
Dependent Claim
19 words
The method of Claim 9, wherein said DNA probe contains a DNA base sequence rich in guanine and cytosine.
Claim 12
Dependent Claim
17 words
The method of Claim 9, wherein said DNA probe comprises a 17-mer with a base sequence of:
Claim 13
Dependent Claim
17 words
The method of Claim 9, wherein said DNA probe comprises a 17-mer with a base sequence of:
Claim 14
Dependent Claim
122 words
The method of Claim 1, wherein said labeled DNA probes are hybridized to said genomic or cDNA library employing an improved method for hybridizing labeled DNA probes to recombinant vectors contained within a host cell and affixed to nitrocellulose filters, the improvement comprising: (a) separating the labeled DNA probe from particulate matter having a solution diameter greater than said labeled DNA probe prior to the addition of said labeled DNA probe to a hybridization solution, (b) adding said labeled DNA probe to the hybridization solution containing an effective amount of dextran sulfate capable of increasing the concentration of the DNA probe in the hybridization solution, and (c) employing a device to simultaneously rotate and incubate the nitrocellulose filters during prehybridization and hybridization.
Claim 15
Dependent Claim
20 words
The method of Claim 13, wherein said device is capable of rotating said nitrocellulose filters 360° in a continuous fashion.
Claim 16
Dependent Claim
38 words
The method of Claim 13, wherein said labeled DNA probe is separated from particulate matter by passing said labeled DNA probe through an inert filter means providing a means for retaining particles in excess of about 0.2 micrometers.
Claim 17
Dependent Claim
17 words
The method fo Claim 13, wherein said effective amount of dextran sulfate is about 10 percent (w/v).
Claim 18
Dependent Claim
30 words
The method of Claim 13, wherein said prehybridization and/or hybridization solution comprises 0.9M NaCl, 6.0mM Na 2 EDTA, 0.9M Tris-HC1, pH 7.5, 0.1% (w/v) SDS and 10% (w/v) dextran sulfate.
Claim 19
Dependent Claim
33 words
The method of Claim 13, wherein said hybridization solution comprises 0.9M NaCl, 6.0mM Na 2 EDTA, 0.09M Tris-Hcl, pH 7.5, 0.1% (w/v) SDS, 10% (w/v) dextran sulfate and said filtered, labeled DNA probe.
Claim 20
Dependent Claim
8 words
A seventeen base DNA fragment of the sequence:
Claim 21
Dependent Claim
8 words
A seventeen base DNA fragment of the sequence:
Claim 22
Dependent Claim
8 words
A seventeen base DNA fragment of the sequence:
Claim 23
Dependent Claim
8 words
A seventeen base DNA fragment of the sequence:
Claim 24
Dependent Claim
21 words
Human alpha-interferon WA gene, the coding strand comprising the base sequence shown in FIG. 4, a gene fragment or allele thereof.
Claim 25
Dependent Claim
21 words
Human alpha-interferon WA gene, the coding strand comprising the base sequence shown in FIG. 4, a gene fragment or allele thereof.
Claim 26
Dependent Claim
20 words
Human alpha-interferon WA-M gene, the coding strand comprising the base sequence shown in FIG.5, a gene fragment or allele thereof.
Claim 27
Dependent Claim
20 words
Human alpha-interferon WA-M gene, the coding strand comprising the base sequence shown in FIG.5, a gene fragment or allele thereof.
Claim 28
Dependent Claim
214 words
A process for the production of an expression vector containing either the human alpha-interferon WA or human alpha-interferon WA-M gene comprising: (a) cloning said human alpha-interferon WA or WA-M gene into a unique restriction endonuclease site on a cloning vector to create a recombinant cloning vector, (b) transfecting a host cell, which is capable of replicating said recombinant cloning vector, with said recombinant cloning vector, (c) replicating said recombinant cloning vector in said host cell to create replicated recombinant cloning vectors, (d) isolating said replicated recombinant cloning vectors, (e) cleaving said replicated recombinant cloning vectors with restriction enzymes capable of cleaving said replicated recombinant cloning vectors at single sites not within said human alpha-interferon WA or WA-M gene so as to generate restriction fragments, (f) identifying said restriction fragments which contain said human alpha-interferon WA or WA-M gene or fragment thereof, (g) isolating said restriction fragments which contain said human alpha-interferon WA or WA-M gene or fragment thereof, and (h) inserting said restriction fragments which contain said human alpha-interferon WA or WA-M gene or fragment thereof into the DNA of an expression vector at a unique restriction endonuclease site on the DN A of said expression vector which allows for the in-phase transcription of said human alpha-interferon WA or WA-M gene or fragment thereof.
Claim 29
Dependent Claim
214 words
A process for the production of an expression vector containing either the human alpha-interferon WA or human alpha-interferon WA-M gene comprising: (a) cloning said human alpha-interferon WA or WA-M gene into a unique restriction endonuclease site on a cloning vector to create a recombinant cloning vector, (b) transfecting a host cell, which is capable of replicating said recombinant cloning vector, with said recombinant cloning vector, (c) replicating said recombinant cloning vector in said host cell to create replicated recombinant cloning vectors, (d) isolating said replicated recombinant cloning vectors, (e) cleaving said replicated recombinant cloning vectors with restriction enzymes capable of cleaving said replicated recombinant cloning vectors at single sites not within said human alpha-interferon WA or WA-M gene so as to generate restriction fragments, (f) identifying said restriction fragments which contain said human alpha-interferon WA or WA-M gene or fragment thereof, (g) isolating said restriction fragments which contain said human alpha-interferon WA or WA-M gene or fragment thereof, and (h) inserting said restriction fragments which contain said human alpha-interferon WA or WA-M gene or fragment thereof into the DNA of an expression vector at a unique restriction endonuclease site on the DN A of said expression vector which allows for the in-phase transcription of said human alpha-interferon WA or WA-M gene or fragment thereof.
Claim 30
Dependent Claim
18 words
The process of Claim 23 wherein the cloning vector comprises bacteriophage M13 mp8 or M 13 mp II.*
Claim 31
Dependent Claim
19 words
The process of Claim 23 wherein the unique restriction endonuclease site on said cloning vector comprises an EcoRI site.
Claim 32
Dependent Claim
34 words
The process of Claim 23 wherein said restriction enzymes capable of cleaving said replicated recombinant cloning vectors at sites not within said human alpha-interferon WA or WA-M or fragment thereof are AluI and EcoRI.
Claim 33
Dependent Claim
67 words
The process of Claim 23 wherein said restriction fragments which contain said human alpha-interferon WA or WA-M or fragment thereof are identified by hybridizing at least one labeled DNA probe comprising sequences homologous to D N A sequences contained within said human alpha-interferon WA or WA-M or fragment thereof to said restriction fragments and thereafter identifying said restriction fragments in a manner consistent with the label chosen.
Claim 34
Dependent Claim
26 words
The process of Claim 23 wherein said restriction fragments which contain said human alpha-interferon WA or WA-M gene or fragment thereof are isolated by gel fractionation.
Claim 35
Dependent Claim
26 words
A recombinant DNA vector comprising a DNA vector having inserted therein at an insertion site at least one human alpha-interferon WA gene or a fragment thereof.
Claim 36
Dependent Claim
26 words
A recombinant DNA vector comprising a DNA vector having inserted therein at an insertion site at least one human alpha-interferon WA gene or a fragment thereof.
Claim 37
Dependent Claim
26 words
A recombinant DNA vector comprising a DNA vector having inserted therein at an insertion site at least one human alpha-interferon WA-M gene or a fragment thereof.
Claim 38
Dependent Claim
26 words
A recombinant DNA vector comprising a DNA vector having inserted therein at an insertion site at least one human alpha-interferon WA-M gene or a fragment thereof.
Claim 39
Dependent Claim
44 words
The recombinant DNA vector of either Claim 29 or 30 wherein said human alpha-interferon WA or WA-M gene or fragment thereof is inserted to allow for in-phase transcription and translation of mRNA corresponding to the inserted gene or gene fragment in a bacterial host.
Claim 40
Dependent Claim
18 words
The recombinant DNA vector of either Claim 29 or 30wherein the DNA vector is bacteriophage or plasmid DNA.
Claim 41
Dependent Claim
15 words
The recombinant DNA vector of Claim 29 wherein said DNA vector is M13 mpll DNA.
Claim 42
Dependent Claim
15 words
The recombinant DNA vector of Claim 29 wherein said DNA vector is M13 mp8 DNA.
Claim 43
Dependent Claim
88 words
A process for replicating human alpha-interferon WA or human alpha-interferon WA-M gene or fragment thereof in a bacterial host, comprising: (a) producing a recombinant DNA vector comprising a DNA vector having inserted therein at an insertion site said human alpha-interferon WA or WA-M gene or fragment thereof; (b) transfecting a bacterial host capable of replicating said recombinant D NA vector with the recombinant DNA vector to generate replicated recombinant DNA vectors, and (c) isolating said replicated recombinant DNA vectors in a manner consistent with the DNA vector chosen.
Claim 44
Dependent Claim
88 words
A process for replicating human alpha-interferon WA or human alpha-interferon WA-M gene or fragment thereof in a bacterial host, comprising: (a) producing a recombinant DNA vector comprising a DNA vector having inserted therein at an insertion site said human alpha-interferon WA or WA-M gene or fragment thereof; (b) transfecting a bacterial host capable of replicating said recombinant D NA vector with the recombinant DNA vector to generate replicated recombinant DNA vectors, and (c) isolating said replicated recombinant DNA vectors in a manner consistent with the DNA vector chosen.
Claim 45
Dependent Claim
12 words
The process of Claim 35 wherein said bacterial host is E. coli.
Claim 46
Dependent Claim
14 words
The process of Claim 35 wherein the DNA vector is bacteriophage or plasmid DNA.
Claim 47
Dependent Claim
17 words
The process of Claim 35 wherein the DNA - vector is M13 mpll or M13 mp8 DNA.
Claim 48
Dependent Claim
164 words
A process for expressing human alpha-interferon WA or human alpha-interferon WA-M gene or fragment thereof in bacteria, comprising: (a) producing a recombinant vector comprising a DNA vector having inserted therein at an insertion site at least one human alpha-interferon WA or WA-M gene or fragment thereof wherein said gene or fragment thereof is inserted in-phase adjacent to a bacterial promoter to allow for in-phase transcription and translation of mRNA corresponding to the inserted gene or gene fragment in bacteria; (b) mixing said bacteria with said recombinant vector under conditions resulting in the infection of said bacteria with said recombinant DNA vector, (c) incubating said infected bacteria in a bacterial growth medium to allow said infected bacteria to grow, (d) inducing said infected bacteria with an inducer specific for the bacterial promoter located adjacent to said gene or fragment thereof, and (e) incubating said induced infected bacteria to allow for the transcription and translation of said human alpha-interferon WA or WA-M gene or fragment thereof.
Claim 49
Dependent Claim
164 words
A process for expressing human alpha-interferon WA or human alpha-interferon WA-M gene or fragment thereof in bacteria, comprising: (a) producing a recombinant vector comprising a DNA vector having inserted therein at an insertion site at least one human alpha-interferon WA or WA-M gene or fragment thereof wherein said gene or fragment thereof is inserted in-phase adjacent to a bacterial promoter to allow for in-phase transcription and translation of mRNA corresponding to the inserted gene or gene fragment in bacteria; (b) mixing said bacteria with said recombinant vector under conditions resulting in the infection of said bacteria with said recombinant DNA vector, (c) incubating said infected bacteria in a bacterial growth medium to allow said infected bacteria to grow, (d) inducing said infected bacteria with an inducer specific for the bacterial promoter located adjacent to said gene or fragment thereof, and (e) incubating said induced infected bacteria to allow for the transcription and translation of said human alpha-interferon WA or WA-M gene or fragment thereof.
Claim 50
Dependent Claim
14 words
40 . The process of Claim 39 wherein said bacteria is E . coli.
Claim 51
Dependent Claim
14 words
The process of Claim 39 wherein said DNA vector is bacteriophage or plasmid DNA.
Claim 52
Dependent Claim
15 words
42 . The process of Claim 41 wherein said DNA vector is M13 mpll DNA.
Claim 53
Dependent Claim
11 words
The process of Claim 39 wherein said bacterial promoter is β-galactosidase.
Claim 54
Dependent Claim
10 words
The process of Claim 43 wherein said inducer is isopropylthiogalactoside.
Claim 55
Dependent Claim
5 words
Human alpha-interferon WA or WA-M.
Claim 56
Dependent Claim
5 words
Human alpha-interferon WA or WA-M.
Claim 57
Dependent Claim
115 words
46 . A method for determining the sequence of a DNA probe useful in identifying a gene or gene fragment thereof by hybridization of said DNA probe to said gene, or gene fragment thereof contained within a cDNA library or genomic library or recombinant vector when said gene or gene fragment thereof is one of a family of genes, said method comprising: comparing the known DNA sequences of know genes or gene fragments in said family of genes and determining at least one oligonucleotide sequence of sufficient length to stably hybridize to said gene or gene fragment thereof which is shared by at least one half of said genes or gene fragments in said family.
Claim 58
Dependent Claim
115 words
46 . A method for determining the sequence of a DNA probe useful in identifying a gene or gene fragment thereof by hybridization of said DNA probe to said gene, or gene fragment thereof contained within a cDNA library or genomic library or recombinant vector when said gene or gene fragment thereof is one of a family of genes, said method comprising: comparing the known DNA sequences of know genes or gene fragments in said family of genes and determining at least one oligonucleotide sequence of sufficient length to stably hybridize to said gene or gene fragment thereof which is shared by at least one half of said genes or gene fragments in said family.
Claim 59
Dependent Claim
14 words
47 . The method of Claim 46 wherein said family of genes is interferon.
Claim 60
Dependent Claim
12 words
The method of Claim 46 wherein said DNA probe comprises the sequence:
Claim 61
Dependent Claim
12 words
The method of Claim 46 wherein said DNA probe comprises the sequence.:
References Cited (Backward Citations)
US4237224A
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