US5096815A — Generation and selection of novel dna-binding pro…

Claim 1

Independent Claim 118 words

A cell culture comprising a plurality of cells, each cell bearing a selection vector, said selection vector comprising a first and a second operon, each comprising at leat one expressible gene, the genes of said first and second operons being different, a copy of the target DNA sequence being included in each operon and positioned therein so that under forward selection conditions the transformed cells enjoy a selective advantage if they express a protein or polypeptide which binds to said copies of the target DNA sequence, said cell culture being transformed with a variegated gene encoding potential DNA-binding proteins or polypeptides, where said cells collectively can express a plurality of different but sequence-related potential DNA-binding proteins or polypeptides.

Claim 2

Dependent Claim 118 words

A cell culture comprising a plurality of cells, each cell bearing a selection vector, said selection vector comprising a first and a second operon, each comprising at leat one expressible gene, the genes of said first and second operons being different, a copy of the target DNA sequence being included in each operon and positioned therein so that under forward selection conditions the transformed cells enjoy a selective advantage if they express a protein or polypeptide which binds to said copies of the target DNA sequence, said cell culture being transformed with a variegated gene encoding potential DNA-binding proteins or polypeptides, where said cells collectively can express a plurality of different but sequence-related potential DNA-binding proteins or polypeptides.

Claim 3

Dependent Claim 12 words

The cell culture of claim 1 wherein the cells are bacterial cells.

Claim 4

Dependent Claim 14 words

The cell culture of claim 2 in which the cells are Escherichia coli cells.

Claim 5

Dependent Claim 19 words

The cell culture of claim 1 wherein the potential DNA-binding protein encoding gene is borne by the selection vector.

Claim 6

Dependent Claim 96 words

A method of obtaining a gene coding on expression for a novel DNA-binding protein or polypeptide that preferentially binds a predetermined DNA target sequence in double stranded DNA, comprising: (a) providing a cell culture according to claim 1; (b) causing the cells of such culture to express said potential DNA-binding proteins or polypeptides; (c) exposing the cells to forward selection conditions to select for cells which express a protein or polypeptide which preferentially binds to said target DNA sequence; and (d) recovering the selected cells bearing a gene coding on expression for such protein or polypeptide.

Claim 7

Dependent Claim 53 words

A method of producing a DNA-binding protein or polypeptide which preferentially binds a predetermined double stranded DNA target, which comprises providing a gene obtained by the method of claim 5 which codes on expression for such protein or polypeptide, expressing the gene in a suitable host cells, and recovering said protein or polypeptide.

Claim 8

Dependent Claim 52 words

A method of obtaining a protein or polypeptide which may be used to specifically repress a coding or regulatory element of interest which comprises identifying an ultimate target sequence within such element and obtaining a protein or polypeptide which preferentially binds to such ultimate target sequence by the method of claim 5.

Claim 9

Dependent Claim 36 words

The method of claim 5, wherein a gene coding on expression for a polypeptide is variegated, said polypeptide having a length of 25-50 amino acids and being capable of lying in the major groove of B-DNA.

Claim 10

Dependent Claim 37 words

The method of claim 5 wherein the gene codes on expression for a molecule comprising a DNA-binding domain of 25-50 amino acids and a custodial demain that substantially inhibits the degradation of the molecule by intracellular enzymes.

Claim 11

Dependent Claim 12 words

The method of claim 5 wherein said protein is a globular protein.

Claim 12

Dependent Claim 25 words

The method of claim 10 wherein a gene coding on expression for a known DNA binding protein having a helix-turn-helix DNA binding motif is variegated.

Claim 13

Dependent Claim 89 words

The method of claim 5 wherein a gene encoding a known DNA binding protein picked from the group consisting of Cro from phage lambda, cI repressor from phage lambda, Cro from phage 434, cI repressor from phage 434, P22 repressor, E. coli tryptophan repressor, E. coli CAP, P22 Arc, P22 Mnt, E. coli lactose repressor, MAT-al-alpha2 from yeast, Polyoma Large T antigen, SV40 Large T antigen, Adenovirus ElA, and TFIIIA from Xenopus laevis is variegated to obtain genes coding on expression for a plurality of potential target DNA-binding proteins.

Claim 14

Dependent Claim 70 words

The method of claim 5 in which the cells prior to transformation are of a GalE - , GalT - , GalK - , Tet S phenotype, the binding marker genes are the tet and galT,K genes, and the forward selection condition is cultivation of the cells in a medium containing galactose, or fusaric acid, or both, or substances metabolized into or catalyzing the production of galactose or fusaric acid.

Claim 15

Dependent Claim 41 words

The method of claim 13 in which the vector further contains an origin of replication and an antibiotic resistance gene, and cells are cultured in medium that further comprises, an antibiotic for which resistance is conferred by said antibiotic resistance gene.

Claim 16

Dependent Claim 22 words

The method of claim 5 in which the forward selection conditions do not result in lytic growth of phage in unselected cells.

Claim 17

Dependent Claim 24 words

The method of claim 15 in which the forward selection conditions are selected from the group consisting of nutritional and chemical resistance selection conditions.

Claim 18

Dependent Claim 42 words

The method of claim 5 wherein at least one variegated codon encodes all twenty amino acids such that the frequency of occurrence of the least favored amino acid is about 0.4 times the frequency of occurrence of the most favored amino acid.

Claim 19

Dependent Claim 23 words

The method of claim 17 wherein for said variegated codon, the frequencies of occurrence of acidic and basic amino acid are about equal.

Claim 20

Dependent Claim 40 words

The method of claim 17 wherein said variegated codon has substantially the following base proportions: ______________________________________ I C A G ______________________________________ base #1 0.26 0.18 0.26 0.30 base #2 0.22 0.16 0.40 0.22 base #3 0.5 0.0 0.0 0.5. ______________________________________

Claim 21

Dependent Claim 24 words

The method of claim 5 wherein for at least one variegated codon, the mixture of bases for at least one base position is non-equimolar.

Claim 22

Dependent Claim 70 words

The method of claim 5 wherein at least one operon comprises a selectable beneficial gene, an occludible promoter operably linked to said beneficial gene and directing its transcription, an occluding promoter occluding transcription of said beneficial gene, and a copy of the target DNA sequence positioned so that the binding of said protein or polypeptide to said copy represses said occluding promoter and thereby facilitates transcription of said beneficial gene.

Claim 23

Dependent Claim 175 words

The method of claim 5 in which said selection vector comprises: (a) a first operon, which operon comprises: (i) a first promoter directing expression of said binding marker gene(s), and (iii) a first copy of the target DNA sequence, where said target DNA sequence interferes substantially with expression of the first gene(s) if and only if a protein expressed by the transformed cell binds to the target DNA sequence, (b) a second operon, which operon comprises: (i) a second binding marker gene(s), (ii) a second promoter directing expression of said binding marker gene(s), and (iii) a second copy of the target DNA sequence, where said target DNA sequence interferes substantially with expression of said gene(s) if and only if a protein expressed by the transformed cell binds to the target DNA sequence, where the binding marker genes of said first and second operons are different, and where, when said cells are exposed to forward selection conditions the gene products of said first and second binding marker genes are deleterious or conditionally lethan to the cell.

Claim 24

Dependent Claim 84 words

The method of claim 22 in which at least one of the operons confers a genotype selected from the group consisting of galT,K + , tetA + , lacZ + , pheS + , argP + , thyA + , crp + , pyrF + , ptsM + , secA + /malE + /lacZ + , ompA + , btuB + , lamB + , tonA + , cir + , tsx + , aroP + , cysK + , and dctA + .

Claim 25

Dependent Claim 38 words

A method of claim 23 wherein the degree of homology between the first and second promoters is less than 50% in the region between the -10 region of the promoter and the base at which transcription is initiated.

Claim 26

Dependent Claim 20 words

The method of claim 24 in which one of said genetic elements is the origin of replication of said vector.

Claim 27

Dependent Claim 13 words

The vector of claim 22 wherein the binding marker genes are functionally unrelated.

Claim 28

Dependent Claim 147 words

The method of claim 26 wherein the first and second operons confer, respectively, a pair of genotypes selected from the group consisting of: (a) galT,K + and tetA + ; (b) argP + and pheS + ; (c) lacZ + and tetA + ; (d) dctA + and cysK + ; (e) crp + and thyA + ; (f) lamB + and thyA + ; (g) SecA + /malE + /lacZ + and pyrF + ; (h) tsx + and cysK + ; (i) dctA + and thyA + ; (j) galT,K + and pheS + ; (k) tetA + and thyA + ; (l) ptsM + and thyA + ; (m) ompA + and pyrF + ; (n) btuB + and pyrF + ; (o) tonA + and galT,K + ; (p) cir + and cysK + ; and (q) aroP + and lacZ + .

Claim 29

Dependent Claim 16 words

The method of claim 22 wherein the promoters of said first and second operons are different.

Claim 30

Dependent Claim 54 words

The method of claim 22,, wherein at least one of said operons comprises a plurality of copies of the target DNA sequences, wherein each copy is positioned so that the target DNA sequence interferes substantially with expression if and only if a protein expressed by the recipient cell binds to the target DNA sequence.

Claim 31

Dependent Claim 70 words

The method of claim 22, further comprising a plurality of genetic elements essential to the maintenance of the vector or the survival of the transformed cells under conditions that select for presence of said vector, said operons and said genetic elements being positioned on said vector so no single deletion event can render non-functional more than one of said operons without also rendering nonfunctional one of said essential genetic elements.

Claim 32

Dependent Claim 48 words

The method of claim 30, wherein at least one of said genetic elements comprises a selectably beneficial or essential gene, and a control promoter operably linked to said beneficial or conditionally essential gene, but where no instance of said target DNA sequence is associated with said genetic element.

Claim 33

Dependent Claim 48 words

The method of claim 22 wherein the control promoter is essentially identical to the promoter of one of said selectable binding marker operons, so that proteins binding to the latter promoter will also bind to the control promoter and thereby inhibit expression of said beneficial or essential gene.

Claim 34

Dependent Claim 27 words

The method of claim 1, wherein under reverse selection conditions the gene products of said binding marker genes are beneficial or conditionally essential to the transformed cells.

Claim 35

Dependent Claim 87 words

The method of claim 33, wherein each of the first and second operons confers a phenotype selected independently but non-identically from the group consisting of: galT,K + , tetA + , lacZ + , pheS + , argP + , thyA + , crp + , pyrF + , ptsM + , secA + /malE + /lacZ + , ompA + , btuB + , lamB + , tonA + , cir + , tsx + , aroP + , cysK + , and dctA + .

Claim 36

Dependent Claim 17 words

The vector of claim 22, wherein said promoter of said gene is an inducible or repressible promoter.

Claim 37

Dependent Claim 45 words

The method of claim 22, further comprising a nondeleterious cloning site so positioned that insertion of a foreign gene at such site does not inactivate said first or second operons or any genetic element of said vector required for its maintenance within the transformed cell.

Claim 38

Dependent Claim 31 words

The method of claim 22 in which the target sequence associated with at least one of said operons is positioned within the RNA-polymerase binding site of the promoter of the operon.

Claim 39

Dependent Claim 31 words

The method of claim 22 in which the target sequence associated with at least one of said operons is positioned upstream of the -35 region of the promoter of the operon.

Claim 40

Dependent Claim 31 words

The method of claim 22 in which the target sequence associated with at least one of said operons in positioned downstream of the -10 region of the promoter of the operon.

Claim 41

Dependent Claim 134 words

A selection vector, said selection vector comprising a first and a second operon, each comprising at least one expressible gene, the genes of said first and second operons being different, a copy of the target DNA sequence being included in each operon and positioned therein so that under forward selection conditions the transformed cells enjoy a selective advantage if they express a protein or polypeptide which binds to said copies of the target DNA sequence, and further comprising a gene coding on expression for a potential DNA-binding protein or polypeptide, said gene comprising: (a) a coding region that codes on expression for a polypeptide, each domain of said polypeptide having at least 50% sequence identify to a known DNA-binding domain, and (b) a promoter operably linked to said coding region for controlling its expression.

Claim 42

Dependent Claim 134 words

A selection vector, said selection vector comprising a first and a second operon, each comprising at least one expressible gene, the genes of said first and second operons being different, a copy of the target DNA sequence being included in each operon and positioned therein so that under forward selection conditions the transformed cells enjoy a selective advantage if they express a protein or polypeptide which binds to said copies of the target DNA sequence, and further comprising a gene coding on expression for a potential DNA-binding protein or polypeptide, said gene comprising: (a) a coding region that codes on expression for a polypeptide, each domain of said polypeptide having at least 50% sequence identify to a known DNA-binding domain, and (b) a promoter operably linked to said coding region for controlling its expression.

Claim 43

Dependent Claim 32 words

A variegated population of vectors according to claim 40, the variegation occurring within the pdbp gene so that said vectors collectively can express a plurality of different but sequence-related potential DNA-binding proteins.

Claim 44

Dependent Claim 26 words

The population of claim 41, wherein the level of variegation is such that from 10 6 to 10 9 different potential DNA-binding proteins can be expressed.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1995-03-17	1995-09-16	1996-03-17	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1999-03-17	1999-09-16	2000-03-17	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2003-03-17	2003-09-16	2004-03-17	$7700.00	Window Closed / Lapsed

Generation and selection of novel dna-binding proteins and polypeptides

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