Vectors for the insertion therein of foreign DNA fragments, according to any translation phase

• US 4,371,625 A
• Filed: 11/13/1979
• Issued: 02/01/1983
• Est. Priority Date: 11/13/1978
• Status: Expired due to Term

• Current Assignee: Institut Pasteur, INST Nat'L de La Sante et Recherche
• Original Assignee: Institut Pasteur, INST Nat'L de La Sante et Recherche
• Inventors: Tiollais, Pierre
• Primary Examiner(s): Tanenholtz, Alvin E.
• Application Number:
  US06/093,270
• Time in Patent Office: 1,176 Days
• Field of Search: 435/172, 435/317, 536/27
• US Class Current: 435/320.1
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00

Abstract

The invention pertains to a set of vectors (or of DNA fragments to be inserted in a vector) which distinguish from one another in that, taking into account one vector in which the number of pairs of bases between the reading initiation point of the vector and a point corresponding to the first pair of bases of a recognition site corresponding to a predetermined restriction enzyme, the two other vectors comprise between the corresponding points additional groups of pairs of bases comprising respectively two pairs of bases on the one hand and either one or four pairs of bases on the other hand, plus possibly whole numbers of triplets. On inserting a determined DNA fragment of which the expression is sought in bacteria in the three vectors, the reading of said DNA fragment will occur in phase as concerns one of the so modified vectors after transfection of bacteria therewith.

Claims

1. A composition of matter for the introduction of foreign DNA into a host whereby the expression of a protein having a desired amino acid sequence is provided, said composition of matter comprising the following three structurally interrelated vectors:

(A) a first vector which comprises;

(i) a preselected bacterial promoter in association with a preselected bacterial gene or part thereof, said preselected bacterial gene or part thereof comprising a translation initiation point,

(ii) a single recognition site for a preselected restriction endonuclease, said site being in association with the preselected bacterial gene or part thereof, and

(iii) a plurality of nucleotide base pair triplets between the translation initiation point of the gene or part thereof and the single restriction endonuclease recognition site associated with the gene or part thereof, wherein the plurality of nucleotide base pair triplets does not contain a nonsense codon, and

(B) a second vector which comprises

(i) (A) (i),

(ii) (A) (ii) and

(iii) the plurality of nucleotide base pair triplets plus two additional nucleotide base pairs between the translation initiation point of the gene or part thereof and the single restriction endonuclease recognition site associated with the gene or part thereof, wherein the plurality of nucleotide base pair triplets does not contain a nonsense codon, and

(C) a third vector which comprises;

(i) (A) (i),

(ii) (A) (ii) and

(iii) the plurality of nucleotide base pair triplets plus 1 or 4 additional nucleotide base pairs between the translation initiation point of the gene or part thereof and the single restriction endonuclease recognition site associated with the gene or part thereof, wherein the plurality of nucleotide base pair triplets does not contain a nonsense codon.

2. The composition of matter as defined in claim 1 wherein the preselected bacterial gene or part thereof is derived from a bacterial operon and the preselected promoter is the promoter associated with the bacterial operon.

3. The composition of matter as defined in claim 2 wherein the single recognition site for the preselected restriction endonuclease is located within the preselected bacterial gene or part thereof or is located in a part of the vector which is adjacent to the end of the gene or part thereof which is farthest from the preselected bacterial promoter.

4. The composition of matter as defined in claim 3 wherein the vector is a phage or a plasmid.

5. The composition of matter as defined in claim 4 wherein the bacterial operon is the lactose operon.

6. The composition of matter as defined in claim 5 wherein the gene or part thereof is the Z gene.

7. The composition of matter as defined in claim 6 wherein the restriction endonuclease is Eco RI.

8. The composition of matter as defined in claim 7 wherein the additional nucleotide base pairs contain bases selected from the group consisting of adenine, thymine, guanine and cytosine.

9. The composition of matter as defined in claim 8 wherein each of the three recited vectors further comprises a preselected foreign DNA fragment which has been inserted into each vector at its single restriction endonuclease recognition site.

10. The composition of matter as defined in claim 9 wherein the host is a bacterium.

11. A bacterial host which has been transformed with the composition of matter as defined in claim 9.

12. A composition of matter for insertion into a preselected vector to make the vector suitable for the introduction of foreign DNA into a host whereby the expression of a protein having a desired amino acid sequence is provided, said composition of matter comprising the following three structurally interrelated DNA insertion fragments:

(A) a first insertion fragment which comprises;

(i) a preselected bacterial promoter in assocation with at least part of a preselected bacterial gene, said preselected bacterial gene or part thereof comprising a translation initiation point

(ii) a cohesive end corresponding to a recognition site for a preselected restriction endonuclease, and

(iii) a plurality of nucleotide base pair triplets between the translation initiation point of the gene or part thereof and the cohesive end corresponding to a recognition site for a preselected restriction endonuclease, wherein the plurality of nucleotide base pair triplets does not contain a nonsense codon, and

(B) a second insertion fragment which comprises

(i) (A) (i),

(ii) (A) (ii) and

(iii) the plurality of nucleotide base pair triplets plus two additional nucleotide base pairs between the translation initiation point of the gene or part thereof and the cohesive end corresponding to a recognition site for a preselected restriction endonuclease, wherein the plurality of nucleotide base pair triplets does not contain a nonsense codon, and

(C) a third insertion fragment which comprises;

(i) (A) (i),

(ii) (A) (ii) and

(iii) the plurality of nucleotide base pair triplets plus 1 or 4 additional nucleotide base pairs between the translation initiation point of the gene or part thereof and the cohesive end corresponding to a recognition site for a preselected restriction endonuclease, wherein the plurality of nucleotide base pair triplets does not contain a nonsense codon.

13. The composition of matter as defined in claim 12 wherein the preselected bacterial gene or part thereof is derived from a bacterial operon and the preselected promoter is the promoter associated with the bacterial operon.

14. The composition of matter as defined in claim 13 wherein the bacterial operon is the lactose operon.

15. The composition of matter as defined in claim 14 wherein the gene or part thereof is the Z gene.

16. The composition of matter as defined in claim 15 wherein the restriction endonuclease is Eco RI.

17. The composition of matter as defined in claim 16 wherein the additional nucleotide base pairs contain bases selected from the group consisting of adenine, thymine, guanine, and cytosine.

18. The composition of matter as defined in claim 17 wherein the host is a bacterium.

19. Composition of matter for the introduction of foreign DNA into a host whereby the expression of a protein having a desired amino acid sequence is provided, said composition of matter comprising the following three structurally interrelated vectors:

(A) a first vector which comprises;

(i) a promoter and gene associated therewith in a preselected bacterial operon or part of said gene,

(ii) a single recognition site for a preselected restriction endonuclease associated with said gene or part of gene and

(iii) a number of nucleotide base pairs between the reading initiation point of the gene and the single restriction endonuclease recognition site associated with said gene

(B) a second vector which comprises;

(i) The promoter and gene or part of gene of said first vector,

(ii) the single recognition site of said first vector, and

(iii) the number of nucleotide base pairs of said first vector plus two additional nucleotide base pairs and, optionally, a whole number of supplementary triplets between said reading initiation point and said single restriction endonuclease recognition site, wherein the bases of said additional nucleotide base pairs and said optional supplementary triplets are selected such as to exclude those which, in the order in which they are placed with respect to said initiation point, would form at least one nonsense codon,

(C) a third vector which comprises;

(i) the promoter and gene or part of gene of said first vector,

(ii) the single recognition site of said first vector, and

(iii) the number of nucleotide base pairs of said first vector, plus one or four additional nucleotide base pairs and, optionally, a whole number of supplementary triplets between said reading initiation point and said single restriction endonuclease recognition site, wherein the bases of said one or four additional nucleotide base pairs and said optional supplementary triplets are selected such as to exclude those which, in the order in which they are placed with respect to said reading initiation point, would form at least one nonsense codon.

20. The composition of claim 19 wherein the bacterial operon is the lactose operon and the gene or part thereof is the Z gene or a part thereof.

21. The composition of claim 20 wherein the restriction site is a EcoRI site.

22. A composition of matter for insertion into a preselected vector to make the vector suitable for the introduction of foreign DNA into a host, whereby the expression of a protein having a desired amino acid sequence is provided, said composition of matter comprising the following three structurally interrelated DNA insertion fragments:

(A) a first insertion fragment which comprises;

(i) a promoter and gene associated therewith in a preselected bacterial operon or part of said gene,

(ii) a single recognition site for a preselected restriction endonuclease associated within said gene or part of gene and

(iii) a number of nucleotide base pairs between the reading initiation point of the gene and the single restriction endonuclease recognition site associated with said gene

(B) a second insertion fragment which comprises;

(i) the promoter and gene or part of gene of said first insertion fragment,

(ii) the single recognition site of said first insertion fragment, and

(iii) the number of nucleotide base pairs of said first insertion fragment plus two additional nucleotide base pairs and, optionally, a whole number of supplementary triplets between said reading initiation point and said single restriction endonuclease recognition site, wherein the bases of said additional nucleotide base pairs and said optional supplementary triplets are selected such as to exclude those which, in the order in which they are placed with respect to said initiation point, would form at least one nonsense codon,

(C) a third insertion fragment which comprises;

(i) the promoter and gene or part of said first insertion fragment,

(ii) the single recognition site of said first insertion fragment, and

(iii) the number of nucleotide base pairs of said first insertion fragment, plus one or four additional nucleotide base pairs and, optionally, a whole number of supplementary triplets between said reading initiation point and said single restriction endonuclease recognition site, wherein the bases of said one or four additional nucleotide base pairs and said optional supplementary triplets are selected such as to exclude those which, in the order in which they are placed with respect to said reading initiation point, would form at least one nonsense codon.

23. The composition of claim 22 wherein the bacterial operon is the lactose operon and the gene or part of said gene is the Z gene or a part thereof.

24. The composition of claim 23 wherein the restriction site is an EcoRI site.