WO1988006601A1 - Represseurs de genes - Google Patents

Represseurs de genes Download PDF

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Publication number
WO1988006601A1
WO1988006601A1 PCT/US1988/000718 US8800718W WO8806601A1 WO 1988006601 A1 WO1988006601 A1 WO 1988006601A1 US 8800718 W US8800718 W US 8800718W WO 8806601 A1 WO8806601 A1 WO 8806601A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
repressor
repressors
lambda
sequence
Prior art date
Application number
PCT/US1988/000718
Other languages
English (en)
Inventor
Robert Charles Ladner
Robert E. Bird
Original Assignee
Genex Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genex Corporation filed Critical Genex Corporation
Publication of WO1988006601A1 publication Critical patent/WO1988006601A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection

Definitions

  • the viral DNA may become integrated into the host genome and cause no change in cell activity for a long time.
  • Such silent viruses are termed temperate. Some future event may cause such a virus to reemerge and lead to rapid growth of virus progeny and cell lysis.
  • 061388 immediately produces progeny and lyses E. coli.
  • class cl supplying and expressing the repressor gene will prevent cell lysis.
  • class vir no natural repressor will prevent cell lysis.
  • the bacteriophages P22 and 434 are closely related to lambda. They too possess CRO and repressor proteins; the exact sequences of these proteins and the DNA sequences to which they bind are different from lambda and from each other.
  • Enzymes may be inhibited by their own products or by the product of a chain of reactions in which they play a part. If the product of an enzyme accumulates, it comes back to the enzyme and says, "Slow down, you're working too hard.” Enzymes may be activated by chemicals which they act upon or by chemicals which will react with their product.
  • the concentration of enzymes may be regulated by the rates at which RNAs are translated into protein.
  • proteins bind to the mRNA which codes for them, thereby reducing the rate of protein synthesis once enough protein is made.
  • the genetic code is redundant; there are several codons for most of the amino acids (only met and trp have unique codons). Not all of the different codons for a given amino acid work equally well. Thus, a given gene will be expressed at a lower level if inefficient codons are used.
  • Positive control sequences bind proteins which can recognize specific DNA sequences. These proteins (called positive regulators) then allow RNA-polymerase (DNA-to-RNA transcriptase) to bind and copy the following DNA into RNA. There may be only one or a few kinds of RNA-polymerase in a cell, but many classes of positive control sequences.
  • the positive regulators act as decoders between DNA control sites and RNA polymerases. There are DNA sequences which bind RNA- polymerase without the assistance of positive regulatory proteins. These sequences are called promoters. Often positive regulatory proteins act to complement partial promoters.
  • Negative control sequences bind proteins which recognize specific DNA sequences. These proteins (called repressors) block the binding or passage of RNA-polymerase and thus prevent transcription of the gene.
  • the negative and positive control sequences need not be disjoint. Indeed, positive regulators and repressors may compete for the same sites.
  • DNA is an antiparallel double helix and that valid genetic information may be read from either strand.
  • a protein may be a repressor for a gene reading in one direction and a positive regulator for a different gene read in the other direction.
  • Fig. 3 shows schematic representations of these three molecules.
  • SUBSTITUTE SHEET of E. coli (or some other suitable bacteria) is repressed.
  • the linker or interface region or regions is or are varied when the gene is made by in vitro DNA synthesis.
  • This population is transfected into E. coli or some other suitable bacteria.
  • First a selection is performed which eliminates unrepressed cells. This must be done because the variation in the framework could produce nonfunctional repressors; such selections are well-known in bacterial genetics.
  • the slowly growing bacteria are supplied with the chemical which will be the signal. Any cells which grow are likely to have one or more binding sites for the chemical signal. Binding the chemical messenger could cause a conformational change in the repressor which abolishes DNA binding.
  • HEET 3 a highly deleterious gene controlled by a hybrid lambda-434 operator
  • step 3080 introduce mutations into the C-domains of both lambda-like repressors.
  • the C-domain of lambda repressor contains 104 residues, judging from other proteins which aggregate, 10% to 20% of these residues will be involved in the dimer interface. Each residue on one side of the interface will touch two or three residues on the other side.
  • the lambda system is highly evolved, so one might assume that the dimer interface is well optimized. Consider a mutation which changes an amino acid in the interface of the C-domain of gene 1. This change will occur in both chains and
  • each amino acid is about 97% correct
  • 95% of each C-domain will be the protein sequence written. 95% of the C-domains will have between 1 and 6 changes. The construction given greatly increases the chance that heterodimers will become much more stable than homodimers.
  • ATCAC gln-ser-ala-ile-asn-lys-ala-ile-his lambda cl TTTAA glu-ser-gln-ile-ser-arg-trp-lys-gly F22 cl
  • CAAGA gln-gln-ser-ile-glu-gln-leu-glu-asn 434R cl
  • step 4010 1024 versions of the plasmid shown in Fig. 12 will be created which have hybrid operators. One-half of each operator will have the natural DNA sequence. The other side of the operator will be systematically varied so that every possible pentanucleotide sequence will appear in the positions of major recognition.
  • SUBSTITUTE SHEET hybrid operators are positioned to repress a highly dele ⁇ terious gene. Thus one can select in vivo (step 4030) working repressors which bind to each of the hybrid operators.
  • the dictionary is compiled simply by sequencing the repressor gene of the winner in each of the 1024 in vivo selections.
  • the parts of the linkers which are least certain are identified by an expert and those parts of the gene for the tetradentate repressor are made intentionally unfaithful. This produces a population of potential tetradentate repressors which vary in the linkage which positions the four DNA-recognizing elements. This population is subjected to in vivo selection.
  • the C-terminal arm (residues 61-67) is assumed to wrap around the DNA.
  • the linker is built by an extension of U.S. Patent Application Serial No. 902,970 as follows. From the C-terminal of chain 1 to the amino-terminal of chain 2 is a wide groove where the two chains come in contact. An expert user viewing this structure on computer graphics decided that an alpha helix would lie in this groove and make many favorable contacts. In addition, a helical linker would make internal hydrogen bonds and so be self-stabilizing. Thus, alpha helices were extrac ⁇ ted from a protein in the Brookhaven Protein Data Bank.

Abstract

Répresseur de gènes comprenant au moins deux domaines de liaison d'ADN à séquence spéficique liés de manière covalente par polypeptide et molécule d'ADN recombinant codant ce même répresseur.
PCT/US1988/000718 1987-03-02 1988-03-02 Represseurs de genes WO1988006601A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2104787A 1987-03-02 1987-03-02
US021,047 1987-03-02

Publications (1)

Publication Number Publication Date
WO1988006601A1 true WO1988006601A1 (fr) 1988-09-07

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1988/000718 WO1988006601A1 (fr) 1987-03-02 1988-03-02 Represseurs de genes

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WO (1) WO1988006601A1 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0436597A1 (fr) * 1988-09-02 1991-07-17 Protein Eng Corp Production et selection de proteines de liaison diversifiees de recombinaison.
WO1991011521A1 (fr) * 1990-01-26 1991-08-08 La Jolla Cancer Research Foundation Polypeptides de codage d'activateurs de la transcription et leurs utilisations
EP0452413A1 (fr) * 1989-01-06 1991-10-23 Dyax Corp. Production et selection de proteines et de polypeptides nouveaux de liaison a l'adn
US5198346A (en) * 1989-01-06 1993-03-30 Protein Engineering Corp. Generation and selection of novel DNA-binding proteins and polypeptides
US5403484A (en) * 1988-09-02 1995-04-04 Protein Engineering Corporation Viruses expressing chimeric binding proteins
EP0684999A1 (fr) * 1992-12-23 1995-12-06 Genelabs Technologies, Inc. Molecules, compositions et procedes de liaison d'adn specifiques a des sequences
EP0700997A1 (fr) 1989-05-10 1996-03-13 Somatogenetics International, Inc. Production d'hémoglobine et de ses analogues par des bactéries et chez la levure
US5545727A (en) * 1989-05-10 1996-08-13 Somatogen, Inc. DNA encoding fused di-alpha globins and production of pseudotetrameric hemoglobin
US5759517A (en) * 1991-11-08 1998-06-02 Somatogen, Inc. Hemoglobins as drug delivery agents
US5871902A (en) * 1994-12-09 1999-02-16 The Gene Pool, Inc. Sequence-specific detection of nucleic acid hybrids using a DNA-binding molecule or assembly capable of discriminating perfect hybrids from non-perfect hybrids
US6869765B2 (en) 1991-06-27 2005-03-22 Genelabs Technologies, Inc. Sequence-directed DNA-binding molecules compositions and methods
US7413537B2 (en) 1989-09-01 2008-08-19 Dyax Corp. Directed evolution of disulfide-bonded micro-proteins
US7893007B2 (en) 1989-09-01 2011-02-22 Dyax Corp. Directed evolution of novel binding proteins
US8114962B2 (en) 1994-12-09 2012-02-14 The Gene Pool, Inc. Method of detection of nucleic acids with a specific sequence composition

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
Cell, Volume 45, issued 1986, (USA), ZINN et al., "Detection on Factors that Interact with the Human Interferon Regulatory Region In Vivo by DNAase I Footprinting", see pages 611-618. *
D. FREIFELDER, "Molecular Biology", published 1983, by Jones and Bartlett, Inc., "A Comprehensive Introduction to Prokaryotes and Eukaryotes", (Boston, MA), see pages 561-570. *
J. Mol. Biol., Volume 162, issued 1982, (London, England), SANGER et al., "Nucleotide Sequence of Bacteriophage DNA", see pages 729-773. *
Molecular and Cellular Biology, Volume 5, issued 1985 (USA), BRADY et al., "Trans Activation of the Simian Virus 40 Late Transcription Unit by T-Antigen", see pages 1391-1399. *
Nucleic Acids Research, Volume 15, issued 1987, (Oxford, England), GROSSMAN et al., "Purification and DNA Binding Properties of the b1aI Gene Product, Repressor for the Lactamase Gene, b1aP, of Bacillus Licheniformis", see pages 6049-6062. *
Proc. Nat. Acad. Sci. USA, Volume 73, issued 1976, (Washington, D.C., USA), BACKMAN et al., "Construction of Plasmids Carrying the cI Gene of Bacteriophage", see pages 4174-4178. *
Proc. Nat. Acad. Sci. USA, Volume 77, issued 1980, (Washington, D.C., USA), SCHMEISSNER et al., "Promoter for the Establishment of Repressor Synthesis in Bacteriophage", see pages 3191-3195. *
Proc. Nat. Acad. Sci., USA, Volume 82, issued 1985, ISACKSON et al., "Dominant Negative Mutations in the Tn10 Tet Repressor: Evidence for Use of the Conserved Helix-Turn-Helix Motif in DNA Binding", see pages 6226-6230. *
The EMBO Journal, Volume 4, issued 1985, (Oxford, England), GUILFOYLE et al., "Two Functions Encoded by Adenovirus Early Region 1A are Responsible for the Activation and Repression of the DNA-Binding Protein Gene", see pages 707-713. *

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5571698A (en) * 1988-09-02 1996-11-05 Protein Engineering Corporation Directed evolution of novel binding proteins
US7118879B2 (en) 1988-09-02 2006-10-10 Dyax Corp. Method of recovering a nucleic acid encoding a proteinaceous binding domain which binds a target material
EP1892296A1 (fr) 1988-09-02 2008-02-27 Dyax Corporation Production et sélection de protéines de liaison diversifiées recombinantes
EP0436597A1 (fr) * 1988-09-02 1991-07-17 Protein Eng Corp Production et selection de proteines de liaison diversifiees de recombinaison.
EP0436597A4 (en) * 1988-09-02 1992-05-20 Protein Engineering Corporation Generation and selection of recombinant varied binding proteins
US5837500A (en) * 1988-09-02 1998-11-17 Dyax, Corp. Directed evolution of novel binding proteins
US6979538B2 (en) 1988-09-02 2005-12-27 Dyax Corp. Directed evolution of novel binding proteins
US5403484A (en) * 1988-09-02 1995-04-04 Protein Engineering Corporation Viruses expressing chimeric binding proteins
EP1997891A1 (fr) 1988-09-02 2008-12-03 Dyax Corporation Production et sélection de protéines de liaison diversifiées recombinantes
EP0768377A1 (fr) 1988-09-02 1997-04-16 Protein Engineering Corporation Production et sélection de protéines de liaison diversifiées de recombinaison
US7208293B2 (en) 1988-09-02 2007-04-24 Dyax Corp. Directed evolution of novel binding proteins
EP0452413A4 (en) * 1989-01-06 1992-06-03 Protein Engineering Corporation Generation and selection of novel dna-binding proteins and polypeptides
US5198346A (en) * 1989-01-06 1993-03-30 Protein Engineering Corp. Generation and selection of novel DNA-binding proteins and polypeptides
US5096815A (en) * 1989-01-06 1992-03-17 Protein Engineering Corporation Generation and selection of novel dna-binding proteins and polypeptides
EP0452413A1 (fr) * 1989-01-06 1991-10-23 Dyax Corp. Production et selection de proteines et de polypeptides nouveaux de liaison a l'adn
US5545727A (en) * 1989-05-10 1996-08-13 Somatogen, Inc. DNA encoding fused di-alpha globins and production of pseudotetrameric hemoglobin
EP0700997A1 (fr) 1989-05-10 1996-03-13 Somatogenetics International, Inc. Production d'hémoglobine et de ses analogues par des bactéries et chez la levure
US6274331B1 (en) 1989-05-10 2001-08-14 Somatogen, Inc. Method of determining a functional linker for fusing globin subunts
US7893007B2 (en) 1989-09-01 2011-02-22 Dyax Corp. Directed evolution of novel binding proteins
US7413537B2 (en) 1989-09-01 2008-08-19 Dyax Corp. Directed evolution of disulfide-bonded micro-proteins
US5580958A (en) * 1990-01-26 1996-12-03 La Jolla Cancer Research Foundation Polypeptides encoding transcriptional activators and uses thereof
US5863737A (en) * 1990-01-26 1999-01-26 La Jolla Cancer Research Foundation Methods of using polypeptides encoding transcriptional activators
US5734036A (en) * 1990-01-26 1998-03-31 The Burnham Institute Nucleic acids encoding transcriptional activators
WO1991011521A1 (fr) * 1990-01-26 1991-08-08 La Jolla Cancer Research Foundation Polypeptides de codage d'activateurs de la transcription et leurs utilisations
US6869765B2 (en) 1991-06-27 2005-03-22 Genelabs Technologies, Inc. Sequence-directed DNA-binding molecules compositions and methods
US5759517A (en) * 1991-11-08 1998-06-02 Somatogen, Inc. Hemoglobins as drug delivery agents
EP0684999A4 (fr) * 1992-12-23 1997-05-07 Genelabs Tech Inc Molecules, compositions et procedes de liaison d'adn specifiques a des sequences.
EP0684999A1 (fr) * 1992-12-23 1995-12-06 Genelabs Technologies, Inc. Molecules, compositions et procedes de liaison d'adn specifiques a des sequences
US5871902A (en) * 1994-12-09 1999-02-16 The Gene Pool, Inc. Sequence-specific detection of nucleic acid hybrids using a DNA-binding molecule or assembly capable of discriminating perfect hybrids from non-perfect hybrids
US8114962B2 (en) 1994-12-09 2012-02-14 The Gene Pool, Inc. Method of detection of nucleic acids with a specific sequence composition

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