Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-09-21
2001-11-13
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S440000, C435S455000
Reexamination Certificate
active
06316261
ABSTRACT:
BACKGROUND OF THE INVENTION
Upon infection of cells by retroviruses, a large-size viral nucleoprotein complex is observed in the cytoplasm. These complexes vary in size from 160S for murine leukemia virus (MLV) to 80S for human immunodeficiency virus (HIV) type-1. The viral nucleoprotein complexes contain newly synthesized blunt-ended linear viral DNA that is subsequently trimmed by two nucleotides at its 3′ termini by the viral integrase(IN). IN can catalyze the concerted integration of the recessed viral DNA termini into exogenous DNA targets (full-site reaction) mimicking the in vivo reaction. Both the trimming and integration of the viral DNA by IN in the nucleoprotein complexes requires the metal cofactor Mg
2+
.
Similar trimming and integration reactions can also be catalyzed by purified IN in vitro. IN derived from bacterial expression systems or purified from avian myeloblastosis virus (AMV) can trim two nucleotides from the termini of oligonucleotides or DNA restriction fragments containing viral long terminal repeat (LTR) sequences . The recessed DNA substrates can then be integrated into other DNA targets by IN. The majority of the observed integration events with these substrates involve the insertion of a single LTR terminus into one strand of the target DNA (half-site reaction). Expressed IN requires Mn
2+
for efficient catalysis of either the trimming or the strand transfer reactions while AMV IN can effectively use Mg
2+
or Mn
2+
for these reactions. To date, expressed IN is not capable of efficiently performing the concerted insertion of viral-like DNA substrates into target DNA using either divalent
DESCRIPTION OF THE INVENTION
The present invention relates to a method and a kit for efficient integration of DNA donor molecules into DNA target molecules using retrovirus integrase , hereinafter referred to as IN. The present invention also relates to a method for studying integrase such as screening of HIV-1 or HIV-2 integrase inhibitors, production of transgenic animals and gene transfer.
According to the present invention, IN purified from virus or IN in virus particles and specifically designed donor substrates, hereinafter referred to as donor DNA, are used to mimic the integration of retrovirus DNA in vivo. This does not exclude the possibility that suitable expressed IN could perform concerted integration. The reaction assay conditions and donor DNA molecules being such that they induce a high cyclization probability for the donor DNA molecules in the reaction solution Concerted integration of a single donor DNA requires the transient formation of a circle molecule prior to this event. The donor DNA molecules could also have regions capable of readily bending in solution and located appropriately to enhance cyclization of the donor DNA molecules in reaction solution. It should be understood that cyclization of donor DNA is necessary for the concerted integration of the two donor termini, in which each termini has a dimer of IN. Furthermore, the condition inducing cyclization may also be influenced by temperature, concentration of reactants, order of addition of reactants, and divalent metal cation.
The donor DNA molecules should have at least one unique restriction site for analysis of successful and efficient concerted integration. This analysis may be readily performed on agarose gels. Preferably, the donor DNA molecules have at least one genetic marker for isolation or characterization of concerted integration products.
Preferably, the donor DNA molecules can be labeled at their 5′ ends, using radioactive probes but not excluding other detection probes.
For efficient concerted integration of a donor DNA molecule into a target molecule, IN and the donor DNA molecule are incubated in reaction buffer which allows formation of transient circles. Preferably, the reaction buffer contains 20 mM MgCl
2
,10% dimethyl sulfoxide, 0.05% Nonidet P-40, 5% polyethylene glycol, and 200 mM NaCl. After formation of transient circles of donor DNA with IN, the target molecule is introduced into the reaction and the integration reaction is allowed to proceed. After the integration reaction is complete, the reaction products may be analyzed for the unique restriction site and the genetic marker on the donor DNA molecule.
Preferably, the donor molecules should be of a optimum size for the cyclization of the DNA to occur in solution. The size can vary with certain DNAs but should be in the 700 bp range, with several hundred base pairs on either side. It should be understood that small size molecules are too rigid for cyclization in assay solution while large size DNA molecules are too flexible for stable cyclization. DNA sequences that readily bend in solution may be introduced in the donor DNA thereby increasing the size of the donor that can be used because its cyclization frequency should be higher.
Preferably, the donor molecule should lack internal DNA sequences, such as A/T rich regions, that IN can readily bind to thereby lowering the concentration of IN in solution. Also, non specific binding of IN to regions other then the donor termini would allow formation of DNA loop structures that inhibit the formation of transient circles by IN.
Preferably, the target DNA molecule for in vitro analysis should be a circle whose size could be easily varied. The circular target DNA and unique restriction site on the donor DNA molecules makes analysis of the concerted integration reaction easy by the use of gel electrophoresis. This invention could be readily applied to the study and identification of HIV-1 or HIV-2 IN inhibitors. HIV-1 virions can readily perform the concerted integration of the appropriate DNA donor that matches the size criteria stated above into a circular target.
Preferably, for the production of transgenics and for the practice of gene transfer, the donor molecule would be complexed with IN prior to their transfer into the animals or cells. As noted below, this formation of IN/donor preintegration complexes is the most efficient method to produce concerted integration recombinants with target DNA. In transgenics and gene transfer the host genome acts as the target DNA.
REFERENCES:
patent: 5612180 (1997-03-01), Brown et al.
patent: 5728551 (1998-03-01), Devine et al.
patent: 5763240 (1998-06-01), Zarling et al.
patent: 5811270 (1998-09-01), Grandgenett
patent: 5830707 (1998-11-01), Bushman et al.
patent: WO 92/03578 (1992-03-01), None
Argos et al. EMBO J. vol. 5(2): 433-440, 1986.*
Kilby et al. Trends in Genetics. vol. 9, pp. 416-421, 1993.*
Fujiwara et al. Proc. Nat'l. Acad. Sci. U.S.A. vol. 86, pp. 3065-3069, May 1989.*
Bushman et al. Science. vol. 249, pp. 1555-1558, Nov. 1990.*
Katz et al. Cell. vol. 63, p. 86-95, Oct. 1990.*
Fitzgerald et al. J. Virology. vol. 66(11), pp. 6257-6263, Nov. 1992.*
Pryciak et al. Cell. vol. 69, pp. 769-780, May 1992.*
Kitamura et al. Proc. Nat'l. Acad. Sci. U.S.A. vol. 89, pp. 5532-5536, Jun. 1992.*
Leavitt et al. The Journal of Biological Chemistry. vol. 268, pp. 2113-2119, Jan. 1993.*
Bushman et al., Retroviral DNA Integration Directed by HIV Integration Protein in Vitro,Science249:1555-1558 (1990).
Fitzgerald et al., Concerted Integration of Viral DNA Termini by Purified Avian Myeloblastosis Virus Integrase,Journal of Virology66(11):6257-6263 (1992).
Fujiwara et al., Integration of mini-retroviral DNA: A cell-free reaction for biochemical analysis of retroviral integration,Proc. Natl. Acad. Sci. USA86:3065-3069 (1989).
Katz et al., The Avian Retroviral IN Protein Is Both Necessary and Sufficient for Integrative Recombination In Vitro,Cell63:87095 (1990).
Vink et al., The human immunodeficiency virus integrase protein,TIG9(12):433-437 (1993).
Howell & Haferkamp L.C.
Saint Louis University
Yucel Remy
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