Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-06-23
2001-02-13
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100, C536S023400, C536S023500
Reexamination Certificate
active
06187584
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a recombinogenic motif capable of transposase activities that is important to the regulation and function of Herpes virus replication, V(D)J recombination, and immunoglobulin class switching which can be used to develop immunosuppressant and anti-viral agents. The present invention also relates to a site-specific DNA binding region for V(D)J and V(D)J-like recombination signals.
BACKGROUND OF THE INVENTION
Recently, a motif shared between retroviral integrases and invertebrate transposase molecules, termed the D35E motif, has been identified. This motif is partially characterized by the first and last amino acid residues of the motif, which are an aspartate (D) and a glutamate (E), respectively. In most transposases that have been characterized, the spacing between the D and E residues is 35 amino acids, however this interval is not absolutely conserved, with spacings of 34 and 39 amino acids also having been identified. This motif is putatively involved in strand cleavage and transfer of targeted DNA, while site-specificity is conferred by a separate region of the molecule.
Progress has been made in understanding the mechanism of invertebrate transposition and retroviral integration to the point that this common D35E catalytic site has been defined in both processes, which in the case of the Tc elements of
C. elegans
has been shown to be a functional requirement for site-specific recombination.
Viruses such as Herpes viruses and the V(D)J recombination pathway of higher vertebrates undergo regulated site-specific recombination. Similarities between terminal and recombination signal sequences suggest that both the Herpes viruses and the immunoglobulin recombination pathway share a conserved recombination mechanism.
In the case of the Herpes viruses, the virus enters the cell in a linear form, which subsequently circularizes to enter a latent state. Following activation of the lytic cycle, the covalently closed genome then replicates via a putative “rolling circle” to yield concatameric intermediates which are then cleaved into infectious linear monomers. The molecules responsible for the Herpes virus recombination events have not been identified. There is no current description of the mechanism that Herpes viruses utilize to form the viral episome from the linear infectious form during the establishment of latency.
In vertebrates, expression of the recombinase activating gene (RAG) proteins has been identified as both necessary and sufficient to direct V(D)J recombination. In this recombination, a regulated series of site-specific recombinations occurs during development of the T and B cell lineages utilizing an interaction between “V(D)J signals” and the recombinase activating genes (RAG), RAG-1 and RAG-2. While it is known that V(D)J recombination is controlled by recombinase activating genes, the mechanism of V(D)J recombination on a molecular level is not understood.
There is a wide spectrum of need for methods and materials to control recombination events of viruses and the immune response. Interaction between recombinogenic viruses such as Herpes viruses and recombinogenic components of the immune system is in fact problematic. However, the complexity of viral life cycles and of the molecular recombination mechanisms in the immune response have hindered development of such methods and materials. Prior to the present invention, a critical component involved in recombination in non-retroviral viral life cycles and in the immune system of higher vertebrates was not appreciated. Thus, there remains a need to elucidate this component and to develop reagents and methods that would have important implications for viral infection related to pathogenesis and autoimmunity, as well as applications for gene therapy and vaccine development.
SUMMARY OF THE INVENTION
The present invention generally relates to the identification and use of peptides derived from a recombinogenic motif that is capable of transposase activities. Such a motif is important to the regulation and function of Herpes virus replication, V(D)J recombination, retroviral integrase function and immunoglobulin class switching, and can be used to develop immunosuppressant, anti-viral agents, and vectors for gene therapy.
Another embodiment of the present invention relates to the identification and use of a site-specific DNA binding region for V(D)J and V(D)J-like recombination signal sequences.
One embodiment of the present invention relates to a method to identify whether a first amino acid sequence includes a recombinogenic amino acid sequence. This method includes the steps of (a) searching the first amino acid sequence to identify at least one amino acid sequence comprising an initial aspartate or glutamate which is followed at least about 30 amino acid residues downstream by a terminal aspartate or glutamate; (b) generating randomizations of at least one of the amino acid sequences; and (c) aligning at least one of the randomizations with a second amino acid sequence to identify at least one first alignment wherein the probability of the first alignment occurring is not consistent with chance. The second amino acid sequence can be a D35E amino acid consensus sequence or a first D35E amino acid sequence. This method can further include the step of identifying second alignments between the first alignment and a second D35E amino acid sequence. This second D35E amino acid sequence can be derived from an organism category which includes a family, genus, or species. This step of identifying includes maximizing sequence similarity between the first alignment and the second D35E sequence using amino acid similarity default values. The default values include the following groups: (i) neutral/weakly hydrophobic residues which include the amino acid residues P, A, G, S and T; acidic/hydrophilic residues which include the amino acid residues Q, N, E, B, D and Z; basic/hydrophilic residues which include the amino acid residues H, K and R;
hydrophobic/aliphatic residues which include the amino acid residues L, I, V and M; hydrophobic/aromatic residues which include the amino acid residues F, Y, and W; and C residues. As used herein, the residues designated by single letters use the standard one-letter nomenclature for amino acid residues known in the art.
One embodiment of the present invention relates to a method to identify whether a first nucleic acid sequence includes a recombinogenic nucleic acid sequence. This method includes the steps of (a) searching the first nucleic acid sequence to identify at least one nucleic acid sequence that encodes an amino acid sequence comprising an initial aspartate or glutamate which is followed at least about 30 amino acid residues downstream by a terminal aspartate or glutamate; (b) generating randomizations of at least one of the nucleic acid sequences; and (c) aligning at least one of the randomizations with a second nucleic acid sequence encoding a second amino acid sequence to identify at least one first alignment wherein the probability of the first alignment occurring is not consistent with chance. The second amino acid sequence can be a D35E amino acid consensus sequence or a first D35E amino acid sequence. This method can further include the step of identifying second alignments between the first alignment and a second D35E nucleic acid sequence. This second D35E nucleic acid sequence can be derived from an organism category which includes a family, genus, or species. This step of identifying includes maximizing sequence similarity between the first alignment and the second nucleic acid sequence using similarity default values for the amino acid residues encoded by the nucleic acid sequences. The default values include the following groups: (i) neutral/weakly hydrophobic residues which include the amino acid residues P, A, G, S and T; acidic/hydrophilic residues which include the amino acid residues Q, N, E, B, D and Z; basic/hydrophilic residues which include the amino acid residues H, K and R; hydrophobic/aliphatic resi
Dreyfus David H.
Gelfand Erwin W.
McKelvey Terry
National Jewish Medical and Research Center
Sheridan & Ross P.C.
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