Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-28
2001-09-25
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S194000, C530S350000, C536S023200, C536S023500
Reexamination Certificate
active
06294334
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular genetics and veterinary medicine. More specifically, the present invention relates to the mutation of a DNA-dependent protein kinase protein which results in equine severe combined immunodeficiency and a diagnostic test to identify carriers of the mutation.
2. Description of the Related Art
V(D)J rearrangement is the molecular mechanism b y which distinct gene segments (V, D, and J) are joined to form the coding sequences of immunoglobulin (Ig) and T cell receptor (TCR) variable regions. The rearrangement process is targeted by simple DNA sequence elements (recombination signal sequences, RSS) found immediately adjacent to all functional immune receptor gene segments and involves two double-stranded DNA cuts and subsequent re-ligations. This process results in the formation of two new DNA joints; coding joints which contain the coding information, and signal joints which contain the two recombination signal sequences. V(D)J rearrangement is mediated by a lymphoid-specific endonuclease (the RAG 1 and RAG 2 proteins) and ubiquitously expressed components of the double strand break repair pathway. The centrality of V(D)J recombination to the development of the vertebrate immune system is evident in situations where the process is defective.
Defective V(D)J recombination results in a complete block of B and T cell lymphopoiesis and the disease severe combined immunodeficiency (SCID). The first example of defective V(D)J recombination was described in 1983 by Bosma and colleagues, relating to a spontaneous mutation in mice that results in severe combined immunodeficiency (C.B-17 mice). In severe combined immunodeficiency mice, the only step in V(D)J recombination that appears to be impaired is resolution of coding ends. Instead of being resolved into functional immune receptors, cleaved coding ends accumulate abnormally in developing severe combined immunodeficiency lymphocytes. However, cleaved signal ends are resolved at a similar rate as in wild type lymphocytes in mice.
In 1990, it was demonstrated that the defect in severe combined immunodeficiency mice not only impairs V(D)J recombination, but also affects the more general process of double strand break repair (DSBR). This observation was the first to link V(D)J recombination and double strand break repair. In recent years it has been shown that at least four factors are required for both V(D)J recombination and double strand break repair: the Ku heterodimer, DNA-dependent protein kinase
catalytic
subunit (PK
CS
), XRCC4, and XRCC6.
Recently, defective DNA-dependent protein kinase
catalytic
subunit has been identified as the determinative factor in C.B-17 severe combined immunodeficiency mice. The DNA-end binding Ku heterodimer interacts with DNA-dependent protein kinase
catalytic
subunit to generate a protein kinase (DNA-PK) that is dependent on linear DNA for activation (i.e., DNA-dependent protein kinase). DNA-dependent protein kinase
catalytic
subunit is related to the phosphatidylinositol 3-kinase family whose members function in a variety of roles such as signal transduction by phosphorylation of phospholipids, control of cell cycle progression, and maintenance of telomere length.
Although DNA-dependent protein kinase
catalytic
subunit has been implicated in a variety of different processes, its precise role is unclear. The factor defective in the double strand break repair mutant CHO cell line XRI. In sum, defects in either the lymphocyte specific components of the V(D)J recombinase (RAG 1 -/- mice, RAG 2-/- mice, RAG-deficient children) or any one of these double strand break repair factors (C.B-17 severe combined immunodeficiency mice, Arabian severe combined immunodeficiency foals, Ku80 -/- mice) results in B and T lymphocyte development being blocked and similar phenotypes are observed.
The occurrence of severe combined immunodeficiency in Arabian foals was initially reported in 1973 by McGuire and Poppie. Recently, it was demonstrated that severe combined immunodeficiency in Arabian foals is explained by a severe block in the generation of specific immune receptors because of defective V(D)J rearrangement. As is the case in murine severe combined immunodeficiency, equine severe combined immunodeficiency cells are hypersensitive to DNA damage because of severely diminished levels of DNA-dependent protein kinase
catalytic
subunit. However, these two genetic defects have important mechanistic differences. Unlike severe combined immunodeficiency mice that are preferentially defective in coding resolution, severe combined immunodeficiency foals are defective in both coding and signal resolution.
The prior art is deficient in the lack of effective means of determining the presence of the genetic deteminant for equine severe combined immunodeficiency in an animal of interest. The present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
Previously, the mechanistic defect responsible for the autosomal recessive disease severe combined immunodeficiency (SCID) in Arabian foals was reported to involve a V(D)J recombination. As with the murine counterpart of SCID, cells from SCID foals have severely depressed levels of DNA dependent protein kinase activity because of a deficiency in the catalytic subunit of the enzyme (DNA-dependent protein kinase
catalytic
subunit). However, unlike SCID mice which are specifically impaired in their ability to resolve immune receptor coding joints, SCID foals are incapable of resolving both coding and signal ends.
The present invention presents the genotypic analysis of the defective DNA-dependent protein kinase
catalytic
subunit allele in Arabian horses and provides the sequence for the normal and mutant DNA-dependent protein kinase
catalytic
subunit gene and protein. These results formally establish the importance of the DNA-dependent protein kinase
catalytic
subunit in signal end resolution during V(D)J rearrangement.
In the equine severe combined immunodeficiency mutation, a frameshift deletion prematurely truncates the DNA-dependent protein kinase
catalytic
subunit at amino acid 3160 of the normal 4127 amino acid polypeptide. This truncation apparently results in a kinase negative version of the protein. In contrast, the DNA-dependent protein kinase
catalytic
subunit mutation responsible for severe combined immunodeficiency in C.B-17 mice may not completely ablate kinase activity. Thus, one explanation for the mechanistic differences in these two DNA-dependent protein kinase
catalytic
subunit defects models is that low levels of DNA-dependent kinase (likely present in severe combined immunodeficiency mice) can support signal end resolution, but normal levels are required to support coding resolution.
In one embodiment of the present invention, there is provided a composition of matter comprising an isolated DNA molecule encoding a DNA-dependent protein kinase
catalytic
subunit protein in Arabian horses having a sequence shown in SEQ ID No. 28.
In another embodiment of the present invention, there is provided a composition of matter comprising an oligonucleotide having a sequence selected from the group of SEQ ID Nos. 24 and 25. These oligonucleotides precisely span the SCID-determinant region of the DNA-PK
CS
gene, and are diagnostic for the normal and SCID alleles, respectively.
In yet another aspect of the present invention, there is provided an isolated DNA sequence having the sequence shown in SEQ ID No: 26 or SEQ ID No: 27.
In yet another aspect of the present invention, there is provided a method of identifying an Arabian horse that is a carrier of equine severe combined immunodeficiency, comprising the step of: determining whether said horse has a mutation in a SCID determinant region of a DNA-dependent protein kinase
catalytic
subunit gene. In one embodiment of this aspect of the present invention, there is provided a method of identifying an Arabian horse that is a carrier of equine severe combined immun
Board of Regents , The University of Texas System
Fulbright & Jaworski
Horlick Kenneth R.
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