Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-12-17
2003-10-28
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C536S023100
Reexamination Certificate
active
06638731
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding either of two alternative splice variants of human sel-10, one of which is expressed in hippocampal cells, and one of which is expressed in mammary cells. The invention also provides isolated sel-10 polypeptides.
BACKGROUND OF THE INVENTION
Alzheimer's disease (AD) is a degenerative disorder of the central nervous system which causes progressive memory and cognitive decline during mid to late adult life. The disease is accompanied by a wide range of neuropathologic features including extracellular amyloid plaques and intra-neuronal neurofibrillary tangles. (Sherrington, R., et al.;
Nature
375: 754-60 (1995)). Although the pathogenic pathway leading to AD is not well understood, several genetic loci are known to be involved in the development of the disease.
Genes associated with early onset Alzheimer's disease (AD) have been identified by the use of mapping studies in families with early-onset AD. These studies have shown that genetic loci on chromosomes 1 and 14 were likely to be involved in AD. Positional cloning of the chromosome 14 locus identified a novel mutant gene encoding an eight-transmembrane domain protein which subsequently was named presenilin-1 (PS-1). (Sherrington, R., et al.;
Nature
375: 754-60 (1995)). Blast search of the human EST database revealed a single EST exhibiting homology to PS-1, designated presenilin-2 (PS-2) which was shown to be the gene associated with AD on chromosome 1. (Levy-Lahad, E. et al.,
Science
269:973-977 (1995); Rogaev, E. I., et al.,
Nature
376: 775-8 (1995); Li, J. et al.,
Proc. Natl. Acad. Sci. U.S.A.
92: 12180-12184 (1995)).
Mutations in PS-1 and PS-2 that are associated with Alzheimer's disease are primarily missense mutations. Both PS-1 and PS-2 undergo proteolytic processing, which can be altered by the point mutations found in familial Alzheimer's disease [Perez-Tur, J. et al.,
Neuroreport
7: 297-301 (1995); Mercken, M. et al.,
FEBS Lett.
389: 297-303 (1996)]. PS-1 gene expression is widely distributed across tissues, while the highest levels of PS-2 mRNA are found in pancreas and skeletal muscle. (Li, J. et al.,
Proc. Natl. Acad. Sci. U.S.A.
92: 12180-12184 (1995); Jinhe Li, personal communication). The highest levels of PS-2 protein, however, are found in brain (Jinhe Li, personal communication). Both PS-1 and PS-2 proteins have been localized to the endoplasmic reticulum, the Golgi apparatus, and the nuclear envelope. (Jinhe Li, personal communication; Kovacs, D. M. et al.,
Nat. Med.
2:224-229 (1996); Doan, A. et al.,
Neuron
17: 1023-1030 (1996)). Mutations in either the PS-1 gene or the PS-2 gene alter the processing of the amyloid protein precursor (APP) such that the ratio of A-beta
1-42
is increased relative to A-beta
1-40
(Scheuner, D. et al.,
Nat. Med.
2: 864-870 (1996)). When coexpressed in transgenic mice with human APP, a similar increase in the ratio of A-beta
1-42
as compared to A-beta
1-40
is observed (Borchelt, D. R. et al.,
Neuron
17: 1005-1013 (1996); Citron, M. et al.,
Nat. Med.
3: 67-72 (1997); Duff, K. et al.,
Nature
383: 710-713 (1996)), together with an acceleration of the deposition of A-beta in amyloid plaques (Borchelt et al.,
Neuron
19: 939 (1997).
Despite the above-described observations made with respect to the role of PS-1 and PS-2 in AD, their biological function remains unknown, placing them alongside a large number of human disease genes having an unknown biological function. Where the function of a gene or its product is unknown, genetic analysis in model organisms can be useful in placing such genes in known biochemical or genetic pathways. This is done by screening for extragenic mutations that either suppress or enhance the effect of mutations in the gene under analysis. For example, extragenic suppressors of loss-of-function mutations in a disease gene may turn on the affected genetic or biochemical pathway downstream of the mutant gene, while suppressers of gain-of-function mutations will probably turn the pathway off.
One model organism that can be used in the elucidation of the function of the presenilin genes is
C. elegans
, which contains three genes having homology to PS-1 and PS-2, with sel-12 having the highest degree of homology to the genes encoding the human presenilins. Sel-12 was discovered in a screen for genetic suppressers of an activated notch receptor, lin-12(d) (Levitan, D. et al.,
Nature
377: 351-354 (1995)). Lin-12 functions in development to pattern cell lineages. Hypermorphic mutations such as lin-12(d), which increase lin-12 activity, cause a “multi-vulval” phenotype, while hypomorphic mutations which decrease activity cause eversion of the vulva, as well as homeotic changes in several other cell lineages (Greenwald, I., et al.,
Nature
346: 197-199 (1990); Sundaram, M. et al.,
Genetics
135: 755-763 (1993)). Sel-12 mutations suppress hypermorphic lin-12(d) mutations, but only if the lin-12(d) mutations activate signaling by the intact lin-12(d) receptor (Levitan, D. et al.,
Nature
377: 351-354 (1995)). Lin-12 mutations that truncate the cytoplasmic domain of the receptor also activate signaling (Greenwald, I., et al.,
Nature
346: 197-199 (1990)), but are not suppressed by mutations of sel-12 (Levitan, D. et al.,
Nature
377: 351-354 (1995)). This implies that sel-12 mutations act upstream of the lin-12 signaling pathway, perhaps by decreasing the amount of functional lin-12 receptor present in the plasma membrane. In addition to suppressing certain lin-12 hypermorphic mutations, mutations to sel-12 cause a loss-of-function for egg laying, and thus internal accumulation of eggs, although the mutants otherwise appear anatomically normal (Levitan, D. et al.,
Nature
377: 351-354 (1995)). Sel-12 mutants can be rescued by either human PS-1 or PS-2, indicating that sel-12, PS-1 and PS-2 are functional homologues (Levitan, D., et al.,
Proc. Natl. Acad. Sci. U.S.A
93: 14940-14944 (1996)).
A second gene, sel-10, has been identified in a separate genetic screen for suppressors of lin-12 hypomorphic mutations. Loss-of-function mutations in sel-10 restore signaling by lin-12 hypomorphic mutants. As the lowering of sel-10 activity elevates lin-12 activity, it can be concluded that sel-10 acts as a negative regulator of lin-12 signaling. Sel-10 also acts as a negative regulator of sel-12, the
C. elegans
presenilin homologue (Levy-Lahad, E. et al.,
Science
269:973-977 (1995)). Loss of sel-10 activity suppresses the egg laying defect associated with hypomorphic mutations in sel-12 (Iva Greenwald, personal communication). The effect of loss-of-function mutations to sel-10 on lin-12 and sel-12 mutations indicates that sel-10 acts as a negative regulator of both lin-12
otch and presenilin activity. Thus, a human homologue of
C. elegans
sel-10 would be expected to interact genetically and/or physiologically with human presenilin genes in ways relevant to the pathogenesis of Alzheimer's Disease.
In view of the foregoing, it will be clear that there is a continuing need for the identification of genes related to AD, and for the development of assays for the identification of agents capable of interfering with the biological pathways that lead to AD.
INFORMATION DISCLOSURE
Hubbard E J A, Wu G, Kitajewski J, and Greenwald I (1997) Sel-10, a negative regulator of lin-12 activity in
Caenorhabditis elegans,
encodes a member of the CDC4 family of proteins. Genes & Dev 11:3182-3193.
Greenwald-I; Seydoux-G (1990) Analysis of gain-of-function mutations of the lin-12 gene of
Caenorhabditis elegans. Nature.
346: 197-9
Kim T-W, Pettingell W H, Hallmark O G, Moir R D, Wasco W, Tanzi R (1997) Endoproteolytic cleavage and proteasomal degradation of presenilin 2 in transfected cells. J Biol Chem 272:11006-11010.
Levitan-D; Greenwald-I (1995) Facilitation of lin-12-mediated signalling by sel-12, a
Caenorhabditis elegans
S182 Alzheimer's disease gene.
Nature.
377: 351-4.
Levitan-D;
Gurney Mark E.
Li Jinhe
Pauley Adele M.
Kerber Lori
Kunz Gary
Pharmacia and Upjohn Company
Rehberg Edward F.
Turner Sharon
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