Wise/Sost nucleic acid sequences and amino acid sequences

a nucleic acid sequence and amino acid sequence technology, applied in the direction of immunoglobulins, peptides, drugs, etc., can solve the problems of specific developmental defects, complex analysis of posteriorizing signals in neural patterning, and clear complex anterior region patterning, so as to increase bone deposition, increase bone deposition, and increase bone deposition

Inactive Publication Date: 2004-02-05
STOWERS INST FOR MEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

0132] The present invention relates to a family of nucleic acid molecules, which encode polypeptides that bind to LRP and likely regulate the Wnt pathway and, resultingly, regulate bone deposition. The polypeptides will also regulate ocular and tooth development. The present invention further relates to proteins and polypeptides, or amino acid sequences, expressed from the family of nucleic acid molecules, which regulate bone deposition through LRP interaction. In particular, a nucleic acid molecule family, which includes the Wise and Sost genes, can be used with the present invention, as well as the family of amino acid sequences expressed therefrom. When the above family of amino acid sequences, including Wise and Sost, are allowed to bind to an LRP protein, bone deposition is regulated. When the family of amino acid sequences are prevented from binding to an LRP protein, deposition of bone will increase.
0133] Antisense RNAs or oligonucleotides can be used to block translation of mRNA related to or translated from the above described nucle

Problems solved by technology

However, patterning of the anterior region is clearly more complicated than a simple default state of neural induction.
Analysis of posteriorizing signals in neural patterning is complicated by the tissue interactions and dynamic morp

Method used

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  • Wise/Sost nucleic acid sequences and amino acid sequences
  • Wise/Sost nucleic acid sequences and amino acid sequences
  • Wise/Sost nucleic acid sequences and amino acid sequences

Examples

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example 1

[0193] Functional screens in Xenopus were performed with the aim of identifying factors derived from tissues surrounding the neural tube that alter A-P patterning in Noggin-treated animal caps. Two clones were isolated, one encoded a truncated .beta.-catenin and the other a novel secreted protein, which was named Wise. Isolation of the two clones is described below.

[0194] FIG. 1A provides an overview of how factors which impacted patterning were determined. Chick embryo somites, which are capable of transforming pre-otic rhombomeres into a more posterior neural tissue were collected together with overlying ectoderm and underlying endoderm. mRNA was collected from the tissue, which was then used to make a cDNA library. This provided a source of putative posteriorizing factors.

[0195] The cDNA library was made from stage 8-13, (Hamburger and Hamilton, 1951) chick embryos using tissues surrounding the neural tube (FIG. 1A) from axial levels capable of inducing Hoxb9 expression in grafti...

example 2

[0202] To isolate a frog clone, Xenopus stage 25 embryos were collected and a cDNA library was formed. This was used as a template for RT-PCR. Using degenerate primers, designed on the basis of conserved regions between chick and mouse Wise, 500 bp fragments were sub-cloned into pBluescriptIIKS (Stratagene) and sequenced. The degenerate primers used were upstream, SEQ ID NO 129: 5'-GCTTT(T / T)AA(A / G)AA(C / T)GATGCCAC-3'; and downstream, SEQ ID NO 130: 5'-GTGAC(T / C)AC(T / G / A)GT(T / G)ATTTTGTA-3'. Two different clones in the frog were identified (XWise-A and XWise-B) presumably resulting from the pseudotetraploid Xenopus genome. For each clone, 5' and 3' flanking sequences were identified by PCR using a Xenopus stage 35 cDNA library. Standard PCR methods are described in U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202; Saiki et al., Science 230:1350-1354 (1985); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif. (1990).

[0203] The predi...

example 3

[0206] A signal sequence motif is present at the N-terminus of Wise, and its secretion was confirmed by Western blotting following expression of an HA-tagged version of the protein in Xenopus oocytes (FIG. 2C) and COS cells. More particularly, Wise was injected in an amount equal to 30 ng / embryo. Western blot analysis detected HA-tagged Wise protein secreted into the medium following RNA injection into oocytes. FIG. 2C, related to the control of uninjected oocytes. Secretion of Wise was confirmed by expression of an HA-tagged version of the protein in Xenopus oocytes and COS cells. The protein was detected in both cell extracts and the culture medium (FIG. 2C). It was observed that Wise encoded a signal sequence motif at its N-terminus, suggesting that the protein is secreted.

[0207] Further, the ability of Wise to posteriorize neural tissue in a cell non-autonomous manner was tested by using a tissue recombination assay in which a Wise-expressing animal cap was combined with a noggi...

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Abstract

The present invention relates to nucleic acid sequences and amino acid sequences which influence bone deposition, the Wnt pathway, ocular development, tooth development, and may bind to LRP. The nucleic acid sequence and polypeptides include Wise and Sost as well as a family of molecules which express a cysteine knot polypeptide. Additionally, the present invention relates to various molecular tools derived from the nucleic acids and polypeptides including vectors, transfected host cells, monochronal antibodies, Fab fragments, and methods for impacting the pathways.

Description

[0001] This application is a non-provisional patent application based on U.S. Provisional Patent Application Serial No. 60 / 388,970, filed Jun. 14, 2002.[0002] The present invention relates to Wise and Sost nucleic acid sequences and related amino acid sequences that can be used to influence bone deposition, the Wnt pathway, tooth development, and ocular development. In particular, the present invention also relates to nucleic acid sequences and amino acid sequences that optionally regulate or suppress bone deposition. The present invention relates to a family of nucleic acid molecules which expresses a family of amino acid sequences, some of which are characterized by a cysteine knot, such as the Wise and Sost proteins. The present invention also relates to resultant molecular biology tools derived from Wise or Sost, including plasmids, transfected host cells, antibodies, tranfected host organisms, and knockout organisms. Finally, the present invention relates to the interaction bet...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61K48/00C07K14/47C07K14/705
CPCA01K2217/05A01K2217/075A01K2227/105A61K38/00A61K48/00C12N15/85C07K14/47C07K14/4702C07K14/705C07K16/22G01N33/74A61K2039/505A61P19/08A61P43/00Y02A50/30
Inventor KRUMLAUF, ROBBELLIES, DEBRA
Owner STOWERS INST FOR MEDICAL RES
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