Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1997-07-10
2001-10-23
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C536S023100, C536S023500, C536S024500, C435S069100, C435S320100, C435S325000, C435S252000, C435S001200, C435S254200
Reexamination Certificate
active
06307019
ABSTRACT:
Throughout this application, various publications are referenced in parenthesis. Full citations for these publications may be found listed at the end of the specification. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein.
INTRODUCTION
A fundamental problem in mammalian embryology is the mechanism by which the egg cylinder, an epithelial cup in which only the dorsal-ventral axis is established, gives rise to an embryo with anterior-posterior (A-P) polarity. In the mouse, the earliest morphological manifestation of the A-P axis is the delamination of mesoderm in the primitive streak at ~E6.5. The position of the streak cannot be predicted by earlier morphological asymmetries in the embryo (Gardner et al., 1992), and the regulative abilities of early mouse embryos appear to rule out axis determination by localized determinants from the egg. While a few secreted factors or transcription factors are expressed asymmetrically in the egg cylinder shortly before primitive streak formation, and thus might be involved in induction of the streak, their roles in this process have not been established (reviewed by Bachvarova, 1996; Conlon and Beddington, 1995).
In the amphibian embryo, the dorsal-ventral axis (the second axis to be specified, analogous to the A-P axis of the mouse) is determined by the point of sperm entry and subsequent cortical rotation. This rotation generates the Nieuwkoop Center, a group of dorsal/vegetal blastomeres that induce formation of the Spemann organizer. Transplantation of the Nieuwkoop Center or the organizer to an ectopic position induces the formation of a complete secondary axis, i.e., notochord, somites, neural tube, and head structures (reviewed by Slack, 1994). Recent studies suggest that the formation of the Nieuwkoop Center depends on activation of components of the Wnt signaling pathway (Carnac et al., 1996; Fagotto et al., 1997; Wylie et al., 1996). The Wnts are a family of secreted polypeptides related to Drosophila wingless, whose receptors are believed to be members of the frizzled family (reviewed by Miller and Moon, 1996). The next known component of the signaling pathway is Dishevelled (Dsh), a cytoplasmic protein that, when activated by a Wnt signal, inhibits the activity of glycogen synthase kinase-3 (GSK-3). In the absence of a Wnt signal, GSK-3 activity leads (directly or indirectly) to the phosphorylation and consequent degradation of &bgr;-catenin. In the presence of a Wnt signal, GSK-3 is inhibited, increasing the cytosolic level of &bgr;-catenin, and promoting its interaction with downstream effectors.
A role for the Wnt signaling pathway in development of the amphibian embryonic axis was revealed by the ability of several Wnts, or downstream factors, to induce an ectopic axis when injected into Xenopus embryos (Miller and Moon, 1996). Furthermore, components of this pathway are required for normal axial development because depletion of maternal &bgr;-catenin mRNA (Heasman et al., 1994), or sequestration of &bgr;-catenin to the plasma membrane (Fagotto et al., 1996), results in ventralized embryos that fail to develop a dorsal axis. However, it is not clear whether a Wnt ligand triggers Nieuwkoop Center formation, or whether downstream components of the Wnt pathway are activated by some other mechanism (Hoppler et al., 1996; Miller and Moon, 1996; Sokol, 1996). The Nieuwkoop Center is thought to induce a Spemann organizer by secreting a (yet to be identified) diffusible signal (Fagotto et al., 1997; Wylie et al., 1996), which may act synergistically with mesoderm-inducing factors, such as Activin and Vg1, to activate the expression of dorsal-specific genes, such as Goosecoid (Watabe et al., 1995). Dorsoventral patterning of the mesoderm is further controlled by opposing signals emanating from the organizer and the ventral mesoderm: a ventral bone morphogenetic protein (BMP) signal represses dorsal genes, while in the dorsal side the secreted factors Noggin, Chordin and Follistatin directly inhibit BMPs (Hogan, 1996).
While little is known about the molecular control of axis formation in mammalian embryos, a potential source of insight is the study of mouse mutants that affect this process (Conlon and Beddington, 1995; St-Jacques and McMahon, 1996), such as Fused (Fu). Two spontaneous alleles of Fu, called Kinky (Fu
Ki
) and Knobbly (Fu
Kb
), and a transgenic insertional allele, Fu
Tg1
(previously called H&egr;46), carry recessive mutations that are lethal at E8-E10 (Gluecksohn-Schoenheimer, 1949; Jacobs-Cohen et al., 1984; Perry et al., 1995). In addition to neuroectodermal and cardiac abnormalities, a remarkable property of many early post-implantation embryos homozygous for these three mutant alleles is a duplication of the embryonic axis. This phenotype, unique among mouse mutants, led nearly 40 years ago to the suggestion that Fu may play a role in the specification of the embryonic axis (Gluecksohn-Schoenheimer, 1949). Kinky, Knobbly and a third spontaneous allele, Fused (Fu
Fu
), but not Fu
Tg1
, also have dominant effects that include transient bifurcations of the fetal tailbud, asymmetric fusion of vertebrae leading to tail kinks, deafness, and neurological defects (Lyon et al., 1996).
The cloning of this locus with the aid of a transgene insertion was previously described (Perry et al., 1995). Here, the isolation and sequence of cDNA clones, and the genomic structure of the wild type (WT) and Fu
Tg1
alleles are reported. Analysis of the Fu
Fu
and Fu
Kb
alleles (Vasicek et al., manuscript submitted) has shown that both are caused by retroviral insertions. Because two mutant alleles causing axial duplications in homozygous embryos, Fu
Tg1
and Fu
Kb
, disrupt production of the major mRNA, the normal gene product may negatively regulate a critical step in the formation of the embryonic axis. This hypothesis is supported by studies in Xenopus embryos, which demonstrate that dorsal injection of WT Fused mRNA blocks axis formation, while ventral injection of a dominant-negative mutant form induces an ectopic axis. Co-injection with factors acting at various steps in axis formation reveals that Fused exerts its effects at a very early stage, by specifically inhibiting signal transduction through the Wnt pathway in the Nieuwkoop Center. Thus, analysis of the Fu locus has identified a novel inhibitor of the Wnt signaling pathway, and suggests that the same pathway regulates an early step in embryonic axis formation in mammals and amphibians. To avoid confusion with the unrelated Drosophila gene fused, applicants have renamed the Fu gene “Axin”, for “axis inhibition”.
SUMMARY OF THE INVENTION
This invention provides an isolated nucleic acid which encodes Axin.
This invention also provides an isolated nucleic acid which encodes a polypeptide comprising the amino acid sequence of Axin.
This invention also provides a polypeptide comprising the amino acid sequence of Axin. This invention also provides a purified wildtype Axin or purified mutant Axin.
This invention also provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a sequence of nucleotides present within a nucleic acid which encodes wildtype Axin without hybridizing to a nucleic acid which encodes mutant Axin, and an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a sequence of nucleotides present within the nucleic acid which encodes mutant Axin without hybridizing to a nucleic acid which encodes wildtype Axin.
This invention also provides a method for determining whether a subject carries a mutation in the axin gene which comprises (a) obtaining an appropriate nucleic acid sample from the subject and (b) determining whether the nucleic acid sample from step (a) is, or is derived from, a nucleic acid which encodes mutant Axin so as to thereby determine whether a subject carries a mutation in the axin gene.
This invention further provides a method
Constantini Franklin
Zeng Li
Achutamurthy Ponnathapu
Cooper & Dunham LLP
The Trustees of Columbia University in the City of New York
Tung Peter P.
White John P.
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