Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-05-16
2001-10-30
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06309821
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of molecular biology and particularly novel polynucleotide and amino acid sequences for a human homolog of the Pac10 gene of Saccharomyces cerevisiae. The present invention provides compositions and methods for the diagnosis and treatment of disease states such as X-linked centronuclear myopathy, schizophrenia and mental retardation.
BACKGROUND OF THE INVENTION
Motor proteins in cells include myosin, which is actin-based, and kinesin, dynein and dynamin, which are microtubule-based (Lillie 1992, Nature 356:358-361). Kinesin has been identified as a transporter of membranous organelles in mammalian neurons. Genes related to kinesin heavy chain have been identified in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Most of the members of the kinesin family are implicated in mechanisms of mitosis or meiosis (Aizawa 1992, J. Cell. Biol. 119:1287-1296).
The Pac10 gene of Saccharomyces cerevisiae, which encodes the heavy chain of members of the kinesin family, is required for viability in the absence of the kinesin-related CIN8 mitotic motor. If deleted with CIN8, an associated family member, the Pac 10/CIN8 double mutation is lethal and presumably prevents separation of the spindle pole bodies (Geiser, J R and Hoyt M A, unpublished data).
Centronuclear myopathy is an X-inked congenital myopathy (MTMI) that has been localized to Xq28 (
Harrison's Principles of Internal Medicine,
13 edition, ed. Isselbacher et al., publisher McGraw-Hill, New York, pg 2387). Dahl et al. (1995 Am. J. Hum. Genet. 56:1108-1115) report that a young girl with a clinically moderate form of myotubular myopathy and associated mental retardation was found to carry a cytogenetically detectable deletion in Xq27-q28. Positional cloning of the MTMI locus has been refined to a 600 kb region between the DX5304 and DX5497 markers. Other diseases linked to the Xp27-q28 locus include schizophrenia, X-linked mental retardation associated with Fragile Site FRAXE and anophthalmos.
The neonatal form of centronuclear myopathy is X-linked and presents with severe hypotonia and weakness at birth. Patients may require respiratory assistance and a feeding tube due to swallowing difficulties. This form of the disease is often fatal. The early childhood form presents without difficulty at birth, but motor milestones, such as walking, running and stair climbing, are delayed. In this form the disease may be static or may progress to weakness. A rare form of the disease has an onset in the second or third decade. The early childhood and adult forms appear to have autosomal dominant or recessive inheritance patterns. Patients with the neonatal form of centronuclear myopathy require carefull management for respiratory support and gastric feeding, and patients with the early childhood disorder often require ambulatory aids and orthotic devices and less often wheelchairs.
Schizophrenic disorders are serious mental illnesses that cause significant social, vocational, and personal disability. In the United States there are about 2 million affected individuals and these individuals account for an estimated loss of 20 billion dollars of lost productivity per year (Harrison's Principles of Internal Medicine supra pg. 2414-2415). X chromosome linked mental retardation is the second most common genetic cause of mental retardation (after Down's syndrome) (Pathophysiology, 2nd Edition, Editors McCance et al., publisher, Mosby, St. Louis pg. 1420). Anopthalmos is a developmental defect characterized by mental retardation and a complete absence of the eyes or by the presence of vestigal eyes.
In view of the severity of the diseases associated with the Xq27-q28 locus, including centronuclear myopathy, myotubular myopathy, schizophrenia, X-linked mental retardation associated with Fragile Site FRAXE and anophthalmos, it would be advantageous to provide an early and accurate method for the detection of such diseases. It would also be advantageous to provide therapeutic compositions and methods for prevention and treatment of such diseases.
SUMMARY
Because the present invention relates to novel nucleotide and amino acid sequences disclosed herein for a PAC 10 human homolog and the nucleotide sequence has been mapped to the Xq27-q28 locus, the present invention also relates to the use of the PAC 10 human homolog in the diagnosis, prevention and treatment of diseases associated with the Xq27-q28 locus, such as centronuclear myopathy, myotubular myopathy, schizophrenia, X-linked mental retardation associated with Fragile Site FRAXE and anophthalmos.
The nucleotide sequence for Incyte clone 41400, encoding the PACIO human homolog, was initially found among the nucleic acid sequences of a cDNA library made from hybrid cells of T-B lymphoblasts from a leukemic cell line. Clone number 41400 was found to have amino acid homology to the Pac10 gene of Saccharomyces cerevisiae which encodes the heavy chain members of the kinesin family that are implicated in mechanisms of mitosis or meiosis. Based upon the results of a BLAST search (which stands for Basic Local Alignment Search Tool (Altschul S F (1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) J Mol Biol 215:403-10)), the PAC10 human homolog appears to have a more distant relationship to the beta cardiac myosin heavy chain (Oryctolagus cuniculus) and the neuronal myosin heavy chain (Rattus rattus). The Pac10 human homolog also shares some nucleotide sequence homology with the serotonin receptors, NCBI Entrez accession numbers GI 177776 and GI 36431.
The present invention is therefore based on the discovery of a novel cytoskeletal gene which maps to the locus Xq27-28 that may be associated with disease states, such as centronuclear myopathy, myotubular myopathy, schizophrenia, X-linked mental retardation associated with Fragile Site FRAXE and anophthalmos. The PAC10 human homolog and nucleotide sequences that encode it and oligonucleotides, peptide nucleic acid (PNA), fragments, portions or antisense molecules thereof, provide the basis for diagnostic methods for the detection and/or quantitation of the Pac10 human homolog that is associated with myopathy and X-linked mental retardation. For example, the nucleotide sequence disclosed herein, which encodes the PAC10 human homolog, or fragments thereof, may be used in hybridization assays of biopsied cells or tissues or bodily fluids, such as amniotic fluid to detect the nucleic acid which may be associated with such disease states.
An abnormal level of the Pac10 human homolog nucleotide sequences or an abnormal transcript size in a biological sample may be characteristic of a regulatory state in which the molecules are over-expressed or under-expressed. Nucleotide sequences encoding the PAC 10 human homolog provide the basis for probes which can be used diagnostically to detect chromosomal aberrations such as deletions, mutations or chromosomal translocations in the gene encoding the molecule. Gene expression may be altered in such disease states or there may be a chromosomal aberration present in the region of the gene encoding the molecule.
The present invention also relates, in part, to expression vectors and genetically engineered host cells comprising nucleotide sequences encoding the Pac10 human homolog for in vitro or in vivo production of the nucleotide sequences.
Additionally, the present invention relates to the use of the PAC10 human homolog polypeptide, or fragment or variant thereof, to produce antibodies and to screen for antagonists or inhibitors of the PAC10 human homolog which can be used diagnostically to detect and quantitate protein levels in disease states.
Peptides or small molecules capable of modulating PAC10 human homolog activity will provide the basis for pharmaceutical compositions for the treatment of disease states associated with the Xq27-28 locus.
The invention further provides diagnostic assays and kits for the detection of the PAC 10 human homolog in cells and tissues comprising the PAC10 human homolog which may be used as
Au-Young Janice
Goold Richard D.
Campbell Eggerton A.
Incyte Genomics Inc.
Incyte Genomics, Inc.
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