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Human methionine synthase reductase: cloning, and methods for evaluating risk of neural tube deffects, cardiovascular disease, cancer and Down's Syndrome

a technology of human methionine synthase and reductase, which is applied in the direction of biochemistry apparatus and processes, enzymes, roofs, etc., can solve the problems of cardiac disease, neural tube defects, cancer, etc., and achieve the effect of increasing or decreasing the stability of mrna or polypeptides

Inactive Publication Date: 2005-09-01
MCGILL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0077] By “positioned for expression” is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a methionine synthase reductase polypeptide, a recombinant protein or a RNA molecule).
[0084] By “neutralizing antibodies” is meant antibodies that interfere with any of the biological activities of a wild-type or mutant methionine synthase reductase polypeptide, for example, the ability of methionine synthase reductase to catalyze the transfer of a methyl group to methionine synthase-cobal(II)amin. The neutralizing antibody may reduce the ability of a methionine synthase reductase polypeptide to catalyze the transfer preferably by 10% or more, more preferably by 25% or more, still more preferably by 50% or more, yet preferably by 70% or more, and most preferably by 90% or more. Any standard assay for the biological activity of methionine synthase reductase may be used to assess potentially neutralizing antibodies that are specific for methionine synthase reductase.
[0092] By “alteration in the level of gene expression” is meant a change in gene activity such that the amount of a product of the gene, i.e., mRNA or polypeptide, is increased or decreased, or that the stability of the mRNA or the polypeptide is increased or decreased.

Problems solved by technology

The presence of mutations in the methionine synthase reductase gene that decrease methionine synthase reductase enzymatic activity are likely to be associated with altered risk for cardiovascular disease, neural tube defects, and cancer.

Method used

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  • Human methionine synthase reductase: cloning, and methods for evaluating risk of neural tube deffects, cardiovascular disease, cancer and Down's Syndrome
  • Human methionine synthase reductase: cloning, and methods for evaluating risk of neural tube deffects, cardiovascular disease, cancer and Down's Syndrome
  • Human methionine synthase reductase: cloning, and methods for evaluating risk of neural tube deffects, cardiovascular disease, cancer and Down's Syndrome

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Experimental program
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example 1

General Methods

Materials

[0130] Radiolabeled compounds were from DuPont (Wilmington, Del.). A human multiple tissue Northern blot and β-actin probe were from Clontech (Palo Alto, Calif.). The random-primed DNA labelling kit was from Boehringer Mannheim (Indianapolis, Ind.). The T / A cloning kit was from Invitrogen (Carlsbad, Calif.), the Geneclean III kit was obtained from Bio101 Inc. (Vista, Calif.), and the Wizard Mini-Preps were from Promega (Madison, Wis.). Taq polymerase, AMV reverse transcriptase, Trizol reagent, and were purchased from Gibco BRL (Gaithersburg, Md.), and restriction enzymes were purchased from Gibco BRL and New England Biolabs (Beverly, Mass.). The Sequenase kits for manual sequencing of crude PCR products or plasmids were from United States Biochemicals (Cleveland, Ohio). The oligonucleotides (SEQ ID NOs: 3-20 and 49-50) were synthesized by ACGT Corporation (Toronto, Canada) or by the Sheldon Biotechnology Centre, McGill University. The sequences of oligonuc...

example ii

Cloning of the Human Methionine Synthase Reductase cDNA

[0144] More than 20 overlapping sequences homologous to the FAD and NADPH-binding domains of cytochrome P450 reductase were identified in an initial survey of the NCBI dbEST database using TblastN. We sequenced clones 550341 (accession #AA085543), 704947 (accession #AA279726) and 31776 (accession #R17835) to confirm the sequence of this part of the cDNA. Reprobing the NCBI databases with this sequence yielded a C. elegans sequence (accession #Z35595) containing binding sites for FMN, FAD and NADPH. We then used the C. elegans sequence to reprobe the dbEST database using TblastN and identified a human sequence (accession #AA192690, clone 628497) containing a putative FMN binding site similar to the one encoded by Z35595. We designed a sense primer based on the FMN binding region of AA192690 and antisense primers corresponding to the FAD / NADPH binding regions of the methionine synthase reductase candidate and amplified a sequence...

example iii

Expression of Human Methionine Reductase mRNA

[0150] A PCR product generated with primers 1902C (SEQ ID NO: 19) and 1812B (SEQ ID NO: 18) was subcloned and used to probe a Northern blot prepared from several human tissues.

[0151]FIGS. 5A and 5B show a Northern blot analysis of methionine synthase reductase expression in human tissues, with the positions of the molecular size (kb) markers indicated at the left. The 1.8 kb probe hybridized to one predominant RNA species of 3.6 kb. Methionine synthase reductase appears to be expressed to some degree in all tissues tested and is particularly abundant in skeletal muscle. In addition to the 3.6 kb band, a 3.1 kb band and a faint 6 kb band were detected in brain mRNA.

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Abstract

The invention features a novel gene encoding methionine synthase reductase. The invention also features a method for detecting an increased likelihood of hyperhomocysteinemia and, in turn, an increased or decreased likelihood of neural tube defects, cardiovascular disease, Down's Syndrome or cancer. The invention also features therapeutic methods for treating and / or reducing the risk of cardiovascular disease, Down's Syndrome, cancer, or neural tube defects. Also provided are the sequences of the human methionine synthase reductase gene and protein and compounds and kits for performing the methods of the invention.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. Ser. No. 09 / 487,841, filed Jan. 19, 2000, which claims priority from U.S. Ser. No. 09 / 371,347, filed Aug. 10, 1999, which claims priority from U.S. Ser. No. 09 / 232,028, filed on Jan. 15, 1999, which claims priority from U.S. Provisional Application No. 60 / 071,622, filed Jan. 16, 1998.FIELD OF THE INVENTION [0002] This invention relates to the diagnosis and treatment of patients at risk for disorders associated with altered methionine synthase activity. BACKGROUND OF THE INVENTION [0003] Methionine is an essential amino acid in mammals that is required for protein synthesis. Methionine also plays a central role in metabolic reactions involving transfer of single-carbon moieties: in its activated form, S-adenosylmethionine, methionine is the methyl donor in hundreds of biological transmethylation reactions. Moreover, methionine is the propylamine donor in polyamine synthesis. The ultimate product...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): B60J7/10C12N9/10
CPCC12N9/1007
Inventor GRAVEL, ROYROZEN, RIMALECLERC, DANIELWILSON, AARONROSENBLATT, DAVID
Owner MCGILL UNIV
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