Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-01-18
1998-01-20
Elliott, George C.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351723, 4352523, 43525233, 435325, 435196, 530350, 536 232, C12N 1512, C12N 121, C12N 1563, C12N 916
Patent
active
057100152
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to cDNA encoding inositol monophosphatase (IMP) which is isolated from brain cells. As used herein, the abbreviation IMP refers to an enzyme which can specifically liberate inositol (Ins) from the naturally occurring substrates Ins(1)P, Ins(3)P and Ins(4)P. IMP is also capable of hydrolyzing certain non-inositol-containing substrates including but not limited to those disclosed in Hallcher and Sherman, (1980), J. Biol. Chem., 224, pp. 10896-10901; Takimoto et al., (1985), J. Biochem, (Tokyo), 98, pp. 363-370; and Gee et al., (1988), Biochem. J., 249, pp. 883-889. (1989), J. Biol. Chem., 265, pp. 5946-5949!. Mammalian cells capable of producing IMP include, but are not limited to, brain tissue cells. Transformed mammalian cell lines which may produce IMP include, but are not limited to, brain derived cell lines such as those available from the American Type Culture Collection listed in the Catalogue of Cell lines & Hybridomas, 7th Edition, 1992. The preferred cells for the present invention include normal human brain-derived tissue cells.
Other cells and cell lines may also be suitable for use in isolating IMP cDNA. Selection of suitable cells may be done by screening for IMP activity in cell extracts or conditioned medium. Methods for detecting IMP 249, pp. 143-148!, and measure the liberation of .sup.14 C-labelled inositol from a substrate. Cells which possess IMP activity in this assay may be suitable for the isolation of IMP cDNA.
Any of a variety of procedures may be used to molecularly clone human IMP cDNA. These methods include, but are not limited to, direct functional expression of the human IMP cDNA following the construction of a human IMP containing cDNA library in an appropriate expression vector system. Another method is to screen a human IMP-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of the purified IMP protein or from the DNA sequence of bovine IMP cDNA. The preferred method consists of screening a human IMP-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a .sup.32 P-labelled cDNA oligonucleotide-primed fragment of the bovine IMP cDNA cDNA library is a commercially available human hippocampal cDNA library in lambdaZAP (Stratagene).
It is readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other cells or cell types, may be useful for isolating IMP-encoding DNA. Other types of libraries include, but are not limited to, cDNA libraries derived from other tissues, cells or cell lines other than human hippocampal cells, and genomic DNA libraries.
It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from cells or cell lines which have IMP activity. The selection of cells or cell lines for use in preparing a cDNA library to isolate IMP cDNA may be done by first measuring cell-associated IMP activity using the .sup.14 C-labelled inositol substrate cleavage
Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found, for example, in Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, New York, 2nd edition, 1989).
It is also readily apparent to those skilled in the art that DNA encoding IMP may also be isolated from a suitable genomic DNA library.
Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techiques can be found in Maniatis et al., supra.
Using the preferred method, cDNA clones encoding human IMP were isolated by cDNA library screening. .sup.32 P-radiolabelled oligonucleotide-primed fragments of the bovine IMP cDNA served as probes for the isolation of full length human IMP cDNA from a commercially available lambdaZAP cDNA library (Stratagene) derived from human hippocamp
REFERENCES:
"Cloning and Expression of Bovine Brain Inositol Monophosphatase", Ronald E. Diehl, et al., Journal of Biological Chemistry, vol. 265, No. 11, Apr. 15, 1990, pp. 5946-5949.
"Purification and Properties of myo-Inositol-1-Phosphatase from Rat Brain", Kouichi Takimoto, Journal of Biochemistry, vol. 98, No. 2, 1985, pp. 363-370.
"cDNA cloning of human and rat brain myo-inositol monophosphatase", George McAllister, et al., The Biochemical Journal, vol. 284, No. 3, Jun. 15, 1992, pp. 749-754.
"Human brain 2'-nucleotidase: partial purification and properties", U. Vogel, et al., Biochemical Society Transactions, vol. 14, No. 2, Apr. 1986, pp. 349-350.
McAllister George
Whiting Paul John
Elliott George C.
Merck Sharp & Dohme Ltd.
North Robert J.
Railey II Johnny F.
Winokur Melvin
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