Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
2000-11-13
2002-11-12
Swartz, Rodney P (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C435S006120, C435S184000, C435S069100, C435S089000, C435S440000, C435S441000, C435S455000, C435S463000, C435S468000, C435S471000, C530S350000, C536S023100, C536S023200, C536S023500, C536S024300
Reexamination Certificate
active
06479628
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to inosine 5′-monophosphate dehydrogenase (IMPDH) and more particularly to purified and isolated DNA sequences that encode proteins possessing the biological properties of eukaryotic IMPDH, to the expression products of these DNA sequences in transformed or transfected host cells, to recombinant and synthetic proteins and peptides having amino acid sequences based on the sequence of amino acids deduced from these DNA sequences, to antibodies specific for such proteins and peptides, to analytical procedures for the detection and quantification of such peptides and proteins and nucleic acids related thereto, to the use of IMPDH-encoding DNA sequences as selectable markers and as tools for gene amplification in recombinant hosts, and to cell lines and organisms displaying enhanced production of IMPDH and/or elevated levels of products such as guanosine monophosphate (GMP), whose synthesis in cells is dependent on the activity of IMPDH.
The enzyme IMPDH (EC 1.2.1.14) catalyzes the formation of xanthine monophosphate (XMP) from inosine monophosphate (IMP). In the purine de novo synthetic pathway, IMPDH is positioned at the branch point in the synthesis of adenine and guanine nucleotides and is thus the rate-limiting enzyme in the de novo synthesis of guanine nucleotides, such as guanosine 5-monophosphate. [Weber,
Cancer Res
., 43:3466-3492 (1983); Weber, et al.,
Adv. Enzyme Regul
., 18:3-26 (1980)]. Inhibition of cellular IMPDH activity results in an abrupt cessation of DNA synthesis [Franklin, et al.,
Biochem. J
., 113:515-524 (1969); Cohen, et al.,
J. Biol. Chem
., 256:8713-8717 (1981); and Duan, et al.,
Cancer Res
., 47:4047-4051 (1987)] and a cell cycle block at the Gl-S interface [Cohen, et al.,
Cancer Res
., 43:1587-1591 (1983)]. Because IMPDH is essential in providing the necessary precursors for DNA and RNA biosynthesis, normal tissues that exhibit increased cell proliferation generally exhibit increased IMPDH activity [Jackson, et al.,
Nature
, 256:331-333 (1975); Jackson, et al.,
Biochem. J
., 166:1-10 (1977), Cooney, et al.,
Anal. Biochem
., 130:339-345 (1983)]. Similarly, increased cell proliferation is accompanied by elevated enzyme activity in certain rat hepatomas with varied growth rates. Weber,
Cancer Res
., 43:3466-3492 (1983). These hepatomas manifest IMPDH activities that are disproportionately higher than those of normal tissues, suggesting that IMPDH is associated with cell proliferation and may be linked to either malignant cell transformation or tumor progression.
To investigate the role of IMPDH in growth regulation and malignancy, attempts have been made to purify the enzyme to homogeneity to allow preparation of specific antibodies thereto and to isolate IMPDH-encoding DNA.
IMPDH isolated from bacterial sources has been determined to vary widely with respect to allosteric properties, size, and subunit composition. IMPDH isolated from
E. coli
has been purified and characterized as a tetramer of identical subunits [Gilbert, et al.,
Biochem. J
., 183:481-494 (1979); and Krishnaiah,
Arch. Biochem. Biophys
., 170:567-575 (1975)]. Unlike mammalian cell enzymes, the
E. coli
IMPDH enzyme is reported to be insensitive to the inhibitory effect of mycophenolic acid [Franklin, et al.,
Biochem. J
., 113:515-524 (1969)]. In
E. coli
, IMPDH has been determined to be the product of the guaB locus and the sequence of the quaB structural gene and surrounding DNA has been determined to span 1.533 kb and to code for an IMPDH subunit sequence of 511 amino acids with a calculated molecular weight 54,512 [Tiedeman, et al.,
Nucleic Acids Research
, 13:1303 (1985)].
Miyagawa, et al.,
Bio/Tech
., 4:225 (1986), have described the cloning of the
Bacillus subtilis
IMPDH gene, which, upon re-introduction into a
B. subtilis
strain that overproduced inosine, resulted in an increased production of guanosine, accompanied by a decreased accumulation of inosine. The IMPDH gene was localized on a 6.5 kb insert and further localized to a Hind III-partially digested 2.9 kb fragment. However, the gene was not reported to have been isolated and no information was provided with respect to the DNA sequence of the gene.
While a number of workers have reported the purification or partial purification of IMPDH from a variety of eukaryotic cell sources, including ascites cells, thymus cells, mouse LS cells, and other mammalian cells, none have been successful in obtaining substantial information about the amino acid sequence of the IMPDH protein, or in establishing the utility of anti-IMPDH antibodies in the characterization of the cellular role of IMPDH.
Eukaryotic IMPDH has been obtained from one plant and several animal species, including cowpea nodule cells [Atkins, et al.,
Arch. Biochem. Biophys
., 236:807-814 (1985)], Yoshida sarcoma ascites cells [Okada, et al.,
J. Biochem
., 94:1605-1613 (1983)], rat hepatoma 3924A cells [Ikegami, et al.,
Life Sci
., 40:2277-2282 (1987) and Yamada, et al.,
Biochem
., 27:2193-2196 (1988)] and Chinese hamster cells [Collart, et al.,
Mol. Cell. Biol
., 7:3328-3331 (1987)]. The disclosures of the last-mentioned publication by the present inventors are specifically incorporated by reference herein. In all of these reports, denaturing polyacrylamide gel electrophoresis was used to assess purity and to estimate molecular weight. The reported molecular weight for all of the above mentioned enzymes was approximately 56,000. A polyclonal antibody raised against the purified protein was prepared for the enzyme isolated from Yoshida sarcoma ascites cells, rat hepatoma 3924A cells, and Chinese hamster cells. As described in detail, infra, only in the case of the antibody prepared against the Chinese hamster enzyme was an antibody determined to be useful in examination of cellular regulation and useful in isolation of eukaryotic IMPDH-encoding DNA.
There continues to exist a need in the art for information regarding IMPDH enzymes of eukaryotic origins (especially of vertebrate and more particularly of mammalian origins) such as can be provided by the isolation, sequencing, and recombinant system utilization of DNA sequences encoding the same. The availability of such materials and information would make possible a vast array of novel systems and methodologies based thereon including methods and materials useful in production of products displaying IMPDH activity.
BRIEF SUMMARY OF THE INVENTION
The present invention provides novel purified and isolated DNA sequences encoding eukaryotic inosine 5′-monophosphate dehydrogenase (IMPDH), which have allowed for the initial determination of the primary structural conformation (i.e., amino acid sequence) of the eukaryotic protein. Specifically provided are sequences encoding human, mouse, and Chinese hamster IMPDH. Provided also are alternate DNA forms such as genomic DNA and DNA manufactured by partial or total chemical synthesis from nucleotides. The association of DNA sequences of the invention with expression regulatory DNA sequences, such as promoters, enhancers and the like, allows for in vivo and in vitro transcription to form messenger RNA, which, in turn, is subject to translation to provide IMPDH protein in large quantities.
Among the multiple aspects of the present invention, therefore, is the provision of (a) novel eukaryotic IMPDH DNA sequences as set out in
FIG. 1A
,
FIG. 1B
,
FIG. 1C
,
FIG. 1D
,
FIG. 2A
,
FIG. 2B
,
FIG. 2C
,
FIG. 2D
,
FIG. 3A
,
FIG. 3B
, and
FIG. 3C
; (b) IMPDH-encoding DNA sequences, which hybridize thereto under hybridization conditions of the stringency equal to or greater than the conditions described herein as used in the initial isolation of cDNAs of the invention, which also encode proteins with IMPDH biological activities; and (c) DNA sequences encoding the same, allelic variant and/or analog IMPDH proteins, which incorporate, at least in part, degenerate codons. Cor
Collart Frank R.
Huberman Eliezer
Arch Development Corporation
Fulbright & Jaworski L.L.P.
Swartz Rodney P
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