Guanosine monophosphate reductase
United States Patent: 7,018,822
Issued: March 28, 2006
Inventors: Hillman; Jennifer L. (San Jose,
Assignee: Incyte Corporation (Wilmington,
Appl. No.: 007306
Filed: January 15, 1998
Executive MBA in Pharmaceutical Management, U. Colorado
The present invention provides a human
guanosine monophosphate reductase (HGMPR) and polynucleotides which
identify and encode HGMPR. The invention also provides genetically
engineered expression vectors and host cells comprising the nucleic acid
sequences encoding HGMPR and a method for producing HGMPR. The invention
also provides for agonists, antibodies, or antagonists specifically
binding HGMPR, and their use, in the prevention and treatment of diseases
associated with expression of HGMPR. Additionally, the invention provides
for the use of antisense molecules to polynucleotides encoding HGMPR for
the treatment of diseases associated with the expression of HGMPR. The
invention also provides diagnostic assays which utilize the
polynucleotide, or fragments or the complement thereof, and antibodies
specifically binding HGMPR.
DESCRIPTION OF THE
The invention is based on the discovery of a novel human guanosine
monophosphate reductase, (HGMPR), the polynucleotides encoding HGMPR, and
the use of these compositions for the diagnosis, prevention, or treatment
of cancer, viral diseases, inflammatory diseases, and immunological
Nucleic acids encoding the human HGMPR of the present invention were first
identified in Incyte Clone 569141 from the macrophage cDNA library
(MMLR3DT010) through a computer-generated search for amino acid
alignments. A consensus sequence, SEQ ID NO:1, was derived from the
following overlapping and/or extended nucleic acid sequences: Incyte
Clones 989/U937NOT01, 43480/TBLYNOT01, 256710/HNT2RAT01, 297670/HIPONOT01,
569141/MMLR3DT01, 971292/MUSCNOT02, 1458673/COLNFET02,1729347/BRSTTUT08,
In one embodiment, the invention encompasses a polypeptide comprising the
amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C,
1D and 1E. HGMPR is 366 amino acids in length and has
cysteine residues representing potential intramolecular cysteine—cysteine
disulfide bridging sites at C86, C113, C145, C204, C223, C240, and C334.
As shown in FIGS. 2A and 2B, HGMPR has chemical and structural homology
with a GMPR from E. coli (GI 473772; SEQ ID NO:3) and human red
blood cells (GI 544455; SEQ ID NO:4). In particular, HGMPR shares 72%
overall identity with the E. coli GMPR, and 77% identity with the
human GMPR. The most significant difference between HGMPR and the two
GMPRs is the presence of an N-terminal sequence in HGMPR that is not
present in the other two GMPR and may represent a novel signal sequence
targeting HGMPR to different tissues. Six of the seven cysteine residues
in HGMPR are shared by one or both of the two GMPRs. As illustrated by
FIGS. 3A, 3B, and 3C, HGMPR and the GMPRs from E. coli
and human have rather similar hydrophobicity plots. Northern analysis
(FIG. 4), together with the libraries mentioned above, indicates that
partial transcripts of the nucleic acid sequence encoding HGMPR are
prominent in various cancerous tissues (prostate, brain, breast, and
blood), and cells associated with inflammatory diseases and the immune
response (mast cells, monocytes, macrophages, lymphocytes, and endothelial
The invention also encompasses HGMPR variants. A preferred HGMPR variant
is one having at least 80%, and more preferably 90%, amino acid sequence
similarity to the HGMPR amino acid sequence (SEQ ID NO:1). A most
preferred HGMPR variant is one having at least 95% amino acid sequence
similarity to SEQ ID NO:1.
The invention also encompasses polynucleotides which encode HGMPR.
Accordingly, any nucleic acid sequence which encodes the amino acid
sequence of HGMPR can be used to generate recombinant molecules which
express HGMPR. In a particular embodiment, the invention encompasses the
polynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 as
shown in FIGS. 1A, 1B, 1C and 1E.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the genetic code, a multitude of nucleotide sequences
encoding HGMPR, some bearing minimal homology to the nucleotide sequences
of any known and naturally occurring gene, may be produced. Thus, the
invention contemplates each and every possible variation of nucleotide
sequence that could be made by selecting combinations based on possible
codon choices. These combinations are made in accordance with the standard
triplet genetic code as applied to the nucleotide sequence of naturally
occurring HGMPR, and all such variations are to be considered as being
Although nucleotide sequences which encode HGMPR and its variants are
preferably capable of hybridizing to the nucleotide sequence of the
naturally occurring HGMPR under appropriately selected conditions of
stringency, it may be advantageous to produce nucleotide sequences
encoding HGMPR or its derivatives possessing a substantially different
codon usage. Codons may be selected to increase the rate at which
expression of the peptide occurs in a particular prokaryotic or eukaryotic
host in accordance with the frequency with which particular codons are
utilized by the host. Other reasons for substantially altering the
nucleotide sequence encoding HGMPR and its derivatives without altering
the encoded amino acid sequences include the production of RNA transcripts
having more desirable properties, such as a greater half-life, than
transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences, or portions
thereof, which encode HGMPR and its derivatives, entirely by synthetic
chemistry. After production, the synthetic sequence may be inserted into
any of the many available expression vectors and cell systems using
reagents that are well known in the art at the time of the filing of this
application. Moreover, synthetic chemistry may be used to introduce
mutations into a sequence encoding HGMPR or any portion thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of hybridizing to the claimed nucleotide sequences, and in
particular, those shown in SEQ ID NO:2, under various conditions of
stringency. Hybridization conditions are based on the melting temperature
(Tm) of the nucleic acid binding complex or probe, as taught in Wahl, G.
M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R.
(1987; Methods Enzymol. 152:507-511), and may be used at a defined
Altered nucleic acid sequences encoding HGMPR which are encompassed by the
invention include deletions, insertions, or substitutions of different
nucleotides resulting in a polynucleotide that encodes the same or a
functionally equivalent HGMPR. The encoded protein may also contain
deletions, insertions, or substitutions of amino acid residues which
produce a silent change and result in a functionally equivalent HGMPR.
Deliberate amino acid substitutions may be made on the basis of similarity
in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or
the amphipathic nature of the residues as long as the biological activity
of HGMPR is retained. For example, negatively charged amino acids may
include aspartic acid and glutamic acid; positively charged amino acids
may include lysine and arginine; and amino acids with uncharged polar head
groups having similar hydrophilicity values may include leucine,
isoleucine, and valine; glycine and alanine; asparagine and glutamine;
serine and threonine and; phenylalanine and tyrosine.
Also included within the scope of the present invention are alleles of the
genes encoding HGMPR. As used herein, an "allele" or "allelic sequence" is
an alternative form of the gene which may result from at least one
mutation in the nucleic acid sequence. Alleles may result in altered mRNAs
or polypeptides whose structure or function may or may not be altered. Any
given gene may have none, one, or many allelic forms. Common mutational
changes which give rise to alleles are generally ascribed to natural
deletions, additions, or substitutions of nucleotides. Each of these types
of changes may occur alone, or in combination with the others, one or more
times in a given sequence.
Methods for DNA sequencing which are well known and generally available in
the art may be used to practice any embodiments of the invention. The
methods may employ such enzymes as the Klenow fragment of DNA polymerase
I, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin
Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of recombinant polymerases and proofreading exonucleases such
as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg,
Md.). Preferably, the process is automated with machines such as the
Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler
(PTC200; MJ Research, Watertown, Mass.) and the ABI 377 DNA sequencers (Perkin
The nucleic acid sequences encoding HGMPR may be extended utilizing a
partial nucleotide sequence and employing various methods known in the art
to detect upstream sequences such as promoters and regulatory elements.
For example, one method which may be employed, "restriction-site" PCR,
uses universal primers to retrieve unknown sequence adjacent to a known
locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). In particular,
genomic DNA is first amplified in the presence of primer to linker
sequence and a primer specific to the known region. The amplified
sequences are then subjected to a second round of PCR with the same linker
primer and another specific primer internal to the first one. Products of
each round of PCR are transcribed with an appropriate RNA polymerase and
sequenced using reverse transcriptase.
Inverse PCR may also be used to amplify or extend sequences using
divergent primers based on a known region (Triglia, T. et al. (1988)
Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO 4.06
Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), or
another appropriate program, to be 22-30 nucleotides in length, to have a
GC content of 50% or more, and to anneal to the target sequence at
temperatures about 68°-72° C. The method uses several restriction enzymes
to generate a suitable fragment in the known region of a gene. The
fragment is then circularized by intramolecular ligation and used as a PCR
Another method which may be used is capture PCR which involves PCR
amplification of DNA fragments adjacent to a known sequence in human and
yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods
Applic. 1:111-119). In this method, multiple restriction enzyme digestions
and ligations may also be used to place an engineered double-stranded
sequence into an unknown portion of the DNA molecule before performing PCR.
Another method which may be used to retrieve unknown sequences is that of
Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER
libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). This
process avoids the need to screen libraries and is useful in finding
When screening for full-length cDNAs, it is preferable to use libraries
that have been size-selected to include larger cDNAs. Also, random-primed
libraries are preferable, in that they will contain more sequences which
contain the 5′ regions of genes. Use of a randomly primed library may be
especially preferable for situations in which an oligo d(T) library does
not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence into the 5′ and 3′ non-transcribed regulatory
Capillary electrophoresis systems which are commercially available may be
used to analyze the size or confirm the nucleotide sequence of sequencing
or PCR products. In particular, capillary sequencing may employ flowable
polymers for electrophoretic separation, four different fluorescent dyes
(one for each nucleotide) which are laser activated, and detection of the
emitted wavelengths by a charge coupled device camera. Output/light
intensity may be converted to electrical signal using appropriate software
(e.g. GENOTYPER and SEQUENCE NAVIGATOR, Perkin Elmer) and the entire
process from loading of samples to computer analysis and electronic data
display may be computer controlled. Capillary electrophoresis is
especially preferable for the sequencing of small pieces of DNA which
might be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or
fragments thereof which encode HGMPR, or fusion proteins or functional
equivalents thereof, may be used in recombinant DNA molecules to direct
expression of HGMPR in appropriate host cells. Due to the inherent
degeneracy of the genetic code, other DNA sequences which encode
substantially the same or a functionally equivalent amino acid sequence
may be produced and these sequences may be used to clone and express HGMPR.
As will be understood by those of skill in the art, it may be advantageous
to produce HGMPR-encoding nucleotide sequences possessing non-naturally
occurring codons. For example, codons preferred by a particular
prokaryotic or eukaryotic host can be selected to increase the rate of
protein expression or to produce a recombinant RNA transcript having
desirable properties, such as a half-life which is longer than that of a
transcript generated from the naturally occurring sequence.
The nucleotide sequences of the present invention can be engineered using
methods generally known in the art in order to alter HGMPR encoding
sequences for a variety of reasons, including but not limited to,
alterations which modify the cloning, processing, and/or expression of the
gene product. DNA shuffling by random fragmentation and PCR reassembly of
gene fragments and synthetic oligonucleotides may be used to engineer the
nucleotide sequences. For example, site-directed mutagenesis may be used
to insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, or introduce mutations, and so
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid sequences encoding HGMPR may be ligated to a heterologous
sequence to encode a fusion protein. For example, to screen peptide
libraries for inhibitors of HGMPR activity, it may be useful to encode a
chimeric HGMPR protein that can be recognized by a commercially available
antibody. A fusion protein may also be engineered to contain a cleavage
site located between the HGMPR encoding sequence and the heterologous
protein sequence, so that HGMPR may be cleaved and purified away from the
In another embodiment, sequences encoding HGMPR may be synthesized, in
whole or in part, using chemical methods well known in the art (see
Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn,
T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the
protein itself may be produced using chemical methods to synthesize the
amino acid sequence of HGMPR, or a portion thereof. For example, peptide
synthesis can be performed using various solid-phase techniques (Roberge,
J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be
achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin
The newly synthesized peptide may be substantially purified by preparative
high performance liquid chromatography (e.g., Creighton, T. (1983)
Proteins, Structures and Molecular Principles, W H Freeman and Co.,
New York, N.Y.). The composition of the synthetic peptides may be
confirmed by amino acid analysis or sequencing (e.g., the Edman
degradation procedure; Creighton, supra). Additionally, the amino acid
sequence of HGMPR, or any part thereof, may be altered during direct
synthesis and/or combined using chemical methods with sequences from other
proteins, or any part thereof, to produce a variant polypeptide.
In order to express a biologically active HGMPR, the nucleotide sequences
encoding HGMPR or functional equivalents, may be inserted into appropriate
expression vector, i.e., a vector which contains the necessary elements
for the transcription and translation of the inserted coding sequence.
Methods which are well known to those skilled in the art may be used to
construct expression vectors containing sequences encoding HGMPR and
appropriate transcriptional and translational control elements. These
methods include in vitro recombinant DNA techniques, synthetic techniques,
and in vivo genetic recombination. Such techniques are described in
Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al.
(1989) Current Protocols in Molecular Biology, John Wiley & Sons,
New York, N.Y.
A variety of expression vector/host systems may be utilized to contain and
express sequences encoding HGMPR. These include, but are not limited to,
microorganisms such as bacteria transformed with recombinant bacteriophage,
plasmid, or cosmid DNA expression vectors; yeast transformed with yeast
expression vectors; insect cell systems infected with virus expression
vectors (e.g., baculovirus); plant cell systems transformed with virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322
plasmids); or animal cell systems.
The "control elements" or "regulatory sequences" are those non-translated
regions of the vector—enhancers, promoters, 5′ and 3′ untranslated
regions—which interact with host cellular proteins to carry out
transcription and translation. Such elements may vary in their strength
and specificity. Depending on the vector system and host utilized, any
number of suitable transcription and translation elements, including
constitutive and inducible promoters, may be used. For example, when
cloning in bacterial systems, inducible promoters such as the hybrid lacZ
promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or
PSPORT1 plasmid (Gibco BRL) and the like may be used. The baculovirus
polyhedrin promoter may be used in insect cells. Promoters or enhancers
derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and
storage protein genes) or from plant viruses (e.g., viral promoters or
leader sequences) may be cloned into the vector. In mammalian cell
systems, promoters from mammalian genes or from mammalian viruses are
preferable. If it is necessary to generate a cell line that contains
multiple copies of the sequence encoding HGMPR, vectors based on SV40 or
EBV may be used with an appropriate selectable marker.
In bacterial systems, a number of expression vectors may be selected
depending upon the use intended for HGMPR. For example, when large
quantities of HGMPR are needed for the induction of antibodies, vectors
which direct high level expression of fusion proteins that are readily
purified may be used. Such vectors include, but are not limited to, the
multifunctional E. coli cloning and expression vectors such as
BLUESCRIPT (Stratagene), in which the sequence encoding HGMPR may be
ligated into the vector in frame with sequences for the amino-terminal Met
and the subsequent 7 residues of β-galactosidase so that a hybrid protein
is produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol.
Chem. 264:5503-5509); and the like. pGEX vectors (Promega, Madison, Wis.)
may also be used to express foreign polypeptides as fusion proteins with
glutathione S-transferase (GST). In general, such fusion proteins are
soluble and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of free
glutathione. Proteins made in such systems may be designed to include
heparin, thrombin, or factor XA protease cleavage sites so that the cloned
polypeptide of interest can be released from the GST moiety at will.
In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha factor,
alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.
(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.
In cases where plant expression vectors are used, the expression of
sequences encoding HGMPR may be driven by any of a number of promoters.
For example, viral promoters such as the 35S and 19S promoters of CaMV may
be used alone or in combination with the omega leader sequence from TMV
(Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters
such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi,
G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science
224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.
17:85-105). These constructs can be introduced into plant cells by direct
DNA transformation or pathogen-mediated transfection. Such techniques are
described in a number of generally available reviews (see, for example,
Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and
Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).
An insect system may also be used to express HGMPR. For example, in one
such system, Autographa californica nuclear polyhedrosis virus (AcNPV)
is used as a vector to express foreign genes in Spodoptera frugiperda
cells or in Trichoplusia larvae. The sequences encoding HGMPR
may be cloned into a non-essential region of the virus, such as the
polyhedrin gene, and placed under control of the polyhedrin promoter.
Successful insertion of HGMPR will render the polyhedrin gene inactive and
produce recombinant virus lacking coat protein. The recombinant viruses
may then be used to infect, for example, S. frugiperda cells or
Trichoplusia larvae in which HGMPR may be expressed (Engelhard, E. K.
et al. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).
In mammalian host cells, a number of viral-based expression systems may be
utilized. In cases where an adenovirus is used as an expression vector,
sequences encoding HGMPR may be ligated into an adenovirus
transcription/translation complex consisting of the late promoter and
tripartite leader sequence. Insertion in a non-essential E1 or E3 region
of the viral genome may be used to obtain a viable virus which is capable
of expressing HGMPR in infected host cells (Logan, J. and Shenk, T. (1984)
Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcription
enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to
increase expression in mammalian host cells.
Specific initiation signals may also be used to achieve more efficient
translation of sequences encoding HGMPR. Such signals include the ATG
initiation codon and adjacent sequences. In cases where sequences encoding
HGMPR, its initiation codon, and upstream sequences are inserted into the
appropriate expression vector, no additional transcriptional or
translational control signals may be needed. However, in cases where only
coding sequence, or a portion thereof, is inserted, exogenous
translational control signals including the ATG initiation codon should be
provided. Furthermore, the initiation codon should be in the correct
reading frame to ensure translation of the entire insert. Exogenous
translational elements and initiation codons may be of various origins,
both natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers which are appropriate for the particular
cell system which is used, such as those described in the literature (Scharf,
D. et al. (1994) Results Probl. Cell Differ. 20:125-162).
In addition, a host cell strain may be chosen for its ability to modulate
the expression of the inserted sequences or to process the expressed
protein in the desired fashion. Such modifications of the polypeptide
include, but are not limited to, acetylation, carboxylation, glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding and/or function. Different host
cells such as CHO, HeLa, MDCK, HEK293, and WI38, which have specific
cellular machinery and characteristic mechanisms for such
post-translational activities, may be chosen to ensure the correct
modification and processing of the foreign protein.
For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which stably express
HGMPR may be transformed using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector. Following the
introduction of the vector, cells may be allowed to grow for 1-2 days in
an enriched media before they are switched to selective media. The purpose
of the selectable marker is to confer resistance to selection, and its
presence allows growth and recovery of cells which successfully express
the introduced sequences. Resistant clones of stably transformed cells may
be proliferated using tissue culture techniques appropriate to the cell
Any number of selection systems may be used to recover transformed cell
lines. These include, but are not limited to, the herpes simplex virus
thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine
phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes
which can be employed in tk- or aprt- cells,
respectively. Also, antimetabolite, antibiotic or herbicide resistance can
be used as the basis for selection; for example, dhfr which confers
resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
77:3567-70); npt, which confers resistance to the aminoglycosides neomycin
and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and
als or pat, which confers resistance to chlorsulfuron and phosphinotricin
acetyltransferase, respectively (Murry, supra). Additional selectable
genes have been described, for example, trpB, which allows cells to
utilize indole in place of tryptophan, or hisD, which allows cells to
utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan
(1988) Proc. Natl. Acad. Sci. 85:8047-51). Recently, the use of visible
markers has gained popularity with such markers as anthocyanins, β
glucuronidase and its substrate GUS, and luciferase and its substrate
luciferin, being widely used not only to identify transformants, but also
to quantify the amount of transient or stable protein expression
attributable to a specific vector system (Rhodes, C. A. et al. (1995)
Methods Mol. Biol. 55:121-131).
Although the presence/absence of marker gene expression suggests that the
gene of interest is also present, its presence and expression may need to
be confirmed. For example, if the sequence encoding HGMPR is inserted
within a marker gene sequence, recombinant cells containing sequences
encoding HGMPR can be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a sequence
encoding HGMPR under the control of a single promoter. Expression of the
marker gene in response to induction or selection usually indicates
expression of the tandem gene as well.
Alternatively, host cells which contain the nucleic acid sequence encoding
HGMPR and express HGMPR may be identified by a variety of procedures known
to those of skill in the art. These procedures include, but are not
limited to, DNA—DNA or DNA-RNA hybridizations and protein bioassay or
immunoassay techniques which include membrane, solution, or chip based
technologies for the detection and/or quantification of nucleic acid or
The presence of polynucleotide sequences encoding HGMPR can be detected by
DNA—DNA or DNA-RNA hybridization or amplification using probes or portions
or fragments of polynucleotides encoding HGMPR. Nucleic acid amplification
based assays involve the use of oligonucleotides or oligomers based on the
sequences encoding HGMPR to detect transformants containing DNA or RNA
encoding HGMPR. As used herein "oligonucleotides" or "oligomers" refer to
a nucleic acid sequence of at least about 10 nucleotides and as many as
about 60 nucleotides, preferably about 15 to 30 nucleotides, and more
preferably about 20-25 nucleotides, which can be used as a probe or
A variety of protocols for detecting and measuring the expression of HGMPR,
using either polyclonal or monoclonal antibodies specific for the protein
are known in the art. Examples include enzyme-linked immunosorbent assay
(ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on HGMPR is preferred, but a
competitive binding assay may be employed. These and other assays are
described, among other places, in Hampton, R. et al. (1990; Serological
Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox,
D. E. et al. (1983; J. Exp. Med. 158:1211-1216).
A wide variety of labels and conjugation techniques are known by those
skilled in the art and may be used in various nucleic acid and amino acid
assays. Means for producing labeled hybridization or PCR probes for
detecting sequences related to polynucleotides encoding HGMPR include
oligolabeling, nick translation, end-labeling or PCR amplification using a
labeled nucleotide. Alternatively, the sequences encoding HGMPR, or any
portions thereof may be cloned into a vector for the production of an mRNA
probe. Such vectors are known in the art, are commercially available, and
may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
These procedures may be conducted using a variety of commercially
available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison
Wis.); and U.S. Biochemical Corp., Cleveland, Ohio). Suitable reporter
molecules or labels, which may be used, include radionuclides, enzymes,
fluorescent, chemiluminescent, or chromogenic agents as well as
substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding HGMPR may be
cultured under conditions suitable for the expression and recovery of the
protein from cell culture. The protein produced by a recombinant cell may
be secreted or contained intracellularly depending on the sequence and/or
the vector used. As will be understood by those of skill in the art,
expression vectors containing polynucleotides which encode HGMPR may be
designed to contain signal sequences which direct secretion of HGMPR
through a prokaryotic or eukaryotic cell membrane. Other recombinant
constructions may be used to join sequences encoding HGMPR to nucleotide
sequences encoding a polypeptide domain which will facilitate purification
of soluble proteins. Such purification facilitating domains include, but
are not limited to, metal chelating peptides such as histidine-tryptophan
modules that allow purification on immobilized metals, protein A domains
that allow purification on immobilized immunoglobulin, and the domain
utilized in the FLAGS extension/affinity purification system (Immunex
Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such
as those specific for Factor XA or enterokinase (Invitrogen, San Diego,
Calif.) between the purification domain and HGMPR may be used to
facilitate purification. One such expression vector provides for
expression of a fusion protein containing HGMPR and a nucleic acid
encoding 6 histidine residues preceding a thioredoxin or an enterokinase
cleavage site. The histidine residues facilitate purification on IMIAC
(immobilized metal ion affinity chromatography) as described in Porath, J.
et al. (1992, Prot. Exp. Purif. 3: 263-281) while the enterokinase
cleavage site provides a means for purifying HGMPR from the fusion
protein. A discussion of vectors which contain fusion proteins is provided
in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).
In addition to recombinant production, fragments of HGMPR may be produced
by direct peptide synthesis using solid-phase techniques (Merrifield J.
(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed
using manual techniques or by automation. Automated synthesis may be
achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin
Elmer). Various fragments of HGMPR may be chemically synthesized
separately and combined using chemical methods to produce the full length
HGMPR bears chemical and structural homology to GMPRs from E. coli
(SEQ ID NO:3) and man (SEQ ID NO:4). Furthermore, GMPRs are known to play
a role in various conditions involving a proliferative cell state such as
cancer, viral diseases, inflammation, or the immune response. Based on
these facts, and the expression of HGMPR in various normal and tumor
tissues and in tissues involved in inflammation and the immune response,
HGMPR is believed to play a role in cancer, viral diseases, inflammatory
diseases, and immunological disorders.
Therefore, in one embodiment, a vector expressing antisense of the
polynucleotide encoding HGMPR may be administered to a subject to treat or
prevent cancer. Cancers may include, but are not limited to, cancers of
the prostate, brain, breast, colon, heart, and leukemias.
In another embodiment, a vector expressing antisense of the polynucleotide
encoding HGMPR may be administered to a subject to treat or prevent viral
diseases. Viral diseases may include, but are not limited to herpes
simplex type I and type II, influenza, rhinovirus, cytomegalovirus,
hepatitis, and human immunodeficency virus (AIDS).
In another embodiment, a vector expressing antisense of the polynucleotide
encoding HGMPR may be administered to a subject to treat or prevent
inflammatory diseases and immunological disorders, which may include, but
are not limited to conditions such as anemias, asthma, systemic lupus,
myasthenia gravis, diabetes mellitus, autoimmune thyroiditis, pancreatitis,
ulcerative colitis, osteoporosis, glomerulonephritis; rheumatoid and
osteoarthritis; and scleroderma.
In another embodiment, antagonists or inhibitors of HGMPR may be
administered to a subject to treat or prevent cancers described above.
In another embodiment, antagonists or inhibitors of HGMPR may be
administered to a subject to treat or prevent viral diseases described
In another embodiment, antagonists or inhibitors of HGMPR may be
administered to a subject to treat or prevent inflammatory diseases and
immunological disorders described above.
Antibodies which are specific for HGMPR may be used directly as an
antagonist, or indirectly as a targeting or delivery mechanism for
bringing a pharmaceutical agent to cells or tissue which express HGMPR.
In other embodiments, any of the therapeutic proteins, antagonists,
antibodies, agonists, antisense sequences or vectors described above may
be administered in combination with other appropriate therapeutic agents.
Selection of the appropriate agents for use in combination therapy may be
made by one of ordinary skill in the art, according to conventional
pharmaceutical principles. The combination of therapeutic agents may act
synergistically to effect the treatment or prevention of the various
disorders described above. Using this approach, one may be able to achieve
therapeutic efficacy with lower dosages of each agent, thus reducing the
potential for adverse side effects.
Antagonists or inhibitors of HGMPR may be produced using methods which are
generally known in the art. In particular, purified HGMPR may be used to
produce antibodies or to screen libraries of pharmaceutical agents to
identify those which specifically bind HGMPR.
Antibodies to HGMPR may be generated using methods that are well known in
the art. Such antibodies may include, but are not limited to, polyclonal,
monoclonal, chimeric, single chain, Fab fragments, and fragments produced
by a Fab expression library. Neutralizing antibodies, (i.e., those which
inhibit dimer formation) are especially preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans, and others, may be immunized by injection with HGMPR
or any fragment or oligopeptide thereof which has immunogenic properties.
Depending on the host species, various adjuvants may be used to increase
immunological response. Such adjuvants include, but are not limited to,
Freund's, mineral gels such as aluminum hydroxide, and surface active
substances such as lysolecithin, pluronic polyols, polyanions, peptides,
oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among
adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are especially preferable.
It is preferred that the peptides, fragments, or oligopeptides used to
induce antibodies to HGMPR have an amino acid sequence consisting of at
least five amino acids, and more preferably at least 10 amino acids. It is
also preferable that they are identical to a portion of the amino acid
sequence of the natural protein, and they may contain the entire amino
acid sequence of a small, naturally occurring molecule. Short stretches of
HGMPR amino acids may be fused with those of another protein such as
keyhole limpet hemocyanin and antibody produced against the chimeric
Monoclonal antibodies to HGMPR may be prepared using any technique which
provides for the production of antibody molecules by continuous cell lines
in culture. These include, but are not limited to, the hybridoma
technique, the human B-cell hybridoma technique, and the EBV-hybridoma
technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al.
(1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl.
Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.
In addition, techniques developed for the production of "chimeric
antibodies", the splicing of mouse antibody genes to human antibody genes
to obtain a molecule with appropriate antigen specificity and biological
activity can be used (Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci.
81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; Takeda,
S. et al. (1985) Nature 314:452-454). Alternatively, techniques described
for the production of single chain antibodies may be adapted, using
methods known in the art, to produce HGMPR-specific single chain
antibodies. Antibodies with related specificity, but of distinct idiotypic
composition, may be generated by chain shuffling from random combinatorial
immunoglobulin libraries (Burton D. R. (1991) Proc. Natl. Acad. Sci.
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population or by screening recombinant immunoglobulin libraries
or panels of highly specific binding reagents as disclosed in the
literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:
3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
Antibody fragments which contain specific binding sites for HGMPR may also
be generated. For example, such fragments include, but are not limited to,
the F(ab′)2 fragments which can be produced by pepsin digestion of the
antibody molecule and the Fab fragments which can be generated by reducing
the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab
expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired specificity (Huse,
W. D. et al. (1989) Science 254:1275-1281).
Various immunoassays may be used for screening to identify antibodies
having the desired specificity. Numerous protocols for competitive binding
or immunoradiometric assays using either polyclonal or monoclonal
antibodies with established specificities are well known in the art. Such
immunoassays typically involve the measurement of complex formation
between HGMPR and its specific antibody. A two-site, monoclonal-based
immunoassay utilizing monoclonal antibodies reactive to two
non-interfering HGMPR epitopes is preferred, but a competitive binding
assay may also be employed (Maddox, supra).
In another embodiment of the invention, the polynucleotides encoding HGMPR,
or any fragment thereof, or antisense molecules, may be used for
therapeutic purposes. In one aspect, antisense to the polynucleotide
encoding HGMPR may be used in situations in which it would be desirable to
block the transcription of the mRNA. In particular, cells may be
transformed with sequences complementary to polynucleotides encoding HGMPR.
Thus, antisense molecules may be used to modulate HGMPR activity, or to
achieve regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligomers or larger fragments, can be
designed from various locations along the coding or control regions of
sequences encoding HGMPR.
Expression vectors derived from retroviruses, adenovirus, herpes or
vaccinia viruses, or from various bacterial plasmids may be used for
delivery of nucleotide sequences to the targeted organ, tissue or cell
population. Methods which are well known to those skilled in the art can
be used to construct recombinant vectors which will express antisense
molecules complementary to the polynucleotides of the gene encoding HGMPR.
These techniques are described both in Sambrook et al. (supra) and in
Ausubel et al. (supra).
Genes encoding HGMPR can be turned off by transforming a cell or tissue
with expression vectors which express high levels of a polynucleotide or
fragment thereof which encodes HGMPR. Such constructs may be used to
introduce untranslatable sense or antisense sequences into a cell. Even in
the absence of integration into the DNA, such vectors may continue to
transcribe RNA molecules until they are disabled by endogenous nucleases.
Transient expression may last for a month or more with a non-replicating
vector and even longer if appropriate replication elements are part of the
As mentioned above, modifications of gene expression can be obtained by
designing antisense molecules, DNA, RNA, or PNA, to the control regions of
the gene encoding HGMPR, i.e., the promoters, enhancers, and introns.
Oligonucleotides derived from the transcription initiation site, e.g.,
between positions -10 and +10 from the start site, are preferred.
Similarly, inhibition can be achieved using "triple helix" base-pairing
methodology. Triple helix pairing is useful because it causes inhibition
of the ability of the double helix to open sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using triplex DNA have been described in the
literature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,
Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,
N.Y.). The antisense molecules may also be designed to block translation
of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the
specific cleavage of RNA. The mechanism of ribozyme action involves
sequence-specific hybridization of the ribozyme molecule to complementary
target RNA, followed by endonucleolytic cleavage. Examples which may be
used include engineered hammerhead motif ribozyme molecules that can
specifically and efficiently catalyze endonucleolytic cleavage of
sequences encoding HGMPR.
Specific ribozyme cleavage sites within any potential RNA target are
initially identified by scanning the target molecule for ribozyme cleavage
sites which include the following sequences: GUA, GUU, and GUC. Once
identified, short RNA sequences of between 15 and 20 ribonucleotides
corresponding to the region of the target gene containing the cleavage
site may be evaluated for secondary structural features which may render
the oligonucleotide inoperable. The suitability of candidate targets may
also be evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection assays.
Antisense molecules and ribozymes of the invention may be prepared by any
method known in the art for the synthesis of nucleic acid molecules. These
include techniques for chemically synthesizing oligonucleotides such as
solid phase phosphoramidite chemical synthesis. Alternatively, RNA
molecules may be generated by in vitro and in vivo transcription of DNA
sequences encoding HGMPR. Such DNA sequences may be incorporated into a
wide variety of vectors with suitable RNA polymerase promoters such as T7
or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA
constitutively or inducibly can be introduced into cell lines, cells, or
RNA molecules may be modified to increase intracellular stability and
half-life. Possible modifications include, but are not limited to, the
addition of flanking sequences at the 5′ and/or 3′ ends of the molecule or
the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in
the production of PNAs and can be extended in all of these molecules by
the inclusion of nontraditional bases such as inosine, queosine, and
wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available
and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo
therapy, vectors may be introduced into stem cells taken from the patient
and clonally propagated for autologous transplant back into that same
patient. Delivery by transfection and by liposome injections may be
achieved using methods which are well known in the art.
Any of the therapeutic methods described above may be applied to any
subject in need of such therapy, including, for example, mammals such as
dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of
a pharmaceutical composition, in conjunction with a pharmaceutically
acceptable carrier, for any of the therapeutic effects discussed above.
Such pharmaceutical compositions may consist of HGMPR, antibodies to HGMPR,
mimetics, agonists, antagonists, or inhibitors of HGMPR. The compositions
may be administered alone or in combination with at least one other agent,
such as stabilizing compound, which may be administered in any sterile,
biocompatible pharmaceutical carrier, including, but not limited to,
saline, buffered saline, dextrose, and water. The compositions may be
administered to a patient alone, or in combination with other agents,
drugs or hormones.
The pharmaceutical compositions utilized in this invention may be
administered by any number of routes including, but not limited to, oral,
intravenous, intramuscular, intra-arterial, intramedullary, intrathecal,
intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions
may contain suitable pharmaceutically-acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the active
compounds into preparations which can be used pharmaceutically. Further
details on techniques for formulation and administration may be found in
the latest edition of Remington's Pharmaceutical Sciences (Maack
Publishing Co., Easton, Pa.).
Pharmaceutical compositions for oral administration can be formulated
using pharmaceutically acceptable carriers well known in the art in
dosages suitable for oral administration. Such carriers enable the
pharmaceutical compositions to be formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions, and the like, for
ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through
combination of active compounds with solid excipient, optionally grinding
a resulting mixture, and processing the mixture of granules, after adding
suitable auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are carbohydrate or protein fillers, such as sugars,
including lactose, sucrose, mannitol, or sorbitol; starch from corn,
wheat, rice, potato, or other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums
including arabic and tragacanth; and proteins such as gelatin and
collagen. If desired, disintegrating or solubilizing agents may be added,
such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a
salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as
concentrated sugar solutions, which may also contain gum arabic, talc,
polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium
dioxide, lacquer solutions, and suitable organic solvents or solvent
mixtures. Dyestuffs or pigments may be added to the tablets or dragee
coatings for product identification or to characterize the quantity of
active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit
capsules made of gelatin, as well as soft, sealed capsules made of gelatin
and a coating, such as glycerol or sorbitol. Push-fit capsules can contain
active ingredients mixed with a filler or binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft capsules, the active compounds may be dissolved or
suspended in suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks'solution, Ringer's solution, or physiologically
buffered saline. Aqueous injection suspensions may contain substances
which increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions
of the active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty oils
such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate
or triglycerides, or liposomes. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the solubility of
the compounds to allow for the preparation of highly concentrated
For topical or nasal administration, penetrants appropriate to the
particular barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
The pharmaceutical compositions of the present invention may be
manufactured in a manner that is known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating, entrapping, or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many acids, including but not limited to, hydrochloric, sulfuric,
acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more
soluble in aqueous or other protonic solvents than are the corresponding
free base forms. In other cases, the preferred preparation may be a
lyophilized powder which may contain any or all of the following: 1-50 mM
histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to
5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed
in an appropriate container and labeled for treatment of an indicated
condition. For administration of HGMPR, such labeling would include
amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein the active ingredients are contained in an effective
amount to achieve the intended purpose. The determination of an effective
dose is well within the capability of those skilled in the art.
For any compound, the therapeutically effective dose can be estimated
initially either in cell culture assays, e.g., of neoplastic cells, or in
animal models, usually mice, rabbits, dogs, or pigs. The animal model may
also be used to determine the appropriate concentration range and route of
administration. Such information can then be used to determine useful
doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active
ingredient, for example HGMPR or fragments thereof, antibodies of HGMPR,
agonists, antagonists or inhibitors of HGMPR, which ameliorates the
symptoms or condition. Therapeutic efficacy and toxicity may be determined
by standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., ED50 (the dose therapeutically effective in 50% of the
population) and LD50 (the dose lethal to 50% of the population). The dose
ratio between therapeutic and toxic effects is the therapeutic index, and
it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions
which exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies is used in formulating a range
of dosage for human use. The dosage contained in such compositions is
preferably within a range of circulating concentrations that include the
ED50 with little or no toxicity. The dosage varies within this range
depending upon the dosage form employed, sensitivity of the patient, and
the route of administration.
The exact dosage will be determined by the practitioner, in light of
factors related to the subject that requires treatment. Dosage and
administration are adjusted to provide sufficient levels of the active
moiety or to maintain the desired effect. Factors which may be taken into
account include the severity of the disease state, general health of the
subject, age, weight, and gender of the subject, diet, time and frequency
of administration, drug combination(s), reaction sensitivities, and
tolerance/response to therapy. Long-acting pharmaceutical compositions may
be administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular formulation.
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a
total dose of about 1 g, depending upon the route of administration.
Guidance as to particular dosages and methods of delivery is provided in
the literature and generally available to practitioners in the art. Those
skilled in the art will employ different formulations for nucleotides than
for proteins or their inhibitors. Similarly, delivery of polynucleotides
or polypeptides will be specific to particular cells, conditions,
In another embodiment, antibodies which specifically bind HGMPR may be
used for the diagnosis of conditions or diseases characterized by
expression of HGMPR, or in assays to monitor patients being treated with
HGMPR, agonists, antagonists or inhibitors. The antibodies useful for
diagnostic purposes may be prepared in the same manner as those described
above for therapeutics. Diagnostic assays for HGMPR include methods which
utilize the antibody and a label to detect HGMPR in human body fluids or
extracts of cells or tissues. The antibodies may be used with or without
modification, and may be labeled by joining them, either covalently or
non-covalently, with a reporter molecule. A wide variety of reporter
molecules which are known in the art may be used, several of which are
A variety of protocols including ELISA, RIA, and FACS for measuring HGMPR
are known in the art and provide a basis for diagnosing altered or
abnormal levels of HGMPR expression. Normal or standard values for HGMPR
expression are established by combining body fluids or cell extracts taken
from normal mammalian subjects, preferably human, with antibody to HGMPR
under conditions suitable for complex formation. The amount of standard
complex formation may be quantified by various methods, but preferably by
photometric means. Quantities of HGMPR expressed in subject, control and
disease, samples from biopsied tissues are compared with the standard
values. Deviation between standard and subject values establishes the
parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding HGMPR
may be used for diagnostic purposes. The polynucleotides which may be used
include oligonucleotide sequences, antisense RNA and DNA molecules, and
PNAs. The polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of HGMPR may be
correlated with disease. The diagnostic assay may be used to distinguish
between absence, presence, and excess expression of HGMPR, and to monitor
regulation of HGMPR levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of
detecting polynucleotide sequences, including genomic sequences, encoding
HGMPR or closely related molecules, may be used to identify nucleic acid
sequences which encode HGMPR. The specificity of the probe, whether it is
made from a highly specific region, e.g., 10 unique nucleotides in the 5′
regulatory region, or a less specific region, e.g., especially in the 3′
coding region, and the stringency of the hybridization or amplification
(maximal, high, intermediate, or low) will determine whether the probe
identifies only naturally occurring sequences encoding HGMPR, alleles, or
Probes may also be used for the detection of related sequences, and should
preferably contain at least 50% of the nucleotides from any of the HGMPR
encoding sequences. The hybridization probes of the subject invention may
be DNA or RNA and derived from the nucleotide sequence of SEQ ID NO:2 or
from genomic sequence including promoter, enhancer elements, and introns
of the naturally occurring HGMPR.
Means for producing specific hybridization probes for DNAs encoding HGMPR
include the cloning of nucleic acid sequences encoding HGMPR or HGMPR
derivatives into vectors for the production of mRNA probes. Such vectors
are known in the art, commercially available, and may be used to
synthesize RNA probes in vitro by means of the addition of the appropriate
RNA polymerases and the appropriate labeled nucleotides. Hybridization
probes may be labeled by a variety of reporter groups, for example,
radionuclides such as 32P or 35S, or enzymatic labels, such as alkaline
phosphatase coupled to the probe via avidin/biotin coupling systems, and
Polynucleotide sequences encoding HGMPR may be used for the diagnosis of
conditions or diseases which are associated with expression of HGMPR.
Examples of such conditions or diseases include cancers of the prostate,
brain, breast, colon, heart, and leukemias, viral diseases such as simplex
type I and type II, influenza, rhinovirus, cytomegalovirus, hepatitis, and
human immunodeficency virus (AIDS), and immunological disorders such as
anemias, asthma, systemic lupus, and myasthenia gravis, diabetes mellitus,
osteoporosis, glomerulonephritis; rheumatoid and osteoarthritis; and
scleroderma. The polynucleotide sequences encoding HGMPR may be used in
Southern or northern analysis, dot blot, or other membrane-based
technologies; in PCR technologies; or in dip stick, pin, ELISA or chip
assays utilizing fluids or tissues from patient biopsies to detect altered
HGMPR expression. Such qualitative or quantitative methods are well known
in the art.
In a particular aspect, the nucleotide sequences encoding HGMPR may be
useful in assays that detect activation or induction of various cancers,
particularly those mentioned above. The nucleotide sequences encoding
HGMPR may be labeled by standard methods, and added to a fluid or tissue
sample from a patient under conditions suitable for the formation of
hybridization complexes. After a suitable incubation period, the sample is
washed and the signal is quantitated and compared with a standard value.
If the amount of signal in the biopsied or extracted sample is
significantly altered from that of a comparable control sample, the
nucleotide sequences have hybridized with nucleotide sequences in the
sample, and the presence of altered levels of nucleotide sequences
encoding HGMPR in the sample indicates the presence of the associated
disease. Such assays may also be used to evaluate the efficacy of a
particular therapeutic treatment regimen in animal studies, in clinical
trials, or in monitoring the treatment of an individual patient.
In order to provide a basis for the diagnosis of disease associated with
expression of HGMPR, a normal or standard profile for expression is
established. This may be accomplished by combining body fluids or cell
extracts taken from normal subjects, either animal or human, with a
sequence, or a fragment thereof, which encodes HGMPR, under conditions
suitable for hybridization or amplification. Standard hybridization may be
quantified by comparing the values obtained from normal subjects with
those from an experiment where a known amount of a substantially purified
polynucleotide is used. Standard values obtained from normal samples may
be compared with values obtained from samples from patients who are
symptomatic for disease. Deviation between standard and subject values is
used to establish the presence of disease.
Once disease is established and a treatment protocol is initiated,
hybridization assays may be repeated on a regular basis to evaluate
whether the level of expression in the patient begins to approximate that
which is observed in the normal patient. The results obtained from
successive assays may be used to show the efficacy of treatment over a
period ranging from several days to months.
With respect to cancer, the presence of a relatively high amount of
transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a means
for detecting the disease prior to the appearance of actual clinical
symptoms. A more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive treatment
earlier thereby preventing the development or further progression of the
Additional diagnostic uses for oligonucleotides designed from the
sequences encoding HGMPR may involve the use of PCR. Such oligomers may be
chemically synthesized, generated enzymatically, or produced from a
recombinant source. Oligomers will preferably consist of two nucleotide
sequences, one with sense orientation (5′->3′) and another with antisense
(3′<-5′), employed under optimized conditions for identification of a
specific gene or condition. The same two oligomers, nested sets of
oligomers, or even a degenerate pool of oligomers may be employed under
less stringent conditions for detection and/or quantitation of closely
related DNA or RNA sequences.
Methods which may also be used to quantitate the expression of HGMPR
include radiolabeling or biotinylating nucleotides, coamplification of a
control nucleic acid, and standard curves onto which the experimental
results are interpolated (Melby, P. C. et al. (1993) J. Immunol. Methods,
159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The speed
of quantitation of multiple samples may be accelerated by running the
assay in an ELISA format where the oligomer of interest is presented in
various dilutions and a spectrophotometric or calorimetric response gives
In another embodiment of the invention, the nucleic acid sequences which
encode HGMPR may also be used to generate hybridization probes which are
useful for mapping the naturally occurring genomic sequence. The sequences
may be mapped to a particular chromosome or to a specific region of the
chromosome using well known techniques. Such techniques include FISH, FACS,
or artificial chromosome constructions, such as yeast artificial
chromosomes, bacterial artificial chromosomes, bacterial PI constructions
or single chromosome cDNA libraries as reviewed in Price, C. M. (1993)
Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.
FISH (as described in Verma et al. (1988) Human Chromosomes: A Manual
of Basic Techniques, Pergamon Press, New York, N.Y.) may be correlated
with other physical chromosome mapping techniques and genetic map data.
Examples of genetic map data can be found in the 1994 Genome Issue of
Science (265:1981f). Correlation between the location of the gene encoding
HGMPR on a physical chromosomal map and a specific disease, or
predisposition to a specific disease, may help delimit the region of DNA
associated with that genetic disease. The nucleotide sequences of the
subject invention may be used to detect differences in gene sequences
between normal, carrier, or affected individuals.
In situ hybridization of chromosomal preparations and physical mapping
techniques such as linkage analysis using established chromosomal markers
may be used for extending genetic maps. Often the placement of a gene on
the chromosome of another mammalian species, such as a mouse, may reveal
associated markers even if the number or arm of a particular human
chromosome is not known. New sequences can be assigned to chromosomal
arms, or parts thereof, by physical mapping. This provides valuable
information to investigators searching for disease genes using positional
cloning or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular genomic
region, for example, AT to 11q22-23 (Gatti, R. A. et al. (1988) Nature
336:577-580), any sequences mapping to that area may represent associated
or regulatory genes for further investigation. The nucleotide sequence of
the subject invention may also be used to detect differences in the
chromosomal location due to translocation, inversion, etc. among normal,
carrier, or affected individuals.
In another embodiment of the invention, HGMPR, its catalytic or
immunogenic fragments or oligopeptides thereof, can be used for screening
libraries of compounds in any of a variety of drug screening techniques.
The fragment employed in such screening may be free in solution, affixed
to a solid support, borne on a cell surface, or located intracellularly.
The formation of binding complexes, between HGMPR and the agent being
tested, may be measured.
Another technique for drug screening which may be used provides for high
throughput screening of compounds having suitable binding affinity to the
protein of interest as described in published PCT application WO84/03564.
In this method, as applied to HGMPR large numbers of different small test
compounds are synthesized on a solid substrate, such as plastic pins or
some other surface. The test compounds are reacted with HGMPR, or
fragments thereof, and washed. Bound HGMPR is then detected by methods
well known in the art. Purified HGMPR can also be coated directly onto
plates for use in the aforementioned drug screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the
peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in
which neutralizing antibodies capable of binding HGMPR specifically
compete with a test compound for binding HGMPR. In this manner, the
antibodies can be used to detect the presence of any peptide which shares
one or more antigenic determinants with HGMPR.
In additional embodiments, the nucleotide sequences which encode HGMPR may
be used in any molecular biology techniques that have yet to be developed,
provided the new techniques rely on properties of nucleotide sequences
that are currently known, including, but not limited to, such properties
as the triplet genetic code and specific base pair interactions.
Claim 1 of 2 Claims
1. A substantially purified
guanosine monophosphate reductase comprising the amino acid sequence of SEQ
ID NO:1 or an enzymatically active fragments thereof.
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