Methods and compositions for the identification, characterization and inhibition of farnesyltransferase

Disclosed are methods and compositions for the identification, characterization and inhibition of mammalian protein farnesyltransferases, enzymes involved in the farnesylation of various cellular proteins, including cancer related ras proteins such as p21.sup.ras. One protein farnesyltransferase which is disclosed herein exhibits a molecular weight of between about 70,000 and about 100,000 upon gel exclusion chromatography. Also disclosed are methods and compositions for the preparation of farnesyltransferase by recombinant means, following the molecular cloning and co-expression of its two subunits, for assay and purification of the enzyme, as well as procedures for using the purified enzyme in screening protocols for the identification of possible anticancer agents which inhibit the enzyme and thereby prevent expression of proteins such as p21.sup.ras. Also disclosed is a families of compounds which act either as false substrates for the enzyme or as pure inhibitors and can therefore be employed for inhibition of the enzyme. The most potent inhibitors are ones in which phenylalanine occurs at the third position of a tetrapeptide whose amino terminus is cysteine.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates generally to improved peptide-based inhibitors of 
farnesyltransferase, the enzyme responsible for the farnesylation of 
p21.sup.ras protein, and more particularly relates to peptide-based "pure" 
inhibitors having improved characteristics and structures. Improvements 
are based on the inventors' discovery of structural characteristics that 
ensure that a peptide inhibitor will exhibit "pure" inhibitor 
characteristics, which provide important guidelines for inhibitor design 
that will allow cellular uptake while preserving inhibitory capabilitites. 
2. Description of the Related Art 
In recent years, some progress has been made in the elucidation of cellular 
events lending to the development or progression of various types of 
cancers. A great amount of research has centered on identifying genes 
which are altered or mutated in cancer relative to normal cells. In fact, 
genetic research has led to the identification of a variety of gene 
families in which mutations can lead to the development of a wide variety 
of tumors. The ras gene family is a family of closely related genes that 
frequently contain mutations involved in many human tumors, including 
tumors of virtually every tumor group (see, e.g., ref. 1 for a review). In 
fact, altered ras genes are the most frequently identified oncogenes in 
human tumors (2). 
The ras gene family comprises three genes, H-ras, K-ras and N-ras, which 
encode similar proteins with molecular weights of about 21,000 (2). These 
proteins, of ten termed p21.sup.ras, comprise a family of GTP-binding and 
hydrolyzing proteins that regulate cell growth when bound to the inner 
surface of the plasma membrane (3,4). Overproduction of P21.sup.ras ras 
proteins or mutations that abolish their GTP-ase activity lead to 
uncontrolled cell division (5). However, the transforming activity of ras 
is dependent on the localization of the protein to membranes, a property 
thought to be conferred by the addition of farnesyl groups (3,6). 
A precedent for the covalent isoprenylation of proteins had been 
established about a decade ago when peptide mating factors secreted by 
several fungi were shown to contain a farnesyl group attached in thioether 
linkage to the C-terminal cysteine (7-9). Subsequent studies with the 
mating a-factor from Saccharomyces cerevisiae and farnesylated proteins 
from animal cells have clarified the mechanism of farnesylation. In each 
of these proteins the farnesylated cysteine is initially the fourth 
residue from the C terminus (3, 4, 10). Immediately after translation, in 
a sequence of events whose order is not yet totally established, a 
farnesyl group is attached to this cysteine, the protein is cleaved on the 
C-terminal side of this residue, and the free COOH group of the cysteine 
is methylated (3, 10, 11, 12). All of these reactions are required for the 
secretion of active a-factor in Saccharomyces (4). 
Most, if not all, of the known p21.sup.ras proteins contain the cysteine 
prerequisite, which is processed by farnesylation, proteolysis and 
COOH-methylation, just as with the yeast mating factor (3, 4, 10, 11, 12). 
The farnesylated p21.sup.ras binds loosely to the plasma membrane, from 
which most of it can be released with salt (3). After binding to the 
membrane, some P21.sup.ras proteins are further modified by the addition 
of palmitate in thioester linkage to cysteines near the farnesylated 
C-terminal cysteine (3) Palmitylation renders the protein even more 
hydrophobic and anchors it more tightly to the plasma membrane. 
However, although it appears to be clear that farnesylation is a key event 
in ras-related cancer development, prior to now, the nature of this event 
has remained obscure. Nothing has been known previously, for example, of 
the nature of the enzyme or enzymes which may be involved in ras 
tumorigenesis or required by the tumor cell to achieve farnesylation. If 
the mechanisms that underlie farnesylation of cancer-related proteins such 
as P21.sup.ras could be elucidated, then procedures and perhaps even 
pharmacologic agents could be developed in an attempt to control or 
inhibit expression of the oncogenic phenotype in a wide variety of 
cancers. It goes without saying that such discoveries would be of 
pioneering proportions in cancer therapy. 
SUMMARY OF THE INVENTION 
The present invention addresses one or more short-comings in the prior art 
through the identification and characterization of an enzyme, termed 
farnesyltransferase, involved in the oncogenic process through the 
transfer of farnesyl groups to various proteins including oncogenic ras 
proteins. The invention relates particularly to the molecular cloning of 
mammalian farnesyltransferase subunits, to the purification of the native 
or recombinant enzyme, to protein and peptide substances that are capable 
of inhibiting the enzyme, and to assay methods for the identification of 
further inhibitory compounds. 
A certain object of the present invention is therefore to provide ready 
means for obtaining farnesyltransferase enzymes, by purification of the 
native enzyme from tissues of choice, or by purification of the 
recombinant enzyme from host cells that express the constituent subunits, 
which methods are proposed to be generally applicable to the purification 
of all such farnesyltransferases. 
It is an additional object of the invention to provide means for obtaining 
these enzymes in a relatively purified form, allowing their use in 
predictive assays for identifying compounds having the ability to reduce 
the activity of or inhibit the farnesyltransferase activity, particularly 
in the context of p21.sup.ras proteins. 
It is a still further object of the invention to identify classes of 
compounds which demonstrate farnesyltransferase inhibiting activity, along 
with a potential application of these compounds in the treatment of 
cancer, particularly ras-related cancers. 
Farnesyltransferase Characterization 
Accordingly, in certain embodiments, the present invention relates to 
compositions which include a purified protein farnesyltransferase enzyme, 
characterized as follows: 
a) capable of catalyzing the transfer of farnesyl to a protein or peptide 
having a farnesyl acceptor moiety; 
b) capable of binding to an affinity chromatography medium comprised of 
TKCVIM coupled to a suitable matrix; 
c) exhibiting a molecular weight of between about 70,000 and about 100,000 
upon gel filtration chromatography; and 
d) having a farnesyltransferase activity that is capable of being inhibited 
by one of the following peptides: 
i) TKCVIM; 
ii) CVIM; or 
iii) KKSKTKCVIM. 
As used herein, the phrase "capable of catalyzing the transfer of farnesol 
to a protein or peptide having a farnesyl acceptor moiety," is intended to 
refer to the functional attributes of farnesyltransferase enzymes of the 
present invention, which catalyze the transfer of farnesol, typically in 
the form of all-trans farnesol, from all-trans farnesyl pyrophosphate to 
proteins which have a sequence recognized by the enzyme for attachment of 
the farnesyl moieties. Thus, the term "farnesyl acceptor moiety" is 
intended to refer to any sequence, typically a short amino acid 
recognition sequence, which is recognized by the enzyme and to which a 
farnesyl group will be attached by such an enzyme. 
Farnesyl acceptor moieties have been characterized by others in various 
proteins as a four amino acid sequence found at the carboxy terminus of 
target proteins. This four amino acid sequence has been characterized as 
-C-A-A-X, wherein "C" is a cysteine residue, "A" refers to any aliphatic 
amino acid, and "X" refers preferably to methionine or serine, with 
glutamine or cysteine being tolerated. While it is believed that other 
substitutes at the "X" position will provide some utility, it has been 
noted that farnesyltransferase will not recognize peptides with leucine in 
this position (35). Of course, the term "aliphatic amino acid" is 
well-known in the art to mean any amino acid having an aliphatic side 
chain, such as, for example, leucine, isoleucine, alanine, methionine, 
valine, etc. While the most preferred aliphatic amino acids, for the 
purposes of the present invention include valine and isoleucine, it is 
believed that virtually any aliphatic amino acids in the designated 
position can be recognized within the farnesyl acceptor moiety. In 
addition, the enzyme has been shown to recognize a peptide containing a 
hydroxylated amino acid (serine) in place of an aliphatic amino acid 
(CSIM). 
Principal examples of proteins or peptides having a farnesyl acceptor 
moiety, for the purposes of the present invention, will be the p21.sup.ras 
proteins, including p21.sup.H-ras, p21.sup.K-rasA, p21.sup.K-rasB and 
p21.sup.N-ras. Thus, in light of the present disclosure, a wide variety of 
peptidyl sequences having a farnesyl acceptor moiety will become apparent. 
As outlined above, the inventors have discovered that the 
farnesyltransferase enzyme is capable of binding to an affinity 
chromatography medium comprised of the peptide TKCVIM, coupled to a 
suitable matrix. This feature of the farnesyltransferase enzyme was 
discovered by the present inventors in developing techniques for its 
isolation. Surprisingly, it has been found that the coupling of a peptide 
such as one which includes CVIM, as does TKCVIM, to a suitable 
chromatography matrix allows for the purification of the protein to a 
significant degree, presumably through interaction and binding of the 
enzyme to the peptidal sequence. A basis for this interaction could be 
posited as due to the apparent presence of a farnesyl acceptor moiety 
within this peptide. 
The phrase "capable of binding to an affinity chromatography medium 
comprised of TKCVIM coupled to a suitable matrix," is intended to refer to 
the ability of the protein to bind to such a medium under conditions as 
specified herein below. There will, of course, be conditions, such as when 
the pH is below 6.0, wherein the farnesyltransferase enzyme will not bind 
effectively to such a matrix. However, through practice of the techniques 
disclosed herein, one will be enabled to achieve this important objective. 
There are numerous chromatography matrixes which are known in the art that 
can be applied to the practice of this invention. The inventors prefer to 
use activated CH-Sepharose 4B, to which peptides such as TKCVIM, or which 
incorporate the CVIM structure, can be readily attached and washed with 
little difficulty. However, the present invention is by no means limited 
to the use of CH-Sepharose 4B, and includes within its intended scope the 
use of any suitable matrix for performing affinity chromatography known in 
the art. Examples include solid matrices with covalently bound linkers, 
and the like, as well as matrices that contain covalently associated 
avidin, which can be used to bind peptides that contain biotin. 
Farnesyltransferase enzymes of the present invention have typically been 
found to exhibit a molecular weight of between about 70,000 and about 
100,000 upon gel filtration chromatography. For comparison purposes, this 
molecular weight was identified for protein farnesyltransferase through 
the use of a Superose 12 column, using a column size, sample load and 
parameters as described herein below. 
It is quite possible, depending on the conditions employed, that different 
chromatographic techniques may demonstrate a farnesyltransferase protein 
that has an apparent molecular weight somewhat different than that 
identified using the preferred techniques set forth in the examples. It is 
intended therefore, that the molecular weight determination and range 
identified for farnesyltransferase in the examples which follow, are 
designated only with respect to the precise techniques disclosed herein. 
It has been determined that the farnesyltransferase can be characterized as 
including two subunits, each having a molecular weight of about 45 to 50 
kDa, as estimated by SDS polyacrylamide gel electrophoresis (PAGE). These 
subunits have been designated as .alpha. and .beta., with the .alpha. 
subunit migrating slightly higher than the .beta. subunit, which suggests 
that the .alpha. subunit may be slightly larger. From tryptic peptide 
sequence analyses and molecular cloning the nature of the .alpha. and 
.beta. subunits as distinct proteins, encoded by separate genes, has been 
confirmed. 
The inventors have found that the holoenzyme forms a stable complex with 
all- trans [.sup.3 H]farnesyl pyrophosphate (FPP) that can be isolated by 
gel electrophoresis. The [.sup.3 H]FFP is not covalently bound to the 
enzyme, and is released unaltered when the enzyme is denatured. When 
incubated with an acceptor such as p21.sup.H-ras, the complex transfers 
[.sup.3 H]farnesyl from the bound [.sup.3 H]FFP to the ras protein. 
Furthermore, crosslinking studies have shown that p21.sup.H-ras binds to 
the .beta. subunit, raising the possibility that the [.sup.3 H]FFP binds 
to the .alpha. subunit. If this is the case, it would invoke a reaction 
mechanism in which the .alpha. subunit act as a prenyl pyrophosphate 
carrier that delivers FPP to p21.sup.H-ras, which is bound to the .beta. 
subunit. Interestingly, the inventors have recently discovered that the 
.alpha. subunit is shared with another prenyltransferase, 
geranylgeranyltransferase, that attaches 20-carbon geranylgeranyl to 
Ras-related proteins. 
An additional property discovered for farnesyltransferase enzymes is that 
they can be inhibited by peptides or proteins, particularly short 
peptides, which include certain structural features, related in some 
degree to the farnesyl acceptor moiety discussed above. As used herein, 
the word "inhibited" refers to any degree of inhibition and is not limited 
for these purposes to only total inhibition. Thus, any degree of partial 
inhibition or relative reduction in farnesyltransferase activity is 
intended to be included within the scope of the term "inhibited." 
Inhibition in this context includes the phenomenon by which a chemical 
constitutes an alternate substrate for the enzyme, and is therefore 
farnesylated in preference to the ras protein, as well as inhibition where 
the compound does not act as an alternate substrate for the enzyme. 
Preparation of Farnesyltransferase 
The present invention is also concerned with techniques for the 
identification and isolation of farnesyltransferase enzymes, and 
particularly mammalian farnesyltransferases. Techniques are herein 
disclosed for the isolation of farnesyltransferase which are believed to 
be applicable to the purification of the native protein, or alternatively, 
to the purification of the recombinant enzyme following the molecular 
cloning and co-expression of the constituent subunits. 
An important feature of the purification scheme disclosed herein involves 
the use of short peptide sequences which the inventors have discovered 
will bind the enzyme, allowing their attachment to chromatography 
matrices, such matrices may, in turn, be used in connection with affinity 
chromatography to purify the enzyme to a relative degree. Thus, in certain 
embodiments, the present invention is concerned with a method of preparing 
a farnesyltransferase enzyme which includes the steps of: 
(a) preparing a cellular extract which includes the enzyme; 
(b) subjecting the extract to affinity chromatography on an affinity 
chromatography medium to bind the enzyme thereto, the medium comprised of 
a farnesyltransferase binding peptide coupled to a suitable matrix; 
(c) washing the medium to remove impurities; and 
(d) eluting the enzyme from the washed medium. 
Thus, the first step of the purification protocol involves simply preparing 
a cellular extract which includes the enzyme. The inventors have 
discovered that the enzyme is soluble in buffers such as low-salt buffers, 
and it is proposed that virtually any buffer of this type can be employed 
for initial extraction of the protein from the tissue of choice or from 
recombinant cells in which the constituent subunits of the enzyme are 
expressed. The inventors prefer a 50 mM Tris-chloride, pH 7.5, buffer 
which includes a divalent chelator (e.g., 1 mM EDTA, 1 mM EGTA), as well 
as protease inhibitors such as phenylmethylsulphonyl fluoride (PMSF) 
and/or leupeptin. Of course, those of skill in the art will recognize that 
a variety of other types of buffers may be employed as extractants where 
desired, so long as the enzyme is extractable in such a buffer and its 
subsequent activity is not adversely affected to a significant degree. 
In embodiments concerning the purification of the native enzyme, the choice 
of tissue from which one will seek to obtain the farnesyltransferase 
enzyme is not believed to be of crucial importance. In fact, it is 
believed that farnesyltransferases are components of virtually all living 
cells. Therefore, the tissue of choice will typically be that which is 
most readily available to the practitioner. In that farnesyltransferase 
action appears to proceed similarly in most systems studied, including, 
cultured hamster cells, rat brain, and even yeast, it is believed that 
this enzyme will exhibit similar qualities, regardless of its source of 
isolation. 
In preferred embodiments, the inventors have isolated the native enzyme 
from rat brains in that this source is readily available. However, 
numerous other sources are contemplated to be directly applicable for 
isolation of the native enzyme, especially mammalian tissues such as 
liver, and human placenta, and also reticulocytes, or even yeast. Those of 
skill in the art, in light of the present disclosure, should appreciate 
that the techniques disclosed herein will be generally applicable to all 
such farnesyltransferases. 
It will also be appreciated that the enzyme may be purified from 
recombinant cells prepared in accordance with the present invention. The 
techniques disclosed for the isolation of native farnesyltransferase are 
believed to be equally applicable to the purification of the protein from 
recombinant host cells, whether bacterial or eukaryotic, in which DNA 
segments encoding the selected constituent subunit has been expressed or 
co-expressed. 
After the cell extract is prepared the enzyme is preferably subjected to 
two partial purification steps prior to affinity chromatography. These 
steps comprise preliminary treatment with 30% saturated ammonium sulfate 
which removes certain contaminants by precipitation. This is followed by 
treatment with 50% saturated ammonium sulfate, which precipitates the 
farnesyltransferase. The pelleted enzyme is then dissolved in a suitable 
buffer, such as 20 mM Tris-chloride (pH 7.5) containing 1 mM DTT and 20 
.mu.M ZnCl.sub.2, dialyzed against the same buffer, and then subjected to 
further purification steps. 
In preferred embodiments, the dialyzed solution containing the enzyme is 
applied to a column containing an ion exchange resin such as Mono Q. After 
washing of the column to remove contaminants, the enzyme is eluted with a 
gradient of 0.25-1.0M NaCl in the same buffer. The enzyme activity in each 
fraction is assayed as described below, and the fractions containing 
active enzyme are pooled and applied to the affinity column described 
below. 
It is, of course, recognized that the preliminary purification steps 
described above are preferred laboratory procedures that might readily be 
replaced with other procedures of equivalent effect such as ion exchange 
chromatography on other resins or gel filtration chromatography. Indeed, 
it is possible that these steps could even be omitted and the crude cell 
extract might be carried directly to affinity chromatography. 
After the preliminary purification steps, the extract may be subjected to 
affinity chromatography on an affinity chromatography medium which 
includes a farnesyltransferase binding peptide coupled to a suitable 
matrix. Typically, preferred farnesyltransferase binding peptides will 
comprise a peptide of at least 4 amino acids in length and will include a 
carboxy terminal sequence of -C-A.sub.1 -A.sub.2 -X, wherein: 
C=cysteine; 
A.sub.1 =any amino acid (aliphatic, aromatic, or hydroxy); 
A.sub.2 =an aliphatic amino acid, preferrably leucine, isoleucine or 
valine; and 
X=preferably methionine or serine, less preferably glutamine or cysteine, 
and even less preferably any other amino acid other than leucine. 
Preferred binding peptides of the present invention which fall within the 
above general formula include structures such as -C-V-I-M, -C-S-I-M and 
-C-A-I-M, all of which structures are found to naturally occur in proteins 
which are believed to be acted upon by protein farnesyltransferases in 
nature. Particularly preferred are relatively short peptides, such as on 
the order of about 4 to about 10 amino acids in length which incorporate 
one of the foregoing binding sequences. Of particular preference is the 
peptide T-K-C-V-I-M, which has been effectively employed by the inventors 
in the isolation of protein farnesyltransferase. 
The next step in the overall general purification scheme involves simply 
washing the medium to remove impurities. That is, after subjecting the 
extract to affinity chromatography on the affinity matrix, one will desire 
to wash the matrix in a manner that will remove the impurities while 
leaving the farnesyltransferase enzyme relatively intact on the medium. A 
variety of techniques are known in the art for washing matrices such as 
the one employed herein, and all such washing techniques are intended to 
be included within the scope of this invention. Of course, for washing 
purposes, one will not desire to employ buffers that will release or 
otherwise alter or denature the enzyme. Thus, one will typically want to 
employ buffers which contain non-denaturing detergents such as 
octylglucoside buffers, but will want to avoid buffers containing, e.g., 
chaotropic reagents which serve to denature proteins, as well as buffers 
of low pH (e.g., less than 7), or of high ionic strength (e.g., greater 
than 1.0M), as these buffers tend to elute the bound enzyme from the 
affinity matrix. 
After the matrix-bound enzyme has been sufficiently washed, for example in 
a medium-ionic strength buffer at essentially neutral pH, the specifically 
bound material can be eluted from the column by using a similar buffer but 
of reduced pH (for example, a pH of between about 4 and 5.5). At this pH, 
the enzyme will typically be found to elute from the preferred affinity 
matrices disclosed in more detail hereinbelow. 
While it is believed that advantages in accordance with the invention can 
be realized simply through affinity chromatography techniques, additional 
benefits will be achieved through the application of additional 
purification techniques, such as gel filtration techniques. For example, 
the inventors have discovered that Sephacryl S-200 high resolution gel 
columns can be employed with significant benefit in terms of protein 
purification. However, the present disclosure is by no means limited to 
the use of Sephacryl S-200, and it is believed that virtually any type of 
gel filtration arrangement can be employed with some degree of benefit. 
For example, one may wish to use techniques such as gel filtration, 
employing media such as Superose, Agarose, or even Sephadex. 
Through the application of various of the foregoing approaches, the 
inventors have successfully achieved farnesyltransferase enzyme 
compositions of relatively high specific activity, measured in terms of 
ability to transfer farnesol from all-trans farnesyl pyrophosphate. For 
the purposes of the present invention, one unit of activity is defined as 
the amount of enzyme that transfers 1 pmol of farnesol from all-trans 
farnesyl pyrophosphate (FPP) into acid-precipitable p21.sup.H-ras per hour 
under the conditions set forth in the Examples. Thus, in preferred 
embodiments the present invention is concerned with compositions of 
farnesyltransferase which include a specific activity of between about 5 
and about 10 units/mg of protein. In more preferred embodiments, the 
present invention is concerned with compositions which exhibit a 
farnesyltransferase specific activity of between about 500 and about 
600,000 units/mg of protein. Thus, in terms of the unit definition set 
forth above, the inventors have been able to achieve compositions having a 
specific activity of up to about 600,000 units/mg using techniques 
disclosed herein. 
Cloning of Farnesyltransferase Subunits 
Important aspects of the present invention concern isolated DNA segments 
and recombinant vectors encoding the .alpha. and .beta. subunits of 
mammalian protein farnesyltransferase, and the creation of recombinant 
host cells through the application of DNA technology, which express one, 
or preferably both, of these polypeptides. 
As used herein, the term "DNA segment" in intended to refer to a DNA 
molecule which has been isolated free of total genomic DNA of a particular 
species. Therefore, a DNA segment encoding a subunit of 
farnesyltransferase is intended to refer to a DNA segment which contains 
such coding sequences yet is isolated away from total genomic DNA of the 
species from which the DNA is obtained. Included within the term "DNA 
segment", are DNA segments which may be employed in the preparation of 
vectors, as well as the vectors themselves, including, for example, 
plasmids, cosmids, phage, viruses, and the like. 
In particular embodiments, the invention concerns isolated DNA segments and 
recombinant vectors incorporating DNA sequences which encode a 
farnesyltransferase subunit that includes within its amino acid sequence 
the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, corresponding to 
rat brain farnesyltransferase subunits .alpha. and .beta., respectively. 
Moreover, in other particular embodiments, the invention concerns isolated 
DNA segments and recombinant vectors incorporating DNA sequences which 
encode a farnesyltransferase subunit that includes within its amino acid 
sequence the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:7, 
corresponding to human farnesyltransferase subunits .alpha. and .beta., 
respectively. Recombinant vectors and isolated segments may therefore 
variously include the .alpha. or .beta. subunit coding regions themselves, 
coding regions bearing selected alterations or modifications in the basic 
coding region or may encode larger polypeptides which nevertheless include 
sequences which will confer farnesyltransferase activity when said 
polypeptide is combined with the alternate subunit. 
However, it will be understood that this aspect of the invention is not 
limited to the particular nucleic acid and amino acid sequences of SEQ ID 
NO:1 and SEQ ID NO:2 and SEQ ID NO:5 and SEQ ID NO:6 (.alpha. subnit) or 
SEQ ID NO:3 and SEQ ID NO:4 and SEQ ID NO:7 and SEQ ID NO:8 (.beta. 
subunit). Accordingly, DNA segments prepared in accordance with the 
present invention may also encode biologically functional equivalent 
proteins or peptides which have variant amino acids sequences. Such 
sequences may arise as a consequence of codon redundancy and functional 
equivalency which are known to occur naturally within nucleic acid 
sequences and the proteins thus encoded. Alternatively, functionally 
equivalent proteins or peptides may be created via the application of 
recombinant DNA technology, in which changes in the protein structure may 
be engineered, based on considerations of the properties of the amino 
acids being exchanged. 
The recombinant cloning of cDNAs encoding the farnesyltransferase .alpha. 
and .beta. subunits was achieved through the use of the peptide sequence 
information set forth above which was used in the preparation of 
subunit-specific oligonucleo-tides. Such oligonucleotides could be 
employed in the direct hybridization screening of a clone bank. However, 
the inventors preferred to use the peptide sequences in the preparation of 
primers for use in PCR amplification and partial sequencing of the 
selected subunit gene to confirm the underlying DNA sequence, and to 
prepare longer and more specific probes for use in clone bank screening. 
In screening for the farnesyltransferase subunit-specific sequences, the 
inventors chose to use a cDNA clone bank prepared from poly A.sup.+ RNA. 
However, it is believed that the type of clone bank used is not crucial 
and that, if desired, one could employ a genomic clone bank. Similarly, in 
that the farnesyltransferase enzyme appears to be fairly ubiquitous in 
nature, it is believed that virtually any eukaryotic cell source may be 
employed for the preparation of RNA from which the clone bank is to be 
generated. One may mention by way of example, yeast, mammalian, plant, 
eukaryotic parasites and even viral-infected types of cells as the source 
of starting poly A.sup.+ RNA. 
As the protein was initially purified from a mammalian source (rat), it is 
contemplated that particular advantages may be found in the use of 
mammalian cells, such as rat or human cell lines, as an RNA source. One 
may, of course, wish to first test such a cell line to ensure that 
relatively high levels of the farnesyltransferase enzyme are being 
produced by the selected cells. Rat brain, PC12 (a rat adrenal tumor cell 
line) and KNRK (a newborn rat kidney cell line) were preferred by the 
present inventors as they exhibited high levels of endogenous 
farnesyltransferase activity. 
The type of cDNA clone bank used in the screening procedure is not believed 
to be particularly critical. However, one will likely find particular 
benefit through the preparation and use of a phage-based bank, such as 
.lambda.gt10 or .lambda.gt11, preferably using a particle packaging 
system. Phage-based cDNA banks are preferred because of the large numbers 
of recombinants that may be prepared and screened will relative ease. The 
manner in which the cDNA itself is prepared is again not believed to be 
particularly crucial. However, the inventors successfully employed both 
oligo dT and randomly primed cDNA, from a consideration of the 
difficulties which may arise in the reverse transcription of a large mRNA 
molecule. 
Once a clone bank has been prepared, it may be screened in a number of 
fashions. For example, as mentioned above, one could employ the subunit 
peptide sequences set forth above for the preparation of nucleotide probes 
with which to directly screen the clone bank. A more preferrable approach 
was found to be to use such sequences in the preparation of primers which 
may were used in PCR-based reactions to amplify and then sequence portions 
of the selected subunit gene, to thereby confirm the actual underlying DNA 
sequence, and to prepare longer and more specific probes for further 
screening. These primers may also be employed for the preparation of cDNA 
clone banks which are enriched for 3' and/or 5' sequences. This may be 
important, e.g., where less than a full length clone is obtained through 
the initially prepared bank. 
If a less than full length clone was obtained on initial screening, the 
entire sequence could be subsequently obtained through the application of 
5' and/or 3' extension technology, as required. The techniques for 
obtaining an extended farnesyltransferase subunit clone will be known to 
those of skill in the art in light of the present disclosure. The 
procedures used are those described in Frohman et al. (34), involving a 
combination of reverse transcription, tailing with terminal 
deoxytransferase and, finally, PCR. 
It is proposed that the DNA segments of the present invention may be 
employed for a variety of applications. For example, a particularly useful 
application concerns the recombinant production of the individual subunits 
or proteins or peptides whose structure is derived from that of the 
subunits, or in the recombinant production of the holoenzyme following 
co-expression of the two subunits. Additionally, the 
farnesyltransferase-encoding DNA segments of the present invention can 
also be used in the preparation of nucleic acid probes or primers, which 
can, for example, be used in the identification and cloning of 
farnesyltransferase genes or related genomic sequences, or in the study of 
subunit(s) expression, and the like. 
Expression of Farnesyltransferase Subunits 
Turning firstly to the expression of the cloned subunits. Once a suitable 
(full length if desired) clone or clones have been obtained, whether they 
be cDNA based or genomic, one may proceed to prepare an expression system 
for the recombinant preparation of one, or preferably both, of the 
subunits. The engineering of DNA segment(s) for expression in a 
prokaryotic or eukaryotic system may be performed by techniques generally 
known to those of skill in recombinant expression. It is believed that 
virtually any expression system may be employed in the expression of 
either or both subunits. Both subunits of the enzyme have been 
successfully co-expressed in eukaryotic expression systems with the 
production of active enzyme, but it is envisioned that bacterial 
expression systems may ultimately be preferred for the preparation of 
farnesyltransferase for all purposes. The cDNAs for both subunits have 
been separately expressed in bacterial systems, with the encoded proteins 
being expressed as fusions with Schistosoma japonicum glutathione 
s-transferase. It is believed that bacterial expression will ultimately 
have numerous advantages over eukaryotic expression in terms of ease of 
use and quantity of materials obtained thereby. Furthermore, it is 
proposed that co-transformation of host cells with DNA segments encoding 
both the .alpha. and .beta. subunits will provide a convenient means for 
obtaining active enzyme. However, separate expression followed by 
reconstitution is also certainly within the scope of the invention. Both 
cDNA and genomic sequences are suitable for eukaryotic expression, as the 
host cell will, of course, process the genomic transcripts to yield 
functional mRNA for translation into protein. 
It is similarly believed that almost any eukaryotic expression system may 
be utilized for the expression of either, or preferably, both of the 
farnesyltransferase subunits, e.g., baculovirus-based, glutamine 
synthase-based or dihydrofolate reductase-based systems could be employed. 
However, in preferred embodiments, it is contemplated that plasmid vectors 
incorporating an origin of replication and an efficient eukaryotic 
promoter, as exemplified by the eukaryotic vectors of the pCMV series, 
such as pCMV5, will be of most use. For expression in this manner, one 
would position the coding sequences adjacent to and under the control of 
the promoter. It is understood in the art that to bring a coding sequence 
under the control of such a promoter, one positions the 5' end of the 
transcription initiation site of the transcriptional reading frame of the 
protein between about 1 and about 50 nucleotides "downstream" of (i.e., 3' 
of) the chosen promoter. 
Where eukaryotic expression is contemplated, one will also typically desire 
to incorporate into the transcriptional unit which includes the enzyme, an 
appropriate polyadenylation site (e.g., 5'-AATAAA-3') if one was not 
contained within the original cloned segment. Typically, the poly A 
addition site is placed about 30 to 2000 nucleotides "downstream" of the 
termination site of the protein at a position prior to transcription 
termination. 
As noted above, it is proposed that in embodiments concerning the 
production of farnesyltransferase enzyme, the .alpha. and .beta. subunits 
may be co-expressed in the same cell. This may be achieved by 
co-transfecting the cell with two distinct recombinant vectors, each 
bearing a copy of either the .alpha. or .beta.-encoding DNA. 
Alternatively, a single recombinant vector may be constructed to include 
the coding regions for both of the subunits, which could then be expressed 
in cells transfected with the single vector. In either event, the term 
"co-expression" herein refers to the expression of both the .alpha. and 
.beta. subunits of farnesyltransferase in the same recombinant cell. 
It is contemplated that virtually any of the commonly employed host cells 
can be used in connection with the expression of one, or preferably both, 
of the farnesyltransferase subunits in accordance herewith. Examples 
include cell lines typically employed for eukaryotic expression such as 
239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7, RIN and MDCK cell 
lines. A preferred line for use in eukaryotic expression embodiments of 
the present invention has been found to be the human embryonic kidney cell 
line, 293. 
In accordance with the general guidelines described above, a preferred 
method for expressing farnesyltransferase DNA has been found to be the 
transfection of human embryonic kidney 293 cells with expression vectors 
termed pFT-.alpha. or pFT-.beta.. The pFT expression vectors are 
constructed from pCMV5, a plasmid that contains the promoter-enhancer 
region of the major immediate early gene of human cytomegalovirus (38). 
Nucleic Acid Hybridization 
The DNA sequences disclosed herein will also find utility as probes or 
primers in nucleic acid hybridization embodiments. As such, it is 
contemplated that oligonucleotide fragments corresponding to the sequences 
of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8 for stretches of 
between about 10 nucleotides to about 30 nucleotides will find particular 
utility, with even longer sequences, e.g., 40, 50, 60, even up to full 
length, being even more particularly preferred. The ability of such 
nucleic acid probes to specifically hybridize to farnesyltransferase 
subunit-encoding sequences will enable them to be of use in a variety of 
embodiments. Most importantly, the probes can be used in a variety of 
assays for detecting the presence of complementary sequences in a given 
sample. However, other uses are envisioned, including the use of the 
sequence information for the preparation of mutant species primers, or 
primers for use in preparing other genetic constructions. 
The use of a hybridization probe of about 10 nucleotides in length allows 
the formation of a duplex molecule that is both stable and selective. 
Molecules having complementary sequences over stretches greater than 10 
bases in length are generally preferred, though, in order to increase 
stability and selectivity of the hybrid, and thereby improve the quality 
and degree of specific hybrid molecules obtained. One will generally 
prefer to design nucleic acid molecules having gene-complementary 
stretches of 15 to 20 nucleotides, or even longer where desired. Such 
fragments may be readily prepared by, for example, directly synthesizing 
the fragment by chemical means, by application of nucleic acid 
reproduction technology, such as the PCR technology of U.S. Pat. No. 
4,603,102 (herein incorporated by reference) or by introducing selected 
sequences into recombinant vectors for recombinant production. 
Accordingly, the nucleotide sequences of the invention may be used for 
their ability to selectively form duplex molecules with complementary 
stretches of farnesyltransferase genes or cDNAs. Depending on the 
application envisioned, one will desire to employ varying conditions of 
hybridization to achieve varying degrees of selectivity of probe towards 
target sequence. For applications requiring high selectivity, one will 
typically desire to employ relatively stringent conditions to form the 
hybrids, e.g., one will select relatively low salt and/or high temperature 
conditions, such as provided by 0.02M-0.15M NaCl at temperatures of 
50.degree. C. to 70.degree. C. Such selective conditions tolerate little, 
if any, mismatch between the probe and the template or target strand, and 
would be particularly suitable for isolating farnesyltransferase genes. 
Of course, for some applications, for example, where one desires to prepare 
mutants employing a mutant primer strand hybridized to an underlying 
template or where one seeks to isolate farnesyltransferase-encoding 
sequences for related species, functional equivalents, or the like, less 
stringent hybridization conditions will typically be needed in order to 
allow formation of the heteroduplex. In these circumstances, one may 
desire to employ conditions such as 0.15M-0.9M salt, at temperatures 
ranging from 20.degree. C. to 55.degree. C. Cross-hybridizing species can 
thereby be readily identified as positively hybridizing signals with 
respect to control hybridizations. In any case, it is generally 
appreciated that conditions can be rendered more stringent by the addition 
of increasing amounts of formamide, which serves to destabilize the hybrid 
duplex in the same manner as increased temperature. Thus, hybridization 
conditions can be readily manipulated, and thus will generally be a method 
of choice depending on the desired results. 
In certain embodiments, it will be advantageous to employ nucleic acid 
sequences of the present invention in combination with an appropriate 
means, such as a label, for determining hybridization. A wide variety of 
appropriate indicator means are known in the art, including radioactive, 
enzymatic or other ligands, such as avidin/biotin, which are capable of 
giving a detectable signal. In preferred embodiments, one will likely 
desire to employ an enzyme tag such a urease, alkaline phosphatase or 
peroxidase, instead of radioactive or other environmental undesirable 
reagents. In the case of enzyme tags, colorimetric indicator substrates 
are known which can be employed to provide a means visible to the human 
eye or spectrophotometrically, to identify specific hybridization with 
complementary nucleic acid-containing samples. 
In general, it is envisioned that the hybridization probes described herein 
will be useful both as reagents in solution hybridization as well as in 
embodiments employing a solid phase. In embodiments involving a solid 
phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a 
selected matrix or surface. This fixed, single-stranded nucleic acid is 
then subjected to specific hybridization with selected probes under 
desired conditions. The selected conditions will depend on the particular 
circumstances based on the particular criteria required (depending, for 
example, on the G+C contents, type of target nucleic acid, source of 
nucleic acid, size of hybridization probe, etc.). Following washing of the 
hybridized surface so as to remove nonspecifically bound probe molecules, 
specific hybridization is detected, or even quantified, by means of the 
label. 
Biological Functional Equivalent Amino Acids 
As mentioned above, modification and changes may be made in the structure 
of the farnesyltransferase subunits and still obtain a molecule having 
like or otherwise desirable characteristics. For example, certain amino 
acids may be substituted for other amino acids in a protein structure 
without appreciable loss of interactive binding capacity with structures 
such as, for example, antigen-binding regions of antibodies or binding 
sites on substrate molecules. Since it is the interactive capacity and 
nature of a protein that defines that protein's biological functional 
activity, certain amino acid sequence substitutions can be made in a 
protein sequence (or, of course, its underlying DNA coding sequence) and 
nevertheless obtain a protein with like or even counterveiling properties 
(e.g., antagonistic v. agonistic). It is thus contemplated by the 
inventors that various changes may be made in the sequence of the peptides 
(or underlying DNA) without appreciable loss of their biological utility 
or activity. 
In making such changes, the hydropathic index of amino acids may be 
considered. The importance of the hydropathic amino acid index, i.e. 
hydrophobicity and charge characteristics, in conferring interactive 
biologic function on a protein is generally understood in the art (43). 
For example, it is known that certain amino acids may be substituted for 
other amino acids having a similar hydropathic index or score and still 
retain a similar biological activity. It is believed that the relative 
hydropathic character of the amino acid determines the secondary structure 
of the resultant protein, which in turn defines the interaction of the 
protein with substrate molecules. Thus, for example, it is proposed the 
isoleucine, which has a hydrophatic index of +4.5, can be substituted for 
valine (+4.2) or leucine (+3.8), and still obtain a protein having similar 
biologic activity. Alternatively, at the other end of the scale, it is 
proposed that lysine (-3.9) can be substituted for arginine (-4.5), and so 
on. 
Amino acid substitutions are generally therefore based on the relative 
similarity of the side-chain substituents, for example, size, 
electrophilic character, charge, and the like. Exemplary substitutions 
which take various of the foregoing characteristics into consideration are 
well known to those of skill in the art and include: alanine, glycine and 
serine; arginine and lysine; glutamate and aspartate; serine and 
threonine; and valine, leucine and isoleucine. 
Inhibitors of Farnesyltransferase 
Of principal importance to the present invention is the discovery that 
proteins or peptides which incorporate a farnesyl acceptor sequence, such 
as one of the farnesyl acceptor sequences discussed above, function as 
inhibitors of farnesyltransferase, and therefore may serve as a basis for 
anticancer therapy. In particular, it has been found that farnesyl 
acceptor peptides can successfully function both as false substrates that 
serve to inhibit the farnesylation of natural substrates such as 
p21.sup.ras, and as direct inhibitors which are not themselves 
farnesylated. Compounds falling into the latter category are particularly 
important in that these compounds are "pure" inhibitors that are not 
consumed by the inhibition reaction and can continue to function as 
inhibitors. Both types of compounds constitute an extremely important 
aspect of the invention in that they provide a means for blocking 
farnesylation of p21.sup.ras proteins, for example, in an affected cell 
system. 
The farnesyltransferase inhibitor embodiments of the present invention 
concern in,a broad sense a peptide or protein other than p21.sup.ras 
proteins, lamin a or lamin b, or yeast mating factor a, which peptide or 
protein includes a farnesyl acceptor sequence within its structure and is 
further capable of inhibiting the farnesylation of p21.sup.ras by 
farnesyltransferase. 
In preferred embodiments, the farnesyltransferase inhibitor of the present 
invention will include a farnesyl acceptor or inhibitory amino acid 
sequence having the amino acids -C-A.sub.1 -A.sub.2 -X, wherein: 
C=cysteine; 
A.sub.1 =any amino acid (aliphatic, aromatic, or hydroxy); 
A.sub.2 =an aliphatic amino acid, preferrably leucine, isoleucine or 
valine; and 
X=preferably methionine or serine, less preferably glutamine or cysteine, 
and even less preferably any other amino acid other than leucine. 
Whereas it was previously proposed that the X amino acid could be any amino 
acid, it has more recently been discovered that one will prefer that x 
will be methionine or serine, less preferably glutamine or cysteine, and 
even less preferably any other amino acid other than leucine. Leucine 
should not be employed as the carboxy terminal amino acid in that such 
peptides are known to be substrates for a separate enzyme, 
geronylgeronyltransferase (35). 
Typically, the farnesyl acceptor or inhibitory amino acid sequence will be 
positioned at the carboxy terminus of the protein or peptide such that the 
cysteine residue is in the fourth position from the carboxy terminus, or 
will simple correspond to a tetrapeptide structure. 
In preferred embodiments, the inhibitor will be a relatively short peptide 
such as a peptide from about 4 to about 10 amino acids in length. To date, 
the most preferred inhibitor tested is a tetrapeptide which incorporates 
the above-mentioned C-A-A-X recognition structure. It is possible that 
even shorter peptides will ultimately be preferred for practice of the 
invention in that the shorter the peptide, the greater the uptake by such 
peptide by biological systems, and the reduced likelihood that such a 
peptide will be destroyed or otherwise rendered biologically ineffective 
prior to effecting inhibition. However, numerous suitable inhibitory 
peptides have been prepared and tested by the present inventors, and shown 
to inhibit enzymatic activities virtually completely, at reasonable 
concentrations, e.g., between about 1 and 3 .mu.M (with 50% inhibitions on 
the order of 0.1 to 0.5 .mu.M). 
While, broadly speaking, it is believed that compounds exhibiting an 
IC.sub.50 of between about 0.01 .mu.M and 10 .mu.M will have some utility 
as farnesyltransferase inhibitors, the more preferred compounds will 
exhibit an IC.sub.50 of between 0.01 .mu.M and 1 .mu.M. The most preferred 
compounds will generally have an IC.sub.50 of between about 0.01 .mu.M and 
0.3 .mu.M. 
Exemplary peptides which have been prepared, tested and shown to inhibit 
farnesyltransferase at an IC.sub.50 of between 0.01 and 10 .mu.M include 
CVIM; KKSKTKCVIM; TKCVIM; RASNRSCAIM; TQSPQNCSIM; CIIM; CVVM; CVLS; CVLM; 
CAIM; CSIM; CCVQ; CIIC; CIIS; CVIS; CVLS; CVIA; CTVA; CVAM; CKIM; CLIM; 
CVLM; CFIM; CVFM; CIFM; CVIF; CEIM; CGIM; CPIM; CVYM; CVWM; CVTM; CVPM; 
CVSM; CVIF; CVIV; CVIP. 
A variety of peptides have been synthesized and tested such that now the 
inventors can point out peptide sequences having particularly high 
inhibitory activity, i.e., wherein relatively lower concentrations of the 
peptides will exhibit an equivalent inhibitory activity (IC.sub.50). 
Interestingly, it has been found that slight changes in the sequence of 
the acceptor site can result in loss of inhibitory activity. Thus, when 
TKCVIM is changed to TKVCIM, the inhibitory activity of the peptide is 
reversed. Similarly, when a glycine is substituted for one of the 
aliphatic amino acids in CAAX, a decrease in inhibitory activity is 
observed. However, it is proposed that as long as the general formula as 
discussed above is observed, one will achieve a structure that is 
inhibitory to farnesyltransferase. 
A particularly important discovery is the finding that the incorporation of 
an aromatic residue such as phenylalanine, tyrosine or tryptophan into the 
third position of the CAAX sequence will result in a "pure" inhibitor. As 
used herein, a "pure" farnesyltransferase is intended to refer to one 
which does not in itself act as a substrate for farnesylation by the 
enzyme. This is particularly important in that the inhibitor is not 
consumed by the inhibition process, leaving the inhibitor to continue its 
inhibitory function unabated. Exemplary compounds which have been tested 
and found to act as pure inhibitors include CVFM, CVWM, CVYM, CIFM, 
CV(pCl-F)M, L-penicillamine-VFM, and L-penicillamine-VIM. Pure inhibitors 
will therefore incorporate an inhibitory amino acid sequence rather than 
an acceptor sequence, with the inhibitory sequence characterized generally 
as having an aromatic moiety associated with the penultimate carboxy 
terminal amino acid, whether it be an aromatic amino acid or another amino 
acid which has been modified to incorporate an aromatic structure. (see 
Goldstein et al., ref. 53) 
Importantly, the pure inhibitor CVFM is the best inhibitor identified to 
date by the inventors. It should be noted that the related peptide, CFIM 
is not a "pure" inhibitor; its inhibitory activity is due to its action as 
a substrate for farnesylation. 
The potency of CVFM peptides as inhibitors of the enzyme may be enhanced by 
attaching substituents such as fluoro, chloro or nitro derivatives to the 
phenyl ring. An example is parachlorophenylalanine, which has been tested 
and found to have "pure" inhibitory activity. It may also be possible to 
substitute more complex hydrophobic substances for the phenyl group of 
phenylalanine. These would include naphthyl ring systems. 
The present inventors propose that additional improvements can be made in 
pharmaceutical embodiments of the inhibitor by including within their 
structure moieties which will improve their hydrophobicity, which it is 
proposed will improve the uptake of peptidyl structures by cells. Thus, in 
certain embodiments, it is proposed to add fatty acid or polyisoprenoid 
side chains to the inhibitor which, it is believed, will improve their 
lipophilic nature and enhance their cellular uptake. 
Other possible structural modifications include the addition of benzyl, 
phenyl or acyl groups to the amino acid structures, preferably at a 
position sufficiently removed from the farnesyl acceptor site, such as at 
the amino terminus of the peptides. It is proposed that such structures 
will serve to improve lypophilicity. In this regard, the inventors have 
found that N-acetylated and N-octylated peptides such as modified CVIM 
retain much of their inhibitory activity, whereas S-acetoamidated CVIM 
appears to lose much of its inhibitory activity. 
Important additional structural characteristics for the preparation of 
"pure" peptide-based inhibitors have been discovered, which should be 
taken into account in designing such inhibitors. In particular, it has 
been found that a positively charged alpha nitrogen at the N-terminus is 
required for the realization of a "pure" inhibitor. Thus, when such a 
positive charge is absent, such as through acylation (e.g., acetyl or 
octanoyl group addition) or through N-terminal amino acid addition, and 
this added structure is not removed intracellularly or in the test system 
employed to reveal a positively charged alpha nitrogen on the N-terminal 
cysteine, the inhibitor is farnesylated and therefore will not serve as a 
pure inhibitor. 
Accordingly, where a pure inhibitor is desired, any modification that is 
made, e.g., to improve cellular uptake, should take into account the 
ultimate need for a positively charged alpha nitrogen on the N-terminal 
cysteine at the site of action. A variety of embodiments are envisioned 
that will serve to preserve or otherwise reveal a positively charged alpha 
nitrogen on the N-terminal cysteine upon entry into a cellular target. In 
general, therefore, it is contemplated that advantages will be realized 
through the addition of groups to the N-terminal cysteine that will either 
retain a positive charge on the alpha nitrogen (e.g., alkyl, substituted 
alkyl, phenyl, benzyl, etc.) or that will reveal such a positively charged 
nitrogen when removed by normal cellular processes, e.g., cellular enzymes 
such as oxo-prolinase, esterases, trypsin, chymotrypsin, aminopeptidases, 
transpeptidases, etc., or by intracellular conditions, such as through 
hydrolysis or deacylation. Of course, charged species cross cellular 
membranes only with some difficulty, if at all. Thus, one may desire to 
employ a carrier composition, such as liposome or carrier molecule, or the 
addition of a group that will temporarily negate the positive charge. 
More particularly, in the case of non-removable N-terminal modifications, 
preferred modifications will be those that retain a positively charged 
nitrogen, yet which increase hydrophobicity of the peptide. Examples 
include structures such as R.sub.1 R.sub.2 R.sub.3 N-peptide, wherein 
R.sub.1 R.sub.2 R.sub.3 =H, alkyl, phenyl, benzyl, substituted phenyl, 
substituted benzyl, etc., or even cyclic aza structures such as: 
##STR1## 
The generation of removable structures may provide particular advantages. 
Such structures might advantageously include modifications of the 
N-terminal cysteine which encompass both the cysteine alpha nitrogen 
and/or the thiol side chain. Examples would include 2-substituted 
thiazolidine-4-carboxylic acids, which would undergo intracellular 
hydrolysis to unmask the cysteine (64). One such example would be: 
##STR2## 
R=H, alkyl, halo-substituted alkyl, phenyl, substituted phenyl, pyridyl or 
the like. 
Other structures within this category would include 
2-oxo-thiazolidine-4-carboxylic acids, that would be cleaved 
intracellularly at the C-S bond by oxo-prolinases to unmask the cysteine 
(65). Structures within this category would include: 
##STR3## 
In other embodiments, the invention contemplates removable modifications at 
the N-terminus based on structures such as: 
##STR4## 
Examples would include acyl modifications of this structure that would be 
removable by enzymatic cleavage, such as those that include N-acetyl 
structures wherein R=CH.sub.3 CO, that are deacylated in vivo or in cell 
culture (65). Alternatively, phenyl carbamates such as wherein 
##STR5## 
would be cleaved by intracellular esterases, liberating the inhibitor and 
CO.sub.2. Other examples would include the inclusion of an N-terminal 
pyroglutamyl group such as 
##STR6## 
that would be cleaved by pyroglutamyl aminopeptidase to release the 
inhibitor. 
Trypsin or chymotrypsin sensitive structures are also contemplated. Trypsin 
sensitive structures would include the addition of an L-arginine or 
L-lysine, or even the addition of a protein or peptide that includes an 
L-lysine or L-arginine carboxy terminus, onto the N-terminus of the 
tetrapeptide cysteine. An example of such a structure would be as follows, 
with the site of trypsin sensitivity shown: 
##STR7## 
Similar structures are envisioned for conferring chymotrypsin sensitivity, 
except these would include the amino terminal introduction of 
L-phenylalanine, L-tyrosine or L-tryptophan moiety, or a peptide or 
protein including these amino acids. 
Similarly, gamma-glutamyl derivatives, e.g., where 
##STR8## 
would be removable by .gamma.-glutamyl transpeptidases. 
Other modifications that would undergo intracellular hydrolysis to release 
a free N-terminus are also contemplated. Examples would include N-mannich 
base structures such as 
##STR9## 
R=phenyl, substituted phenyl, alkyl, substituted alkyl, and the like or 
Schiff bases such as 
##STR10## 
The invention also contemplates that modifications can be made in the 
structure of inhibitory proteins or peptides to increase their stability 
within the body, such as modifications that will reduce or eliminate their 
susceptibility to degradation, e.g., by proteases. For example, the 
inventors contemplate that useful structural modifications will include 
the use of amino acids which are less likely to be recognized and cleaved 
by proteases, such as the incorporation of D-amino acids, or amino acids 
not normally found in proteins such as ornithine or taurine. Other 
possible modifications include the cyclization of the peptide, 
derivatization of the NH groups of the peptide bonds with acyl groups, 
etc. 
Assays for Farnesyltransferase 
In still further embodiments, the invention concerns a method for assaying 
protein farnesyltransferase activity in a composition. This is an 
important aspect of the invention in that such an assay system provides 
one with not only the ability to follow the isolation and purification of 
native or recombinant farnesyltransferase enzymes, but it also forms the 
basis for developing a screening assay for candidate inhibitors of the 
enzyme, discussed in more detail below. The assay method generally 
includes determining the ability of a composition suspected of having 
farnesyltransferase activity to catalyze the transfer of farnesol to an 
acceptor protein or peptide. As noted above, a farnesyl acceptor protein 
or peptide is generally defined as a protein or peptide which will act as 
a substrate for farnesyltransferase and which includes a recognition site 
such as -C-A-A-X, as defined above. 
Typically, the assay protocol is carried out using all-trans farnesyl 
pyrophosphate as the farnesol donor in the reaction. Thus, one will find 
particular benefit in constructing an assay wherein a label is present on 
the farnesyl moiety of all-trans farnesyl pyrophosphate, in that one can 
measure the appearance of such a label, for example, a radioactive label, 
in the farnesyl acceptor protein or peptide. 
As with the characterization of the enzyme discussed above, the farnesyl 
acceptor sequence which are employed in connection with the assay can be 
generally defined by -C-A-A-X, with preferred embodiments including 
sequences such as -C-V-I-M, -C-S-I-M, -C-A-I-M, etc., all of which have 
been found to serve as useful enzyme substrates. It is believed that most 
proteins or peptides that include a carboxy terminal sequence of -C-A-A-X 
can be successfully employed in protein farnesyltransferase assays. For 
use in the assay a preferred farnesyl acceptor protein or peptide will be 
a p21.sup.ras protein. This is particularly true where one seeks to 
identify inhibitor substances, as discussed in more detail below, which 
function either as "false acceptors" in that they divert farnesylation 
away from natural substrates by acting as substrates in and or themselves, 
or as "pure" inhibitors which are not in themselves farnesylated. The 
advantage of employing a natural substrate such as p21.sup.ras is several 
fold, but includes the ability to separate the natural substrate from the 
false substrate to analyze the relative degrees of farnesylation. 
However, for the purposes of simply assaying enzyme specific activity, 
e.g., assays which do not necessarily involve differential labeling or 
inhibition studies, one can readily employ short peptides as a farnesyl 
acceptor in such protocols, such as peptides from about 4 to about 10 
amino acids in length which incorporate the recognition signal at their 
carboxy terminus. Exemplary farnesyl acceptor protein or peptides include 
but are not limited to CVIM; KKSKTKCVIM; TKCVIM; RASNRSCAIM; TQSPQNCSIM; 
CIIM; CVVM; and CVLS. 
Assays for Candidate Substances 
In still further embodiments, the present invention concerns a method for 
identifying new farnesyltransferase inhibitory compounds, which may be 
termed as "candidate substances." It is contemplated that this screening 
technique will prove useful in the general identification of any compound 
that will serve the purpose of inhibiting farnesyltransferase. It is 
further contemplated that useful compounds in this regard will in no way 
be limited to proteinaceous or peptidyl compounds. In fact, it may prove 
to be the case that the most useful pharmacologic compounds for 
identification through application of the screening assay will be 
non-peptidyl in nature and, e.g., which will be recognized and bound by 
the enzyme, and serve to inactivate the enzyme through a tight binding or 
other chemical interaction. 
Thus, in these embodiments, the present invention is directed to a method 
for determining the ability of a candidate substance to inhibit a 
farnesyltransferase enzyme, the method including generally the steps of: 
(a) obtaining an enzyme composition comprising a farnesyltransferase enzyme 
that is capable of transferring a farnesyl moiety to a farnesyl acceptor 
substance; 
(b) admixing a candidate substance with the enzyme composition; and 
(c) determining the ability of the farnesyltransferase enzyme to transfer a 
farnesyl moiety to a farnesyl acceptor substrate in the presence of the 
candidate substance. 
An important aspect of the candidate substance screening assay hereof is 
the ability to prepare a native or recombinant farnesyltransferase enzyme 
composition in a relative purified form, for example, in a manner as 
discussed above. This is an important aspect of the candidate substance 
screening assay in that without at least a relatively purified 
preparation, one will not be able to assay specifically for enzyme 
inhibition, as opposed to the effects of the inhibition upon other 
substances in the extract which then might affect the enzyme. In any 
event, the successful isolation of the farnesyltransferase enzyme now 
allows for the first time the ability to identify new compounds which can 
be used for inhibiting this cancer-related enzyme. 
The candidate screening assay is quite simple to set up and perform, and is 
related in many ways to the assay discussed above for determining enzyme 
activity. Thus, after obtaining a relatively purified preparation of the 
enzyme, either from native or recombinant sources, one will desire to 
simply admix a candidate substance with the enzyme preparation, preferably 
under conditions which would allow the enzyme to perform its 
farnesyltransferase function but for inclusion of a inhibitory substance. 
Thus, for example, one will typically desire to include within the 
admixture an amount of a known farnesyl acceptor substrate such as a 
p21.sup.ras protein. In this fashion, one can measure the ability of the 
candidate substance to reduce farnesylation of the farnesyl acceptor 
substrate relatively in the presence of the candidate substance. 
Accordingly, one will desire to measure or otherwise determine the activity 
of the relatively purified enzyme in the absence of the added candidate 
substance relative to the activity in the presence of the candidate 
substance in order to assess the relative inhibitory capability of the 
candidate substance. 
Methods of Inhibiting Farnesyltransferase 
In still further embodiments, the present invention is concerned with a 
method of inhibiting a farnesyltransferase enzyme which includes 
subjecting the enzyme to an effective concentration of a 
farnesyltransferase inhibitor such as one of the family of peptidyl 
compounds discussed above, or with a candidate substance identified in 
accordance with the candidate screening assay embodiments. This is, of 
course, an important aspect of the invention in that it is believed that 
by inhibiting the farnesyltransferase enzyme, one will be enabled to treat 
various aspects of cancers, such as ras-related cancers. It is believed 
that the use of such inhibitors to block the attachment of farnesyl groups 
to ras proteins in malignant cells of patients suffering with cancer or 
pre-cancerous states will serve to treat or palliate the cancer, and may 
be useful by themselves or in conjunction with other cancer therapies, 
including chemotherapy, resection, radiation therapy, and the like.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The following examples illustrate techniques discovered by the inventors 
for the identification and purification of mammalian protein 
farnesyltransferase enzymes, as well as techniques for their assay and for 
the screening of new compounds which may be employed to inhibit such 
enzymes. These studies also demonstrate a variety of peptidyl compounds 
which themselves can be employed to inhibit these enzymes. It should be 
appreciated by those of skill in the art that the techniques disclosed in 
the examples which follow represent laboratory techniques discovered by 
the inventors to function well in the practice of the invention, and thus 
can be considered to constitute preferred modes for its practice. However, 
those of skill in the art should, in light of the present disclosure, 
appreciate that many changes can be made in the specific embodiments which 
are disclosed and still obtain a like or similar result without departing 
from the spirit and scope of the invention. 
Example I 
Preparation and Characterization of Farnesyltransferase 
1. Materials 
Peptides were obtained from Peninsula Laboratories or otherwise synthesized 
by standard techniques. All peptides were purified on HPLC, and their 
identity was confirmed by amino acid analysis. Just prior to use, each 
peptide was dissolved at a concentration of 0.8 mM in 10 mM dithiothreitol 
(DTT), and all dilutions were made in 10 mM DTT. Unlabeled all-trans 
farnesyl pyrophosphate (FPP) was synthesized by the method of Davisson, et 
al. (13). [1-.sup.3 H]Farnesyl pyrophosphate (20 Ci/mmol) was custom 
synthesized by New England Nuclear. Geraniol and farnesol (both all-trans) 
were obtained from Aldrich Chemical. All-trans geranylgeraniol was a gift 
of R. Coates (University of Illinois). 
Recombinant wild type human p21.sup.H-ras protein was produced in a 
bacterial expression system with pAT-rasH (provided by Channing J. Der, La 
Jolla Cancer Research Foundation, La Jolla, Calif.), an expression vector 
based on pXVR (14). The plasmid was transformed into E. coli JM105, and 
the recombinant p21.sup.H-ras protein was purified at 4.degree. C. from a 
high speed supernatant of the bacterial extracts by sequential 
chromatography on DEAE-Sephacel and Sephadex G-75. Purity was .about.90% 
as judged by Coomassie blue staining of SDS gels. Purified p21.sup.H-ras 
was concentrated to 15 mg/ml in 10 mM Tris-chloride (pH 7.5) containing 1 
mM DTT, 1 mM EDTA, 3 mM MgCl.sub.2, and 30 .mu.M GDP and stored in 
multiple aliquots at -70.degree. C. 
2. Assay for FarnesylTransferase Activity 
Farnesyltransferase activity was determined by measuring the amount of 
.sup.3 H-farnesol transferred from all-trans .sup.3 H]farnesyl 
pyrophosphate ([.sup.3 H]FPP) to p21.sup.H-ras protein. The standard 
reaction mixture contained the following concentrations of components in a 
final volume of 25 .mu.l: 50 mM Tris-chloride (pH 7.5), 50 .mu.M 
ZnCl.sub.2, 20 mM KCl, 1 mM DTT, and 40 .mu.M p21.sup.H-ras. The mixture 
also contained 10 pmoles of [.sup.3 H]FPP (.about.30,000 dpm/pmol) and 
1.8-3.5 .mu.g of partially purified farnesyltransferase (see below). After 
incubation for 1 hour at 37.degree. C. in 12.times.75-mm borosilicate 
tubes, the reaction was stopped by addition of 0.5 ml of 4% SDS and then 
0.5 ml of 30% trichloroacetic acid (TCA). 
The tubes were vortexed and left on ice for 45-60 min, after which 2 ml of 
a 6% TCA/2% SDS solution were added. The mixture was filtered on a 2.5-cm 
glass fiber filter with a Hoefer filtration unit (FH 225). The tubes were 
rinsed twice with 2 ml of the same solution, and each filter was washed 
five times with 2 ml of 6% TCA, dried, and counted in a scintillation 
counter. One unit of activity is defined as the amount of enzyme that 
transfers 1 pmol of [.sup.3 H]farnesol from [.sup.3 H]FPP into 
acid-precipitable p21.sup.H-ras per hour under the standard conditions. 
3. Purification of Farnesyltransferarase 
All steps were carried out at 4.degree. C. except where indicated: 
Step 1--Ammonium Sulfate Fractionation 
Brains from 50 male Sprague-Dawley rats (100-150 g) were homogenized in 100 
ml of ice-cold buffer containing 50 mM Tris-chloride (pH 7.5), 1 mM EDTA, 
1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mM 
leupeptin, and the extract was spun at 60,000.times.g for 70 min. The 
supernatant was brought to 30% saturation with solid ammonium sulfate, 
stirred for 30 min on ice, and centrifuged at 12,000.times.g for 10 min to 
remove precipitated proteins. The resulting supernatant was adjusted to 
50% saturation with ammonium sulfate, and the resulting pellet was 
dissolved in .about.20 ml of 20 mM Tris-chloride (pH 7.5) containing 1 mM 
DTT and 20 .mu.M ZnCl.sub.2 and dialyzed for 4 hours against 4 liters of 
the same buffer and then 4 liters of fresh buffer of the same composition 
for 12 hours. The dialyzed material was divided into multiple aliquots and 
stored at -70.degree. C. 
Step 2--Ion-exchange Chromatography 
A portion of the 30-50% ammonium sulfate fraction (200 mg protein) was 
chromatographed on a Mono Q 10/10 column using an FPLC system (Pharmacia 
LKB Biotechnology). The column was run as described in the legend to FIG. 
5. Fractions eluting between 0.3 and 0.4M NaCl contained the majority of 
the transferase activity. These fractions were pooled, divided into 
multiple aliquots, and stored at -70.degree. C. 
Step 3--Affinity Chromatography 
An affinity column containing a peptide corresponding to the COOH-terminal 
six amino acids of p21.sup.K-ras-B protein was prepared as follows. 
Fifteen mg of the peptide TKCVIM were coupled to 1 g of activated 
CH-Sepharose 4B (Pharmacia LKB Biotechnology) according to the 
manufacturer's instructions. The resulting 2.5-ml slurry was poured into a 
column, and excess uncoupled peptide was removed by 10 cycles of 
alternating washes, each consisting of 40 column volumes of 0.1M sodium 
acetate (pH 4.0) and then 0.1M Tris-chloride (pH 8.0). Both buffers 
contained 1M NaCl and 10 mM DTT. The column was stored at 4.degree. C. in 
20 mM Tris-chloride (pH 7.2) and 0.02% sodium azide. Fifteen mg of Mono 
Q-purified material in 10 ml were applied to a 1-ml peptide column 
equilibrated in 50 mM Tris-chloride (pH 7.5) containing 0.1M NaCl and 1 mM 
DTT (Buffer A). The enzyme-containing solution was cycled through the 
column three times at room temperature. The column was washed with 20 ml 
of Buffer A containing 0.2% (w/v) octyl-.beta.-D-glucopyranoside (Buffer 
B). The enzyme was eluted with 20 ml of 50 mM Tris-succinate (pH 5.0) 
containing 1 mM DTT, 0.1M NaCl, and 0.2% octyl-.beta.-D-glucopyranoside. 
The pH 5 eluate was concentrated and washed twice with a 10-fold excess of 
Buffer B in a CF25 Centriflo ultrafiltration cone (Amicon) and brought to 
1 ml (10-fold concentration relative to the starting material). 
Step 4--Gel Filtration 
Affinity-purified farnesyltransferase (.about.1 .mu.g) was chromatographed 
on a Superose 12 column as described in the legend to FIG. 7A and FIG. 7B. 
In the enzyme characterization experiments of FIG. 1A, FIG. 1B, FIG. 2A, 
FIG. 2B, FIG. 3, FIG. 4A, FIG. 4B, FIG. 8 and FIG. 9, a partially purified 
fraction of farnesyltransferase enzyme was prepared by Steps 1 and 2 as 
described above, after which 6 mg of the Mono Q-purified material was 
concentrated to 2 ml and then loaded onto a 1.6.times.50-cm Sephacryl 
S-200 high resolution gel filtration column (Pharmacia LKB Biotechnology). 
The column was equilibrated with 50 mM Tris-chloride (pH 7.5) containing 1 
mM DTT, 0.2M NaCl, 20 .mu.M ZnCl.sub.2, and 0.2% 
octyl-.beta.-glucopyranoside and eluted with the same buffer at a flow 
rate of 15 ml/hour. Only the peak fraction, containing 1 mg protein and 
40% of initial activity, was used for studies. 
4. Identification of .sup.3 H-Isoprenoid Transferred from [.sup.3 H]FPP 
A modification of the procedure described by Casey et al. (6) was employed 
as follows: Briefly, two standard transferase reactions of 25-.mu.l each 
were conducted for 1 hour at 37.degree. C. The mixtures were then pooled, 
and a 25-.mu.l aliquot from the 50-.mu.l pooled sample was diluted to 250 
.mu.l with 2% (w/v) SDS. This mixture was precipitated with an equal 
volume of 30% TCA, filtered through nitrocellulose, (7 mm disc), washed 
twice with 250 .mu.l 6% TCA/2% SDS followed by five washes with 5% TCA, 
digested with 8 .mu.g trypsin, and subjected to cleavage with methyl 
iodide. The released .sup.3 H-isoprenoids were extracted into 
chloroform/methanol and chromatographed on a reverse-phase HPLC system as 
described in the legend to FIG. 4A and FIG. 4B. 
5. Other Methods 
SDS polyacrylamide gel electrophoresis was carried out as described by 
Laemmli (16). Gels were calibrated with high range SDS-PAGE standards 
(Bio-Rad). Protein content of extracts was measured by the method of 
Lowry, et al. (17) except for that of the affinity-purified material, 
which was estimated by comparison to the bovine serum albumin marker 
(M.sub.r 66,000) following SDS gel electrophoresis and Coomassie staining. 
6. Results and Discussion 
As an initial attempt to identify a protein farnesyltransferase enzyme, rat 
brain cytosol was fractionated with ammonium sulfate and the active 
fraction subjected to ion exchange chromatography on a Mono Q column 
followed by gel filtration on Sephacryl S-200. FIG. 1A and FIG. 1B shows 
that the active fraction from this column incorporated radioactivity from 
[.sup.3 H]farnesol into trichloroacetic acid precipitable p21.sup.H-ras in 
a time-dependent fashion at 37.degree. C. The incorporated radioactivity 
could be visualized as a band of the expected molecular weight of 
.about.21 kDa on SDS polyacrylamide gels (inset). The concentration of 
[.sup.3 H]farnesyl pyrophosphate that gave half-maximal reaction velocity 
was approximately 0.5 .mu.M (FIG. 2A). The half-maximal concentration for 
p21.sup.H-ras was approximately 5 .mu.M, and there was no difference when 
the p21.sup.H-ras was equilibrated with a nonhydrolyzable GTP or ATP 
analogue or with GDP (FIG. 2B). 
With p21.sup.H-ras as a substrate, the transferase reaction was inhibited 
by 0.15 mM EDTA, and this inhibition was reversed by 0.1 to 1.0 mM 
concentrations of zinc or magnesium chloride (FIG. 3). At higher 
concentrations of zinc chloride, inhibition was observed. 
To confirm that the transferred material was [.sup.3 H]farnesol, the washed 
trichloracetic acid-precipitated material was digested with trypsin, the 
radioactivity released with methyl iodide, and the products subjected to 
reverse-phase HPLC. The methyl iodide-released material co-migrated with 
an authentic standard of all-trans farnesol (C.sub.15) (FIG. 4A). Some 
radioactivity emerged from the column prior to the geranol standard 
(C.sub.10), but this was the same in the presence and absence of methyl 
iodide treatment. This early-eluting material was believed to represent 
some tryptic peptides whose radioactivity was not released by methyl 
iodide. 
FIG. 5 shows the elution profile of farnesyltransferase activity from a 
Mono Q column. The activity appeared as a single sharp peak that eluted at 
approximately 0.35M sodium chloride. 
The peak fractions from the Mono Q column were pooled and subjected to 
affinity chromatography on a column that contained a covalently-bound 
peptide corresponding to the carboxyl-terminal 6-amino acids of 
p21.sup.K-rasB. All of the farnesyltransferase activity was adsorbed to 
the column, and about 50% of the applied activity was recovered when the 
column was eluted with a Tris-succinate buffer at pH 5.0. 
Table I summarizes the results of a typical purification procedure that 
started with 50 rat brains. After ammonium sulfate precipitation, mono Q 
chromatography, and affinity chromatography, the farnesyltransferase was 
purified approximately 61,000-fold with a yield of 52%. The final specific 
activity was about 600,000 units/mg. 
FIG. 6A shows the SDS gel electrophoretic profile of the proteins at each 
stage of this purification as visualized by silver staining. The peptide 
affinity column yielded a single protein band with an apparent subunit 
molecular weight of 50,000. When the purified enzyme was subjected to SDS 
gel electrophoresis under more sensitive conditions, the 50-kDa protein 
could be resolved into two closely spaced bands that were visualized in 
approximately equimolar amounts (FIG. 6B). 
To confirm that the 50-kDa band was the farnesyltransferase enzyme, the 
affinity column purified material was subjected to gel filtration. FIG. 7A 
and FIG. 7B shows that the farnesyltransferase activity and the 50-kDa 
band co-eluted from this column at a position corresponding to an apparent 
molecular weight of 70-100 kDa as determined from the behavior of markers 
of known molecular weight. 
TABLE I 
______________________________________ 
PURIFICATION OF FARNESYL:PROTEIN TRANSFERASE FROM 
RAT BRAIN 
Specific Total Purifi- 
Protein Activity Activity 
cation 
Recovery 
Fraction mg units/mg units 
fold % 
______________________________________ 
30-50% 712 9.7.sup.a 
6906 1 100 
Ammonium 
Sulfate 
Mono Q 30 275 8250 28 119 
Affinity Column 
.about.0.006.sup.b 
600,000 3600 61,855 
52 
______________________________________ 
The purification procedure was started with 50 rat brains. 
.sup.a One unit of enzyme activity is the amount of enzyme that transfers 
1 pmol of [.sup.3 H]farnesol from [.sup.3 H]FPP into acidprecipitable 
p21.sup.Hras per h under the standard conditions. 
.sup.b Protein concentration was estimated by coomassie blue staining of 
SDS polyacrylamide gel using various amounts (0.5 to 2 .mu.g) of bovine 
serum albumin as a reference standard. 
The adherence of the farnesyltransferase to the peptide affinity column 
suggested that the enzyme was capable of recognizing short peptide 
sequences. To test for the specificity of this peptide recognition, the 
ability of various peptides to compete with p21.sup.H-ras for the 
farnesyltransferase activity was measured. The peptide that was used for 
affinity chromatography corresponded to the carboxyl terminal six amino 
acids of P21.sup.K-rasB (TKCVIM). As expected, this peptide competitively 
inhibited farnesylation of P21.sup.H-ras (open circles in FIG. 8). The 
terminal 4-amino acids in this sequence (CVIM) (closed circles) were 
sufficient for competition. These two short peptides were no less 
effective than a peptide that contained the final 10-amino acids of the 
sequence (KKSKTKCVIM) (open triangles). The simple transposition of the 
cysteine from the fourth to the third position from the COOH-terminus of 
the hexapeptide (TKVCIM) (closed triangles) severely reduced inhibitory 
activity. An irrelevant peptide (closed squares) also did not inhibit. 
FIG. 9 compares the inhibitory activities of four peptides of 10-amino 
acids each, all of which contain a cysteine at the fourth position from 
the COOH-terminus. The peptides corresponding to the COOH-terminus of 
human p21.sup.K-rasB and human lamin A and lamin B all inhibited 
farnesylation. All of these peptides are known to be prenylated in vivo 
(6, 15). On the other hand, the peptide corresponding to the sequence of 
rat Gi.alpha.1, a 40 kDa G protein that does not appear to be farnesylated 
in vivo (Casey, P., unpublished observations), did not compete for the 
farnesyltransferase reaction. 
In data not shown it was found that the 10-amino acid peptide corresponding 
to the COOH-terminus of p21.sup.H-ras (CVLS), p21.sup.N-ras (CVVM), and 
p21.sup.H-rasA (CIIM) all competed for the farnesylation reaction. 
Example II 
Further Characterization of Farnesyltransferase 
In the present Example, a series of tetrapeptides were tested for their 
ability to bind to the rat brain p21.sup.H-ras farnesyltransferase as 
estimated by their ability to compete with p21.sup.H-ras in a farnesyl 
transfer assay. Peptides with the highest affinity had the structure 
Cys-A1-A2-X, where A1 and A2 are aliphatic amino acids and X is a 
C-terminal methionine, serine, or phenylalanine. Charged residues reduced 
affinity slightly at the A1 position and much more drastically at the A2 
and X positions. Effective inhibitors included tetrapeptides corresponding 
to the COOH-termini of all animal cell proteins known to be farnesylated. 
In contrast, the tetrapeptide CAIL, which corresponds to the COOH-terminus 
of the only known examples of geranylgeranylated proteins (neural G 
protein .gamma. subunits) did not compete in the farnesyl transfer assay, 
suggesting that the two isoprenes are transferred by different enzymes. A 
biotinylated hexapeptide corresponding to the COOH-terminus of 
p21.sup.K-rasB was farnesylated, suggesting that at least some of the 
peptides serve as substrates for the transferase. The data are consistent 
with a model in which a hydrophobic pocket in the farnesyltransferase 
recognizes tetrapeptides through interactions with the cysteine and the 
last two amino acids. 
1. Materials and Methods 
a. Peptides 
Peptides were prepared by established procedures of solid-phase synthesis 
(18) Tetrapeptides were synthesized on the Milligen 9050 Synthesizer using 
Fmoc chemistry. After deprotection of the last residue, a portion of the 
resin was used to make the N-acetyl-modified version of CVIM. This was 
done off-line in a solution of acetic anhydride and dimethylformamide at 
pH 8 (adjusted with diisopropylethylamine). The acetylated and 
unacetylated peptides were cleaved with 50 ml of trifluoroacetic 
acid:phenol (95:5) plus approximately 1 ml of ethanedithiol added as a 
scavenger. The N-octyl-modified version of CVIM was synthesized on an 
Applied Biosystems Model 430 Synthesizer using tBoc chemistry. The octyl 
group was added in an amino acid cycle using octanoic acid. The peptide 
was cleaved from the resin at 0.degree. C. with a 10:1:1 ratio of HF 
(mls):resin (g):anisole (ml). The peptides were purified by high pressure 
liquid chromatography (HPLC) on a Beckman C18 reverse phase column (21.1 
cm.times.15 cm), eluted with a water-acetonitrile gradient containing 0.1% 
(v/v) trifluouroacetic acid. Identity was confirmed for all peptides by 
fast atom bombardment (FAB) mass spectrometry. Just prior to use, each 
peptide was dissolved at a concentration of 0.8 mM in 10 mM dithiothreitol 
(DTT), and all dilutions were made in 10 mM DTT. 
Biotinylated KTSCVIM was synthesized on an Applied Biosystems 430A 
Synthesizer. The biotin group was added after removal of the N-terminal 
protecting group before cleavage of the peptide from the resin. 
Specifically, a 4-fold molar excess of biotin 4-nitrophenyl ester was 
added to the 0.5 g resin in 75 ml dimethylformanide at pH 8 and reacted 
for 5 hours at room temperature. Cleavage, identification, and 
purification were carried out as described above. 
To synthesize S-acetoamido CVIM, purified CVIM was dissolved at a final 
concentration of 1 mM in 0.1 ml of 0.5M Tris-chloride (pH 8.0) containing 
15 mM DTT. The tube was flushed with nitrogen for 2 min, sealed, and 
incubated for 2.5 hours at 37.degree. C. to reduce the cysteine residue, 
after which iodoacetamide was added to achieve a final concentration of 35 
mM. After incubation for 15 min at 37.degree. C., the reaction was stopped 
by addition of 10 mMDTT. Complete alkylation of CVIM was confirmed by FAB 
spectrometry and HPLC. The molecular weight of the product corresponded to 
the expected molecular mass of S-acetoamido CVIM. 
b. Assay for Farnesyltransferase 
The standard assay involved measuring the amount of [.sup.3 H]farnesyl 
transferred from all-trans [.sup.3 H]FPP to recombinant human 
p21.sup.H-ras as described in Example I. Each reaction mixture contained 
the following concentrations of components in a final volume of 25 .mu.l: 
50 mM Tris-chloride (pH 7.5), 50 .mu.M ZnCl.sub.2, 30 mM KCl, 1 mM DTT, 30 
or 40 .mu.M p21.sup.H-ras, 15 pmol [.sup.3 H]FPP (12-23,000 dpm/pmol), 4 
to 7.5 .mu.g of partially purified farnesyltransferase (Mono Q fraction, 
see Example I), and the indicated concentration of competitor peptide 
added in 3 .mu.l of 10 mM DTT. After incubation for 30-60 min at 
37.degree. C., the amount of [.sup.3 H]farnesyl present in trichloroacetic 
acid-precipitable p21.sup.H-ras was measured by a filter assay as 
described in Example I. A blank value (&lt;0.6% of input [.sup.3 H]FPP) was 
determined in parallel incubations containing no enzyme. This blank value 
was subtracted before calculating "% of control" values. 
C. Transfer of [.sup.3 H]Farnesyl from [.sup.3 H]FPP to Biotinylated 
KTSCVIM Peptide 
This assay takes advantage of the fact that peptides containing the Cys-AAX 
motif of ras proteins can serve as substrates for prenylation by 
farnesyltransferase. A heptapeptide containing the terminal four amino 
acids of p21.sup.K-rasB was chosen as a model substrate since it has a 20 
to 40-fold higher affinity for the enzyme than does the COOH-terminal 
peptide corresponding to p21.sup.H-ras. A biotinylated peptide is used as 
substrate so that the reaction product, [.sup.3 H]farnesylated peptide, 
can be trapped on a solid support such as streptavidinagarose. The bound 
[.sup.3 H]farnesylated peptide can then be washed, separated from 
unincorporated [.sup.3 H]FPP, and subjected to scintillation counting. 
The biotin-modified KTSCVIM is synthesized on an Applied Biosystems 430A 
Synthesizer using established procedures of solid phase peptide synthesis. 
The biotin group is added after deprotection of lysine and before cleavage 
of the peptide from the resin. The identity and purity of the biotinylated 
peptide is confirmed by quantitative amino acid analysis and fast atom 
bombardment (FAB) mass spectrometry. 
An aliquot of biotinylated KTSCVIM (0.4 mg) is dissolved in 0.6 ml of 10 mM 
sodium acetate (pH 3) buffer containing 1 mM DTT and 50% ethanol to give a 
final concentration of 0.67 mg/ml or 601 .mu.M. This solution can be 
stored at 4.degree. C. for at least 1 month. Immediately prior to use, the 
peptide solution is diluted with 1 mM DTT to achieve a peptide 
concentration of 18 .mu.M. The standard reaction mixture contains the 
following components in a final volume of 25 .mu.l: 50 mM Tris-chloride 
(pH 7.5), 50 .mu.M ZnCl.sub.2, 20 mM KCl, 1 mM DTT, 0.2% (v/v) 
octyl-.beta.-glucopryranoside, 10-15 pmol of [.sup.3 H]FPP (15-50,000 
dpm/pmol), 3.6 .mu.M biotinylated KTSCVIM, and 2-4 units of enzyme. After 
incubation at 37.degree. C. for 30-60 min in 0.5-ml siliconized microfuge 
tubes, the reaction is stopped by addition of 200 .mu.l of 20 mM 
Tris-chloride (pH 7.5) buffer containing 2 mg/ml bovine serum albumin, 2% 
SDS, and 150 mM NaCl. A 25-.mu.l aliquot of well mixed 
streptavidin-agarose (Bethesda Research Laboratories, Cat. No. 5942SA) is 
then added, and the mixture is gently shaken for 30 min at room 
temperature to allow maximal binding of the [.sup.3 H]farnesylated peptide 
to the beads. 
The beads are then collected by spinning the mixture for 1 min in a 
microfuge (12,500 rpm). The supernatant is removed, and the beads are 
washed three times with 0.5 ml of 20 mM Tris-chloride (pH 7.5) buffer 
containing 2 mg/ml bovine serum albumin, 4% SDS, and 150 mM NaCl. The 
pellet is resuspended in 50 .mu.l of the same buffer and transferred to a 
scintillation vial using a 200-.mu.l pipettor in which the tip end has 
been cut off at an angle. The beads remaining in the tube are collected by 
rinsing the tube with 25 .mu.l of the above buffer and adding it plus the 
pipettor to the vial. A blank value, which consists of the radioactivity 
adhering to the beads in parallel incubations containing no enzyme, should 
be less than 0.5% of the input [.sup.3 H]FPP. 
2. Results 
To screen peptides for their affinity for the farnesyltransferase, studies 
were conducted wherein the ability of the peptides to compete with 
p21.sup.H-ras for acceptance of [.sup.3 H]farnesyl from [.sup.3 H]FPP as 
catalyzed by a partially purified rat brain farnesyltransferase was 
tested. As a reference point for the peptides, the tetrapeptide CVIM 
corresponding to the COOH-terminal sequence of p21.sup.K-rasB was 
employed. FIG. 10A, FIG. 10B and FIG. 10C shows a series of typical 
experiments in which alanine (FIG. 10A), lysine (FIG. 10B), or leucine 
(FIG. 10C) was systematically substituted at each of the three positions 
following cysteine in CVIM. In each experiment the results were compared 
with those obtained with CVIM. Alanine and lysine were tolerated only at 
the A1 position. Insertion of these amino acids at the A2 or X positions 
decreased the affinity for the enzyme by more than 30-fold as estimated by 
the concentration required for 50% inhibition. Leucine was tolerated at 
the A2 position, but it decreased the affinity when inserted at the X 
position. 
The substitution of phenylalanine for isoleucine at the A2 position 
increased the affinity for the enzyme by 6-fold, with half-maximal 
inhibition occurring at 25 nM (FIG. 11). No such effect was observed when 
phenylalanine was inserted at either of the other two positions. 
In addition to performing assays with p21.sup.H-ras as a substrate, assays 
were also performed in which the substrate was a biotinylated 
heptapeptide, KTSCVIM, which contains the COOH-terminal four amino acids 
of p21.sup.H rasB (2). The biotin was attached to the NH.sub.2 -terminus 
by coupling to the resin-attached peptide. The [.sup.3 H]farnesylated 
product was isolated by allowing it to bind to beads coated with 
streptavidin as described in section c. above. 
FIG. 12A and FIG. 12B shows that the peptide CVFM was more potent than CVIM 
when either p21.sup.H-ras or the biotinylated heptapeptide was used as 
acceptor (FIG. 12A and FIG. 12B, respectively). In contrast to the other 
studies, which were conducted with a partially purified enzyme, the 
studies of FIG. 12A and FIG. 12B were carried out with a homogeneous 
preparation of affinity-purified farnesyltransferase. 
The free sulfhydryl group for the cysteine is likely required for 
tetrapeptide inhibition, as indicted by the finding that derivitization 
with iodoacetamide abolished inhibitory activity (FIG. 13A). A blocked 
NH.sub.2 -terminus is not required, as indicated by similar inhibitory 
activity of N-acetyl CVIM and N-octyl CVIM (FIG. 13B) as compared to that 
of CVIM (FIG. 13A). 
FIG. 14 summarizes the results of all competition assays in which 
substitutions in the CVIM sequence were made. The results are presented in 
terms of the peptide concentration required for 50% inhibition. Table II 
summarizes the results of other experiments in which tetrapeptides 
corresponding to the COOH-termini of 19 proteins were studied, many of 
which are known to be farnesylated. The implications of these studies are 
discussed below in Section 3. 
TABLE II 
______________________________________ 
Inhibition of Rat Farnesyltransferase by 
COOH-Terminal Tetrapeptides Corresponding to Known Proteins 
Concentration 
for 50% 
COOH-Terminal 
Inhibition 
Protein Species Tetrapeptide 
.mu.M 
______________________________________ 
*p21.sup.K-rasB 
Human, mouse 
CVIM 0.15 
*p21.sup.K-rasA 
Human CIIM 0.15 
p21.sup.N-ras 
Human CVVM 0.15 
p21.sup.N-ras 
Mouse CVLM 0.15 
*Lamin B Human, CAIM 0.15 
Xenopus laevis 
Lamin A Human, CSIM 0.20 
Xenopus laevis 
Retinal cGMP 
Bovine CCVQ 0.35 
phosphodiesterase, 
.alpha. subunit 
*ras1 S. cerevisciae 
CIIC 0.35 
*ras2 S. cerevisciae 
CIIS 0.35 
*.gamma.-Subunit of 
Bovine CVIS 1.0 
transducin 
p21.sup.H-ras 
Chicken CVIS 1.0 
p21.sup.H-ras 
Human, rat CVLS 3.0 
*a-Mating S. cerevisciae 
CVIA 5.0 
factor 
rap2b Human CVIL 11 
Dras Dictostelium 
CLIL 17 
rapla/krevl 
Human CLLL 22 
*Mating factor 
R. Toruloides 
CTVA 30 
.gamma.-Subunit of 
Bovine CAIL 100 
G protein 
HMG CoA S. cerevisciae 
CIKS &gt;100 
reductase-1 
______________________________________ 
Enzyme activity was measured in the presence of the indicated tetrapeptid 
as described in the legend to FIG. 10. Each tetrapeptide was tested at 
seven different concentrations ranging from 0.03 to 100 .mu.M. The 
concentration giving 50% inhibition was calculated from the inhibition 
curve. 
*Shown to be farnesylated in vivo. 
3. Discussion 
The current data extend the observations on the p21.sup.ras 
farnesyltransferase set forth in Example I, and further indicate that the 
recognition site for this enzyme is restricted to four amino acids of the 
Cys-A1-A2-X type. As a reference sequence for these studies, the peptide 
CVIM was used. This peptide inhibited the farnesyltransferase by 50% at a 
concentration of 0.15 .mu.M. Substitution of various amino acids into this 
framework yielded peptides that gave 50% inhibitions at a spectrum of 
concentrations ranging from 0.025 .mu.M (CVFM) to greater than 50 .mu.M 
(FIG. 14). 
In general, the highest inhibitory activities were achieved when the A1 and 
A2 positions were occupied with nonpolar aliphatic or aromatic amino 
acids. This stringency was more severe at the A2 than at the A1 position. 
Thus, peptides containing lysine or glutamic acid at the A1 position gave 
50% inhibition at 0.7 and 1.5 .mu.M, respectively. When these two residues 
were inserted at the A2 position, the affinity for the enzyme declined by 
more than 50-fold. Glycine and proline lowered inhibitory activity 
moderately at the A1 position (50% inhibition at 4 and 8 .mu.M) and 
somewhat more severely at the A2 position (8 and 20 .mu.M). 
The X position showed the highest stringency. In the context of CVIx, 
methionine was the preferred residue but phenylalanine and serine were 
tolerated with only modest losses in activity (0.5 and 1 .mu.M, 
respectively). Aliphatic resides and proline were disruptive at this 
position, with 50% inhibitions in the range of 5-11 .mu.M. Glutamic acid, 
lysine, and glycine were not tolerated at all; 50% inhibition required 
concentrations above 40 .mu.M. 
A study of tetrapeptides corresponding to the COOH-termini of known 
proteins (Table II) gave results that were generally in keeping with those 
obtained with the substituted CVIM peptides. They provided the additional 
information that glutamine and cysteine are well tolerated at the X 
position (CCVQ and CIIC). All of the proteins that are known to be 
farnesylated in intact cells (indicated by the asterisks in Table II) 
followed the rules outlined above, and all inhibited farnesylation at 
relatively low concentrations (5 .mu.M or below) with the exception of the 
CTVA sequence, R. toruloides (19). This peptide inhibited the rat brain 
farnesyltransferase by 50% only at the high concentrations of 30 .mu.M. It 
is likely that the farnesyltransferase in this fungal species has a 
different specificity than that of the rat brain. 
The peptide CAIL, which corresponds to the COOH-terminus of the 
.gamma.-subunit of bovine brain G proteins (20,21), did not compete 
efficiently with p21.sup.H-ras for farnesylation (Table II). A 50% 
inhibition at the highest concentration tested (100 .mu.M) was observed. 
The inhibitory activity was lower than that of CVIL (12 .mu.M) or CAIM 
(0.15 .mu.M). Thus, the combination of alanine at the A1 position and 
leucine at the X position is more detrimental than either single 
substitution. This finding is particularly relevant since the gamma 
subunit of G proteins from human brain (22) and rat PC12 cells (23) have 
been shown to contain a geranylgeranyl rather than a farnesyl. These 
findings suggest the existence of a separate geranylgeranyl transferase 
that favors CAIL and perhaps other related sequences. 
The studies with the biotinylated heptapeptide (FIG. 12B) confirm that at 
least some of the short peptides act as substrates for the enzyme. The 
saturation curves relating reaction velocity to the concentration of 
either p21.sup.H-ras or the biotinylated heptapeptide are complex and 
sigmoidal. The inhibition curves with the various peptides differ from 
classic competitive inhibition curves. Finally, as mentioned in Example I, 
the maximal velocity of the purified enzyme is relatively low. These 
findings suggest that the binding of the peptides to the enzyme is not a 
simple equilibrium reaction. Rather, there may be a slow binding that 
requires conformational change. 
The observation that the A1 position shows a relaxed amino acid specificity 
suggests that the residue at this position may not contact the 
farnesyltransferase directly. Rather, the contacts may involve only the 
cysteine and the residues at the A2 and X positions. A working model for 
the active site of the farnesyltransferase places the peptide substrate in 
an extended conformation with a largely hydrophobic pocket of the enzyme 
interacting with the X group of the CAAX-containing substrate. 
Example III 
Recombinant Cloning of the Farnesyltransferase .alpha. and .beta. Subunit 
cDNAs 
This example demonstrates the recombinant cloning of cDNAs corresponding to 
both the .alpha. and .beta. subunit of rat farnesyltransferase. The method 
employed by the inventors involved the application of the peptide sequence 
information, as detailed above, to prepare specific primers for PCR-based 
sequencing, which sequences were then used for the construction of probes 
with which to screen cDNA libraries. The cloning of each of these cDNAs by 
the inventors' laboratory has recently been reported (36, 36a). 
1. Methods 
a. General Methods 
General molecular biological techniques were employed in connection with 
the cloning reactions described below, as set forth in Sambrook et al., 
(ref 24). cDNA clones were subcloned into bacteriophage M13 or plasmid pUC 
vectors and sequenced by the dideoxy chain termination method (25) using 
the M13 universal sequencing primer or gene specific internal primers. 
Sequencing reactions are preferably performed using a modified 
bacteriophage T7 DNA polymerase (26) with .sup.35 S-labeled nucleotides, 
or Taq polymerase with fluorescently labeled nucleotides on an Applied 
Biosystems Model 370A DNA Sequencer. 
For the isolation of total cellular RNA from rat tissues, the inventors 
preferred to employ the guanidinium thiocyanate/CsCl centrifugation 
procedure (27). Whereas for the isolation of RNA from cell lines, the 
guanidinium HCl method was found to be preferrable (28). The isolation of 
poly A.sup.+ RNA by oligo(dT)-cellulose chromatography was achieved by the 
methods described in Refs. 24 and 29. Northern blot hybridization using 
single-stranded .sup.32 P-labeled probes was carried out as described by 
Lehrman et al. (30). A cDNA probe for rat glyceraldehyde-3-phosphate 
dehydrogenase was kindly provided by Karl Normington, (University of Texas 
Southwestern Medical Center at Dallas). 
Polyclonal antisera, specific for either the .alpha. or .beta. subunit of 
farnesyltransferase, were prepared by immunising rabbits with synthetic 
peptides derived from each specific subunit. Antibody Y533 was raised 
against a synthetic peptide with the sequence LQSKHSRESDIPASV, derived 
from the predicted amino acid sequence of a cDNA clone of the .alpha. 
subunit. Antibody X287 was raised using the synthetic peptide 
IQATTHFLQKPVPGFEE, derived from a tryptic digest of the .beta. subunit. 
Each peptide was coupled to Keyhole Limpet hemocyanin using 
maleimidobenzoic acid N-hydrosuccinimide ester (Signa Chemical Co.) (40). 
For each antibody, three New Zealand White rabbits were immunised with 600 
.mu.g of coupled peptide in Freund's complete adjuvant. Immunoblot 
analysis was performed as described in (35, 36). 
Rat PC12 pheochromocytoma cells, rat KNRK cells (CRL 1569), and human 
embryonic kidney 293 cells were obtained, respectively, from Thomas Sudhof 
(University of Texas Southwestern Medical Center at Dallas), the American 
Type Culture Collection, and Arnold J. Berk (University of California, Los 
Angeles). 
b. PCR and Probe Synthesis 
To derive a sequence for constructing an appropriate probe, rat genomic DNA 
may be used as a template for PCR as described by Saiki et al. (31) and 
Lee et al. (32). The approach used by the inventors was to sequence a 
portion of the .alpha. or .beta. subunit genes through the use of 
appropriate PCR primers, based on a consideration of the peptide 
sequences. Thus, PCR was used to obtain the rat genomic DNA sequences that 
encoded tryptic peptides derived from either the purified .alpha. or 
.beta. subunits of rat farnesyltransferase (FIG. 16A and FIG. 16B). For 
the both the .alpha. and .beta. sequences, the PCR primers were 
synthesized based on the NH.sub.2 - and COOH-terminal sequences of the 
peptides shown in FIG. 16A and FIG. 16B, and included the degenerate 
inosine codons indicated (FIG. 16). PCR primers were end-labeled with 
[.gamma.-.sup.32 P]ATP. Each of the amplified DNA fragments were eluted 
from 12% acrylamide gels and sequenced by the method of Maxam and Gilbert 
(33). Translation of the nucleotide sequences between the two primers 
yielded peptides with amino acid sequences identical to those of the 
peptides shown (FIG. 16A and FIG. 16B). 
Using the DNA sequences of the PCR products, the inventors then synthesized 
an oligonucleotide probe that would hybridize with the region 
corresponding to the peptide, for use in the direct screening of the 
library. For the .alpha. subunit, a 38-mer probe with the nucleotide 
sequence: 5'-ATIGAGTTAAACGCAGCCAACTATACGGTCTGGCACTT-3', was synthesised. 
Whereas for the .beta. subunit, two primers, designated primer .beta.3 and 
primer .beta.4 were synthesised with the respective nucleotide sequences: 
5'-GCGTACTGTGCGGCCTC-3' and 5'-GGCCTCAGTAGCCTCTCTCACCAAC-3'. 
The primers for the .beta. subunit were used for 3'-end amplification of 
cDNA as described by Frohman et al. (34). Poly(A).sup.+ RNA from rat KNRK 
cells was reverse transcribed using a (dT).sub.17 -adaptor, 
5'-GACTCGAGTCGACATCGA(T).sub.17 -3'. The 50 .mu.l reaction mixture, 
containing 4 .mu.g poly(A).sup.+ RNA, 2.5 .mu.g (dT).sub.17 -adaptor, and 
100 units of Moloney murine leukemia virus reverse transcriptase (Bethesda 
Research Laboratories), was incubated at 37.degree. C. for 1 hour. Reverse 
transcribed cDNA was diluted 50-fold with 10 mM Tris-HCl at pH 8.0, 1 mM 
EDTA, and subjected to specific PCR amplification as follows. 10 .mu.l of 
diluted cDNA, 25 pmol of adaptor primer (5'-GACTCGAGTCGACATCG-3'), and 25 
pmol of primer 3 were boiled, after which PCR was carried out for 40 
cycles (95.degree. C., 40 sec; 58.degree. C., 1 min; 72.degree. C., 3 min) 
with TaqI polymerase. Amplified PCR products were subjected to 
electrophoresis on an agarose gel, transferred to a nylon membrane, and 
probed with .sup.32 P-labeled primer 4. The hybridizing DNA fragment was 
eluted, extracted with phenol/chloroform, and used as a template for a 
second PCR reaction. The reaction using 25 pmol each of adaptor primer and 
primer 4 was carried out with the same amplification protocol as described 
above. The reamplified DNA fragment was gel-purified, cleaved with RsaI or 
TaqI, and subcloned into an M13 vector for DNA sequencing and for 
subsequent generation of the single-stranded M13 probe that is referred to 
as Probe B. The DNA sequence of the PCR-derived clone was also used to 
generate a 50-mer oligonucleotide probe that is designated Probe A. Probes 
A and B were then used to screen cDNA libraries in order to obtain a 
full-length .beta. subunit cDNA (see .beta. subunit cloning section, 
below). 
c. cDNA Libraries and Cloning 
Rat PC12 cell poly(A.sup.+) RNA and oligo (dT)-primed KNRK cell 
double-stranded cDNA libraries were constructed in bacteriophage 
.lambda.gt10, using a cDNA synthesis kit from Invitrogen. These cells were 
preferred because the inventors believed them to be rich in 
farnesyltransferase mRNA. Although numerous convenient methods are known 
for the construction of cDNA libraries, the inventors utilised the above 
kit from Invitrogen as they thought it to be a particularly convenient 
method. The cDNA itself was prepared using both oligo(dT)- and random 
hexamer-primed cDNA, then ligated to a suitable linker, with the 
EcoR1/Not1 linker being preferred in this case. cDNAs larger than 1 kb 
were isolated by size fractionation using a 1% agarose gel and ligated 
into EcoR1-cleaved .lambda.gt10 DNA (Stratagene), in order to complete the 
construction of the cDNA-containing vectors for library preparation. After 
in vitro packaging of the recombinant lambda phage with a DNA packaging 
extract (Stratagene), phage were plated out on host strain E. coli C600 
hfl.sup.- cells. 
.alpha. subunit cloning. Approximately 1.times.10.sup.6 plaques of the rat 
brain library were screened. Duplicate filters were hybridized in 
6.times.SSC (1.times.SSC=150 mM NaCl/15 mM Na citrate, at pH 7.0) with 
1.times.10.sup.6 cpm/ml of .sup.32 P-labeled probe (see above). One 
positive clone, .lambda.RB-17, with an insert of 1.4 kb was identified and 
plaque purified. Phage DNA from a plate lysate was subcloned into 
bacteriophage M13 and pBluescript vectors for DNA restriction mapping and 
sequencing (37). 
As the clone first obtained was not a full-length clone, 5'-end 
amplification was employed to produce the complete sequence, as described 
in Ref 34. Firstly, an M13 probe corresponding to the 5' end of 
.lambda.RB-17 was used to screen the KNRK cell library. Replicate filters 
were hybridized in 50% (v/v) formamide containing 1.times.10.sup.6 cpm/ml 
of the probe. Positive clones were analyzed by PCR, and the clone with the 
longest insert (.lambda.KNRK-3) was purified and subcloned for analysis. A 
5' Rapid Amplification of cDNA End procedure (5' RACE) (34) was used to 
extend the 5' end of .lambda.KNRK-3. An M13 probe derived from the 
amplification product (RACE-5') was then used to screen a rat testis 
library (purchased from Clontech), yielding .lambda.RTH, which extended to 
nucleotide position 53. 
To obtain the extreme 5' end of the cDNA, a primer-extension .lambda.gt10 
library was constructed from rat testis poly(A).sup.+ RNA. First stand 
synthesis was primed with an oligonucleotide corresponding to a sequence 
near the 5' end of RACE-5' using Maloney murine leukemia virus reverse 
transcriptase. After incubation at 37.degree. C. for 1 h, the reaction was 
heated at 70.degree. C. for 5 min. Five units of Thermostable rTth 
Transcriptase (Perkin-Elmer) was then added, and the reaction continued at 
70.degree. C. for 30 min. After second strand synthesis, the cDNAs were 
ligated to an EcoRI/NotI linker. Excess linkers were removed by Centricon 
100 Microconcentrator (Amicon). Approximately 5.times.10.sup.5 plaques 
were screened with a .sup.32 P-labeled probe corresponding to nucleotides 
54-104, which was obtained from the sequence of .lambda.RTH. Twenty-five 
positive clones were identified. Phage DNA was prepared from plate 
lysates, and the insert from one of the longest clones, .lambda.PE-7, was 
subcloned for sequencing (37). 
.beta. subunit cloning. Approximately 5.times.10.sup.5 plaques were 
transferred to replicate filters. One filter was hybridized in 10% (v/v) 
formamide with 1.times.10.sup.6 cpm/ml of a .sup.32 P-labeled 50-mer 
oligonucleotide probe (Probe A; described above). The other filter was 
hybridized in 50% formamide with 1.times.10.sup.6 cpm/ml of a 
single-stranded M13 probe (Probe B; described above). One positive clone 
(.lambda.dT-7) with an insert of .about.2.3 kb was identified with both 
probes and plaque purified. Phage DNA isolated from the plate lysate of 
.lambda.dT-7 was subcloned into M13 and pUC vectors for sequencing and 
restriction mapping. 
To obtain the extreme 5' end of the cDNA, an M13 probe corresponding to the 
5' end of .lambda.dT-7 was used to screen a rat brain "5'-stretch" cDNA 
library (purchased from Clontech). Replicate filters were hybridized in 
50% formamide containing 1.times.10.sup.6 cpm/ml of the probe. Of the 
5.times.10.sup.5 plaques screened, six positive clones were plaque 
purified and eluted in 0.2 ml buffer containing 100 mM NaCl, 8 mM 
MgSO.sub.4, 50 mM Tris-HCl at pH 7.5, and 0.01% (w/v) gelatin. A primer 
corresponding to the right arm or left arm of .lambda.gt10 sequences 
flanking the unique EcoR1 cloning site was used in combination with a 
primer derived from the 5' end of the rat protein farnesyltransferase cDNA 
(.lambda.dT-7) for a PCR reaction. PCR products were analyzed on an 
agarose gel, and the clone containing the longest extension, 
.lambda.RB-23, was subcloned for further analysis. 
d. Expression Vectors 
Expression vectors for the .alpha. subunit of rat farnesyltransferase were 
constructed in pCMV5, a plasmid that contains the promoter-enhancer region 
of the major immediate early gene of human cytomegalovirus (38). A PvuII 
fragment containing 20 base pairs of the 5' untranslated region and the 
entire coding region was excised from clone .lambda.RTH-B and ligated into 
SmaI-digested pCMV5 in both orientations. Plasmid .lambda.RTH-B is 
identical to .lambda.RTH except for the presence of an intron in the 
5'-untranslated region at nucleotide position 39, upstream of the PvuII 
site at position 37-42. The resulting plasmids designated pFT-.alpha. 
(correct orientation) and pFT-.alpha.rev (reverse orientation), were 
characterized by restriction mapping. 
Expression vectors for the .beta.-subunit of rat farnesyltransferase were 
also constructed in pCMV5 (38). An EcoR1 fragment containing the entire 5' 
untranslated region and the coding region of farnesyltransferase .beta. 
subunit cDNA was excised from clone .lambda.RB-23 and ligated into 
EcoR1-digested pCMV5 in both orientations. The resulting plasmids, 
designated pFT-.beta.1 (correct orientation) and pFT-.beta.1rev (reverse 
orientation), were characterized by restriction mapping. 
e. DNA Transfection 
Human embryonic kidney 293 cells were grown in monolayer at 37.degree. C. 
in medium A (Dulbecco's modified Eagle medium supplemented with 10% (v/v) 
fetal calf serum, 100 units/ml of penicillin, and 100 .mu.g/ml 
streptomycin). On day 0, 6.times.10.sup.5 cells/100-mm dish were seeded in 
medium A. On day 1, each dish of cells was transfected with 3 .mu.g of the 
indicated plasmid plus 1 .mu.g of pVA (a plasmid encoding adenovirus VA 
RNA.sub.I) (39) by the calcium phosphate method (24). On day 2, the cells 
received fresh medium A. On day 4, the cells from two dishes were 
harvested, pooled, and disrupted by repeated aspiration at 4.degree. C. 
through a 25-gauge needle in 0.4 ml buffer containing 50 mM Tris-HCl at pH 
7.5, 50 .mu.M ZnCl.sub.2, 3 mM MgCl.sub.2, 20 mM KCl, 1 mM dithiothreitol, 
and 0.4% (w/v) octyl-.beta.-glucopyranoside. A cytosolic extract was 
obtained by centrifugation at 100,000.times.g for 1 h at 4.degree. C., 
after which 0.16 to 5.4 .mu.g of the supernatant fraction were assayed for 
farnesyltransferase activity by measuring the amount of [.sup.3 H]farnesyl 
transferred from [.sup.3 H]farnesyl pyrophosphate to p21.sup.H-ras protein 
as described above. 
2. Results 
a. .alpha. Subunit Cloning and Sequence Analysis 
Degenerate oligonucleotide probes encoding the 5' and 3' ends of a tryptic 
peptide derived from the farnesyltransferase .alpha. subunit were used as 
primers in a PCR employing rat genomic DNA (FIG. 16A). The sequence of the 
amplified product was used as a probe to screen a random 
hexanucleotide-primed rat brain cDNA library cloned in .lambda.gt10. This 
procedure yielded .lambda.RB-17, which extended from a poly A tract up to 
nucleotide position 345 (this position refers to the final sequence of the 
mRNA, as in SEQ ID NO:2). 
The 5'-end of the mRNA encoding the .alpha. subunit was found to contain a 
sequence extremely rich in GC basepairs (76% GC from nucleotides 71 to 205 
and 90% GC from nucleotides 116 to 145). Multiple attempts to traverse 
this region by primer extension using reverse transcriptase gave products 
that terminated prematurely, or that encoded unspliced introns. Therefore, 
other strategies were employed in order to obtain the 5'-end of the mRNA 
(see above methods section for detailed protocols). A composite of the 
cDNA sequences thus obtained was used to generate the overall sequence of 
the mRNA (SEQ ID NO:2). 
The mRNA was found to encode a protein of 377 amino acids (SEQ ID NO:1) 
with a calculated molecular weight of 44053. Although the cDNA sequence 
did not contain a terminator codon upstream of the first methionine codon, 
it is believed that this methionine represented the true initiator codon. 
This is supported by transfection studies, in which the recombinant 
protein produced was observed to have a molecular weight that was 
indistinguishable on immunoblots from that of the purified rat brain 
.alpha. subunit (see below and FIG. 20A and FIG. 20B). If the cDNA were 
incomplete, the initiator methionine must be upstream of the 5' end of the 
sequence obtained, and therefore the protein produced by the cDNA should 
be at least 2 kDa smaller than the authentic protein. Such a difference 
should have been detected in gel electrophoresis experiments. 
The most remarkable feature of the .alpha. subunit cDNA was determined to 
be a string of 9 consecutive proline residues near the NH.sub.2 -terminus 
(underlined in SEQ ID NO:2), whose codons accounted for much of the 
extreme GC-richness of this region. The mRNA contained sequences 
corresponding to sequences of the peptides obtained following tryptic 
digestion of the purified .alpha. subunit. Discrepancies only occured at 
positions that were assigned tentatively in sequencing trace amounts of 
protein. Some slight homology has been noted between the rat 
.alpha.-subunit amino acid sequence and yeast RAM2, the sequence of which 
is reported in He et al., reference 51 (see FIG. 17). 
Recently, Kohl et al. have reported the cloning of a partial cDNA clone 
corresponding to the bovine .alpha.-subunit of farnesyltransferase (52). 
The 329 amino acids encoded by this partial clone are 95% identical to the 
corresponding region in the .alpha.-subunit of the rat 
farnesyltransferase. Comparison of the complete amino acid sequence of rat 
farnesyltransferase .alpha.-subunit (377 amino acids) with that of the 
yeast RAM2 gene product (316 amino acids) disclosed by He et al. reveals 
that the two proteins are 39% identical in the COOH-terminal 211 residues, 
suggesting that RAM2 is the yeast counterpart of the .alpha.-subunit of 
mammalian farnesyltransferase. 
b. .beta. subunit Cloning and Analysis 
A unique DNA sequence encoding a portion of the .beta.subunit of the rat 
farnesyltransferase was obtained by the polymerase chain reaction (PCR) 
with rat genomic DNA and degenerate oligonucleotide primers (primers 
.beta.1 and .beta.2) corresponding to potential sequences encoding a 
tryptic peptide obtained from the purified rat brain enzyme (FIG. 16B). 
Two unique oligonucleotides (primers .beta.3 and .beta.4) were synthesized 
based on the sequence of the amplified product (FIG. 16B). These primers 
were then used in a 3'-end amplification strategy (34) to obtain an 
amplified fragment from cDNA prepared from mRNA isolated from cultured rat 
kidney cells (KNRK cells). This fragment was used to generate probes that 
identified a bacteriophage containing a near full-length cDNA 
(.lambda.dT-7) from a cDNA library prepared from rat pheochromocytoma PC12 
cells. Finally, a fragment from the 5'-end of .lambda.dT-7 was used to 
identify a clone containing a full-length farnesyltransferase .beta. 
subunit cDNA (.lambda.RB-23) from a rat brain cDNA library (SEQ ID NO:4). 
The cDNA for the rat brain farnesyltransferase .beta. subunit contains 59 
base pairs of 5' untranslated region followed by protein-coding region of 
1314 base pairs and a 3' untranslated region of 1091 base pairs (SEQ ID 
NO:4). The cDNA encoded a protein of 437 amino acids and contained 
sequences corresponding to sequences of the peptides obtained following 
tryptic digestion of the purified rat brain farnesyltransferase .beta. 
subunit. Although certain minor discrepancies in sequence between the 
protein and the cDNA were apparent, these occurred near the COOH-termini 
of the peptides and were attributed to ambiguities in sequencing the trace 
amounts of peptide that were available. 
The cDNA clones did not contain an inframe terminator codon upstream of the 
first methionine (amino acid residue in SEQ ID NO:3). This is believed to 
be the initiator methionine as it lies in a good context for initiation 
according to Kozak.times.s rules (41) and because the cDNA encode a 
protein of the same size as the .beta.-subunit when transfected into 
animal cells (see below). Although .lambda.dT-7 was obtained from an 
oligo-dT primed cDNA library, the clone did not contain a poly A tract, 
nor did it contain a consensus polyadenylation sequence. However, RNA blot 
hybridization experiments and expression studies (see below) suggested 
that the clone is essentially full-length. 
The molecular weight of the .beta. subunit of the rat brain 
farnesyltransferase was calculated to be 48,679. The amino acid 
composition did not show any particularly remarkable features and the 
calculated isoelectric point was 5.99. An analysis of the hydrophobicity 
plots did not reveal any extensive hydrophobic sequences. 
A search of the GenBank and EMBL data banks revealed significant 
resemblance to two proteins, the DPR1-RAM1 protein of yeast Saccharomyces 
cerevisiae and a yeast open reading frame of unidentified function (ORF2). 
Extensive stretches of identity were evident between the .beta. subunit 
protein sequence and the yeast DPR1-RAM1 gene product (FIG. 18). Sequence 
conservation was observed throughout the two proteins (overall identity: 
37%), but was found to lessen at both ends, and the yeast protein was 
shorter by six amino acids. 
In a recent article by Kohl et al. (52), in a note added in proof, it is 
indicated that the .beta.-subunit of bovine farnesyltransferase has been 
cloned and that it shares 96% homology to the rat .beta.-subunit. However, 
no actual sequences corresponding to the .beta.-subunit are disclosed by 
Kohl et al. 
c. Northern Blotting Analyses 
Northern RNA blot analysis with .sup.32 P-labelled probes derived from the 
.alpha. subunit cDNA revealed a single mRNA of .about.1.75 kb in multiple 
rat tissues, including lung, heart, kidney, brain, adrenal, and testis 
(FIG. 19A). The amount of mRNA in testis was several-fold higher than in 
any other tissue, an observation that was repeated on several occasions. 
An mRNA of the same size was also observed in two lines of cultured rat 
cells derived from kidney (KNRK cells) and adrenal medulla (PC12 cells) 
(FIG. 19B). 
Northern RNA blot analysis with .sup.32 P-labelled probes derived from the 
.beta. subunit cDNA revealed a hybridising mRNA of .about.2.5 kb in all 
rat tissues examined except liver and spleen (FIG. 19C). Adequate amounts 
of mRNA from these tissues were applied to the filter as confirmed by 
hybridization with control probes for cyclophilin and 
glyceraldehyde-3-phosphate dehydrogenase. The brain and adrenal gland 
appeared to have somewhat more mRNA for farnesyltransferase .beta.-subunit 
than did the other tissues. More quantitative studies will be required to 
determine whether the variations shown in FIG. 19C are significant. 
The mRNA for the farnesyltransferase .beta.-subunit was also found in the 
two cultured rat cell lines from which cDNA sequences had been obtained 
(FIG. 19D). PC12 cells had the 2.5-kb transcript, whereas the KNRK cells 
had two transcripts, one of which was smaller than the 2.5-kb mRNA (FIG. 
19D). It was not determined whether the smaller transcript represented 
another gene product that cross-hybridized with the .beta.-subunit probe, 
or whether this mRNA represented alternative processing of an allelic 
transcript. 
d. Co-Expression and Stability 
The cDNA coding regions of both the .alpha. and .beta. subunits were cloned 
into pCMV mammalian expression vectors in either the correct or the 
reverse orientation. The cDNAs were introduced into human kidney 293 cells 
by calcium phosphate mediated transfection, and the proteins were detected 
by immunoblotting with specific antibodies against the .alpha. and .beta. 
subunits. In both cases, the cDNA directed the expression of proteins with 
molecular weights that were indistinguishable on immunoblots from those of 
the purified rat brain farnesyltransferase .alpha. and .beta. subunits 
(FIG. 20 and FIG. 20B). 
The accumulation of detectable amounts of .alpha. subunit required 
simultaneous transfection with a properly oriented cDNA encoding the 
.beta.-subunit (FIG. 20A). Similarly, the amount of detectable 
.beta.-subunit was increased by transfection with the .alpha. subunit cDNA 
in the correct orientation (FIG. 20B). Transfection with the two cDNAs in 
the correct orientation was also required in order to produce significant 
amounts of p21.sup.ras farnesyltransferase activity as measured in 
cytosolic extracts (FIG. 21). 
3. Discussion 
The delineation of the amino acid sequence of the .alpha. subunit has not 
yet allowed its catalytic role to be precisely identified. Homology 
searches of protein databases failed to reveal significant resemblance of 
the .alpha. subunit to other proteins except for proteins that contain 
long stretches of prolines. These include such apparently unrelated 
proteins as the catalytic subunits of rat and human protein phosphatase 
2B, mouse retinoblastoma-associated protein pp105, and Dictyostelium 
discoideum protein tyrosine kinase-1. The .alpha. subunit does not bear 
significant structural resemblance to known prenyltransferases such as 
mammalian farnesyl pyrophosphate synthetase or yeast hexaprenyl 
pyrophosphate synthetase. 
Present evidence suggests that the .alpha. subunit may be shared with 
another prenyltransferase with a different .beta. subunit that exhibits 
geranylgeranyltransferase activity (35). If the shared .alpha. subunit is 
stable only as a complex with one of several .beta. subunits, this 
mechanism would assure that cells maintain only enough .alpha. subunits to 
satisfy all of the .beta. subunits, thereby avoiding accumulation of 
excess .alpha. subunits, which might be toxic (36a). 
The above data reveal that the .alpha. and .beta. subunits of the rat 
farnesyltransferase do not exhibit farnesyltransferase activity when 
expressed by themselves in transfected human 293 cells. However, 
co-expression of the two subunits results in the production of an active 
enzyme. Such expression data provides support for the previous conclusion 
that the farnesyltransferase is a heterodimer that requires both the 
.alpha. and .beta. subunits for catalytic activity (35). 
Furthermore, the transfection experiments indicate that mammalian cells 
will not accumulate high levels of either subunit of the 
farnesyltransferase unless the other subunit is present. This is 
particularly true for the .alpha. subunit, whose accumulation was nearly 
completely dependent on co-expression of the .beta. subunit. It is likely 
that the .alpha. subunit is rapidly degraded unless the .beta. subunit is 
present. However, until pulse-chase labeling experiments are performed, 
the possibility of control at the level of mRNA stability or translation 
cannot be ruled out. 
The similarity between the rat .beta. subunit and the previously reported 
sequence of the yeast DPR1-RAM1 gene product (42) indicates that the 
latter is the yeast equivalent of the peptide-binding subunit of the 
mammalian farnesyltransferase. These findings confirm the previous 
suspicion that the yeast gene encodes one of the subunits of the 
farnesyltransferase and explains the failure of this protein to exhibit 
farnesyltransferase activity when expressed alone in E. coli (44,45). 
Mutations at a second locus, designated RAM2, also disrupt 
farnesyltransferase activity in yeast (44). The defect in the RAM2 cells 
is complemented by mating with the DPR1-RAM1 mutant. This finding suggests 
that the RAM2 gene product is the .alpha. subunit of the yeast 
farnesyltransferase. A more recent report of He et al (51) indicates that 
coexpression of the RAM1 and RAM2 genes in E. coli provided extracts that 
farnesylated synthetic a-factor substrate. However, when extracts from 
separate clones were mixed, only partial farnesyltransferase activity, on 
the order of about 3.5%, was recovered. 
An inspection of the conserved sequences in the rat .beta. subunit and the 
DPR1-RAM1 protein fails to reveal any obvious candidates for the peptide 
binding site. The rat protein does contain the sequence LXDDXXE (residues 
35-41), which resembles a sequence in four polyprenyl synthetases 
(I,L,orV)XDDXXD that is believed to be a prenyl pyrophosphate binding site 
(46). This sequence is not found in the same position in the DPR1-RAM1 
protein, and its significance in the .beta. subunit is uncertain. Although 
the farnesyltransferase reaction requires two divalent cations (Mg.sup.++ 
and Zn.sup.++), the sequence of the .beta. subunit does not reveal any 
obvious metal binding sites. 
Recently, the inventors have explored the separate catalytic roles of 
Zn.sup.2+ and Mg.sup.2+ and the specificity of the prenyl pyrophosphate 
binding site of the rat brain protein farnesyltransferase, using a 
purified enzyme preparation. In summary, it was found that the binding of 
p21.sup.H-ras to the enzyme was abolished by dialysis against EDTA and 
restored by addition of ZnCl.sub.2 as demonstrated by chemical 
crosslinking. The binding of the other substrate, all-trans farnesyl 
pyrophosphate, was independent of divalent cations, as demonstrated by gel 
filtration. Transfer of the enzyme-bound farnesyl group to the bound 
p21.sup.H-ras required Mg.sup.2+. Geranylgeranyl pyrophosphate bound to 
the prenyl pyrophosphate binding site with an affinity equal to that of 
farnesyl pyrophosphate, but the geranylgeranyl group was not transferred 
efficiently to P21.sup.H-ras. It also was not transferred to a modified 
p21.sup.H-ras containing COOH-terminal leucine, a protein that was shown 
previously to be a good substrate for a rat brain 
geranylgeranyltransferase (35). The inventors conclude that the protein 
farnesyltransferase is a metalloenzyme that most likely contains Zn.sup.2+ 
at the peptide-binding site. It thus resembles certain metallopeptidases, 
including carboxy-peptidase A and the angiotensin-converting enzyme. 
Strategies previously developed to screen for inhibitors of those enzymes 
will likely aid in the search for inhibitors of the protein 
farnesyltransferase. 
Thus, these data establish several new points about the enzymology of the 
protein farnesyltransferase from rat brain: 1) the enzyme contains a 
tightly bound divalent cation, most likely Zn.sup.2+, that can be removed 
by dialysis against EDTA; 2) Zn.sup.2+ is essential for binding of the 
peptide substrate, and therefore it is probably attached to the 
.beta.-subunit; 3) the enzyme binds FPP and GGPP with comparable 
affinities, but transfers only the farnesyl group and only to an acceptor 
whose CaaX sequence ends in methionine, serine, glutamine, or cysteine, 
but not leucine; 4) binding of prenyl pyrophosphates does not require any 
cation; and 5) transfer of the bound farnesyl group to the bound peptide 
acceptor requires Mg.sup.2+. 
The reaction sequence for the EDTA-treated protein farnesyltransferase is 
summarized graphically in FIG. 22. The EDTA-treated enzyme binds FPP 
without a requirement for prior Zn.sup.2+ binding. Peptide binding 
requires Zn.sup.2+, but is independent of FPP binding. After both 
substrates are bound, the transfer reaction occurs in a Mg.sup.2+ 
-dependent fashion. In the cell the enzyme is expected to be 
constitutively complexed with Zn.sup.2+. Under these conditions the 
mechanism is a simple random-ordered, two-substrate reaction in which the 
FPP and peptide acceptor can bind to the enzyme in any order. 
The requirement for Zn.sup.2+ in peptide binding is reminiscent of the 
requirement for Zn.sup.2+ in certain metallopeptidases, such as 
carboxypeptidase A (54). In this case the Zn.sup.2+ coordinates with the 
carbonyl and amino groups in the peptide bond that will be broken. In the 
farnesyltransferase the Zn.sup.2+ is likely to coordinate with the 
cysteine sulfhydryl group on the acceptor peptide. If this postulated 
mechanism is correct, inhibitors that mimic peptides that coordinate with 
Zn.sup.2+ might be effective inhibitors of the farnesyltransferase. This 
strategy would be very similar to the strategy followed in the design of 
inhibitors of the angiotensin-converting enzyme, a zinc metalloenzyme that 
is mechanistically similar to carboxypeptidase A (55). 
The ability of GGPP to compete with FPP for the prenyl pyrophosphate 
binding site on the protein farnesyl-transferase creates potential 
regulatory problems for the cell. If the intracellular concentrations of 
FPP and GGPP are similar, then the farnesyltransferase might be 
competitively inhibited at all times. It seems likely that the 
concentration of GGPP in the cell is lower than that of FPP. FPP is an 
intermediate in the synthesis of cholesterol, which is the bulk product of 
the pathway (56). GGPP, on the other hand, is not known to be converted 
into any other metabolites in animal cells, and indeed its existence in 
animal cells was not appreciated prior to the discovery of 
geranylgeranyl-modified proteins (57,58). Thus, it seems likely that cells 
avoid GGPP competition by maintaining the FPP concentration at a higher 
level than the GGPP concentration. 
If the .alpha.-subunit is involved in prenyl phrophosphate binding and if 
the .alpha.-subunit of the farnesyltransferase is identical to that of the 
leucine-recognizing geranyl-geranyltransferase, then the .alpha.-subunit 
must behave differently when it is part of the geranylgeranly-transferase. 
It seems unlikely that the geranylgeranyl-transferase would be inhibited 
by FPP because this would render the enzyme functionally inactive in the 
cell. Resolution of this issue will require the purification of the 
leucine-recognizing geranylgeranyltransferase and the determination as to 
whether its .alpha.-subunit is identical to, or merely similar to, the 
.alpha.-subunit of the farnesyltransferase. 
The binding of prenyl pyrophosphates to the farnesyltransferase appears to 
be independent of divalent cations. In this regard the farnesyltransferase 
resembles the prenyltransferase that catalyzes the condensation of 
isopentenyl pyrophosphate with allylic pyrophosphates to form FPP (59). 
The two enzymes also resemble each other in the requirement for a divalent 
cation (Mg.sup.2+ or Mn.sup.2+) in the transfer reaction. In studies not 
shown, it was found that Mn.sup.2+ will substitute for Mg.sup.2+ in the 
protein farnesyltransferase reaction. The two enzymes differ in that the 
FPP synthetase is a homodimer and it shows no requirement for Zn.sup.2+ 
(60). 
Turning to the issue of the yeast counterpart prenyl transferases, very 
recently two additional putative .beta. subunits of yeast 
prenyltransferases have been identified, BET2 (47) and CAL1 (48). Both 
sequences resemble the DPR1/RAM1 gene product and the .beta. subunit of 
the rat brain farnesyltransferase. A mutation in the BET2 gene prevents 
the membrane attachment of two small GTP binding proteins (YPT1 and SEC4) 
that direct vesicular traffic in the yeast secretory pathway (47). These 
proteins terminate in the sequence CC, which has recently been shown to be 
geranylgeranylated in animal cells (49). The second putative 
.beta.-subunit, encoded by the CAL1 gene, is necessary for yeast to 
control the cell cycle when deprived of calcium. Based on a genetic 
argument, it has been suggested that the targets for this 
prenyltransferase are two proteins that end in a Cys-X-X-Leu sequence and 
are believed to be geranylgeranylated (48). 
Considered together, the yeast and animal experiments suggest the existence 
of a family of closely related .beta. subunits that mediate peptide 
binding to a variety of prenyltransferases. Whether all of these enzymes 
have the same .alpha. subunit, or whether a family of such subunits also 
exists, remains to be determined. 
Example IV 
Recombinant Cloning of the Human Farnesyltransferase .alpha. and .beta. 
Subunit cDNAs 
The inventors have now succeeded in cloning the cDNAs for the human 
counterpart of both the .alpha. and .beta. subunits of the 
farnesyltransferase gene. This was carried out using standard molecular 
cloning techniques with the aid of the information gained from the rat 
farnesyltransferase gene disclosed above. 
To clone the human .alpha.-subunit cDNA, an M13 probe of 200 to 300 
nucleotides corresponding to the 5' end of the cDNA for the rat 
farnesyltransferase was used to screen a human retinal .lambda.gt10 cDNA 
library. Approximately 1.0.times.10.sup.6 plaques were screened, and 27 
positives were identified. Positive clones were analyzed by polymerase 
chain reaction (PCR), and the clone with the largest insert was purified 
and subcloned for DNA sequencing. The resulting nucleotide sequence, and 
corresponding deduced amino acid sequence, obtained for the human 
.alpha.-subunit is set forth as SEQ ID NO:6 and SEQ ID NO:5, respectively. 
To clone the human .beta.-subunit, a cDNA first strand synthesis of human 
prostate poly(A).sup.+ RNA was first carried out using standard 
procedures. This cDNA was then employed as a template in a PCR reaction 
using a primer developed from the rat .beta.-subunit sequence. The 
PCR-amplified product, which represented about 300 basepairs of the human 
farnesyltransferase .beta.-subunit was used as a probe to screen a human 
retinal .lambda.gt10 cDNA library. Approximately 1.5.times.10.sup.6 
plaques were screened, and 10 positive clones identified. Purified clones 
that hybridized to the probe were subcloned into M13 for DNA sequence 
analysis. The nucleotide sequence obtained for the human .beta.-subunit 
cDNA, and deduced amino acid sequence is set forth below as SEQ ID NO:8 
and SEQ ID NO:7, respectively. 
A comparison of the human nucleotide sequence to that of rat and yeast 
revealed the following: 
Percent Identical Nucleotides in Coding Region of cDNAs 
______________________________________ 
.alpha. Subunit 
.beta. Subunit 
______________________________________ 
Rat v. yeast 37.2 41.8 
Human v. yeast 35.9 37.3 
Human v. rat 88.2 90.1 
______________________________________ 
Example V 
NH.sub.2 -Terminal Positive Charge Create Pure Inhibitors 
The examples above demonstrate that protein farnesyltransferase transfers 
farnesyl residues to cysteine residues in tetrapeptides that conform to 
the sequence Cys-A.sub.1 -A.sub.2 -X, where A.sub.1 and A.sub.2 are 
aliphatic amino acids and X is methionine or serine. When the A.sub.2 
residue is aromatic (e.g. phenylalanine as in Cys-Val-Phe-Met), the 
tetrapeptide continues to bind to the enzyme, but it can no longer accept 
a farnesyl group, and it becomes a pure inhibitor. The studies of the 
present example demonstrate that this resistance to farnesylation also 
requires a positive charge on the cysteine NH.sub.2 -group. Derivatization 
of this group with acetyl, octanoyl, or cholic acid residues, or extension 
of the peptide with an additional amino acid, restores the ability of 
phenylalanine-containing peptides to accept a farnesyl residue. The same 
result was obtained when the NH.sub.2 -group of cysteine was deleted 
(mercaptopropionic acid-Val-Phe-Met). These data suggest that the positive 
change on the cysteine amino group acts in concert with an aromatic 
residue in the A.sub.2 position to render peptides resistant to 
farnesylation. Therefore, it can be concluded from these studies that a 
nonfarnesylated tetrapeptide inhibitor of this type must contain both an 
aromatic residue at the A.sub.2 position and a free NH.sub.2 -terminus. 
1. Methods 
a. Peptides 
Peptides were prepared by manual solid phase methodology (61) using either 
t-butyloxycarbonyl (Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc) 
chemistries, purified by reverse-phase high performance liquid 
chromatography (Amicon C18, 0.1% TFA/H.sub.2 O/MeCN or 10 mM TEAA/H.sub.2 
O/MeCN), and analyzed by fast atom bombardment mass spectrometry. Cholic 
acid (Aldrich) was activated using 
benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate 
(BOP) in N, N-dimethylacetamide. Octanoyl chloride was used to prepare the 
octanoyl peptide. Immediately before use, each peptide was dissolved at a 
concentration of 1 mM in dimethyl sulfoxide/10 mM dithiothreitol. All 
dilutions were made in water containing 10 mM dithiothreitol. 
b. Protein Farnesyltransferase 
Protein farnesyltransferase was purified to apparent homogeneity from rat 
brain homogenates by sequential ammonium sulfate fractionation, Mono Q 
ion-exchange chromatography, and peptide affinity chromatography as 
previously described (62,63). 
C. Transfer of [.sup.3 H]Farnesyl from [.sup.3 H]Farnesyl Pyrophosphate to 
Peptides 
Each 25-.mu.l reaction mixture contained the following concentrations of 
components: 50 mM Tris-chloride (pH 7.5), 50 .mu.M ZnCl.sub.2, 3 mM 
MgCl.sub.2, 20 mM KCl, 1 mM dithiothreitol, 0.2% (v/v) octyl 
.beta.-D-glucoside, either 0.6 or 2.4 .mu.M all-trans-[.sup.3 H]farnesyl 
pyrophosphate (8,000-16,000 dpm/pmol, Dupont-New England Nuclear), the 
indicated concentration of peptide, and .about.5 ng of affinity-purified 
protein farnesyltransferase. After incubation at 37.degree. C. for 15 or 
30 min, the reaction was stopped by addition of 2 .mu.l of 250 mM EDTA, 
and the entire reaction mixture was subjected to thin layer chromatography 
as previously described (64). The origin (2-cm strip) and 12 sequential 
1-cm fractions of each thin layer sheet were cut out and subjected to 
scintillation counting in 10 ml of 3a70B scintillation mixture (Research 
Products International). The amount of [.sup.3 H]farnesyl attached to 
peptide was calculated by summing the radioactivity in the peak fractions 
(typically fractions 10 to 12 or 9 to 11, depending on the peptide). Blank 
values were determined in parallel incubations that contained either no 
peptide or a tetrapeptide (SVIM) that is not a substrate for farnesylation 
(64). 
d. Assay for Protein Farnesyltransferase Activity 
The amount of [.sup.3 H]farnesyl transferred from all-trans-[.sup.3 
H]farnesyl pyrophosphate to recombinant p21.sup.H-ras was measured in a 
filter assay as previously described (62). 
2. Results 
As set forth in examples above, the attachment of [.sup.3 H]farnesyl to 
peptides is preferably measured by thin layer chromatography to determine 
which inhibitors were also good substrates for the enzyme. FIG. 23A and 
FIG. 23B compares two peptides, CVFM and N-AcCVFM, in their ability to 
inhibit the transfer of [.sup.3 H]farnesyl to p21.sup.H-ras produced in E. 
coli (FIG. 23A) and their ability to act as acceptors for [.sup.3 
H]farnesyl in a direct transfer assay (FIG. 23B) when incubated with 
purified protein farnesyltransferase isolated from rat brain. 
Incorporation of [.sup.3 H]farnesyl into p21.sup.H-ras was measured 
following precipitation with trichloroacetic acid. Both peptides inhibited 
farnesylation of p21.sup.H-ras with relatively high affinity. The 
concentrations giving 50% inhibition were 0.07 and 0.27 .mu.M for CVFM and 
N-AcCVFM, respectively. In the thin layer chromatography assay used in 
FIG. 23B, [.sup.3 H]farnesylated peptides migrate near the solvent front, 
and unincorporated [.sup.3 H]FPP remains near the origin (53). In the 
presence of CVIM nearly all of the .sup.3 H-radioactivity migrated with 
the peptide. As before, aromatic substitution at A.sub.2 (i.e., CVFM) 
blocked farnesyl transfer. Acetylation of the NH.sub.2 -terminus (i.e., 
N-AcCVFM) restored farnesylation of this potent inhibitor (FIG. 23B). 
FIG. 24A and FIG. 24B shows that the same discrepancy in inhibitory 
activity and acceptor function was also seen for CIFM and N-AcCIFM, which 
differ from the peptides in FIG. 23A and FIG. 23B by the substitution of 
isoleucine for valine at the A.sub.1 position. Again, the two peptides 
inhibited farnesylation of p21.sup.H-ras, but only the acetylated peptide 
(which was the less potent inhibitor of farnesylation) was farnesylated. 
It is interesting that two spatially separate domains of the peptide, the 
NH.sub.2 -terminus and A.sub.2 aromatic side chain, together block 
farnesyl transfer, while either alone yields fully functional substrate. 
Also, the data in Table III (see below) show little correlation between 
inhibitor potency and substrate activity, indicating that peptide binding 
and farnesyltransferase activity are defined by distinct peptide-protein 
interactions. 
Table III compares the farnesyl acceptor activity and the 
farnesyltransferase inhibitory activity of a series of N-modified peptides 
at a high peptide concentration (3.6 .mu.M). The assays were performed on 
several occasions with different preparations of purified 
farnesyltransferase. In order to standardize the results, in each study 
the farnesylation of a standard peptide CVIM (which corresponds to the 
COOH-terminus of p21.sup.K-rasB) was measured and the results expressed as 
a ratio of [.sup.3 H]farnesyl incorporated into the test peptide vs. the 
standard peptide. The data show that the ability of N-acetylation to 
restore farnesylation was not restricted to peptides containing 
phenylalanine. The same type of result was obtained with tryptophan (CVWM 
and N-AcCVWM). Moreover, the N-substituent was not restricted to the 
acetyl group. A similar result was obtained when the substituent on CVFM 
was an octanoyl or a cholic acid residue. Farnesylation was also enabled 
when the N-substituent was an amino acid, creating a pentapeptide such as 
KCVFM or CCVFM. The N-acetylated derivatives of these and other 
pentapeptides that contained CVFM were also farnesylated. 
TABLE III 
______________________________________ 
Interaction of N-Modified Peptides 
With Protein Farnesyltransferase 
Concentration 
Ratio of for 50% 
[.sup.3 H]Farnesyl 
Inhibition of 
Incorporated into 
Farnesylation of 
Peptide Peptide/CVIM 
p21.sup.H-ras (.mu.M) 
______________________________________ 
CVIM 1.0 0.15 
N-AcCVIM 1.8 0.15 
CVWM 0 0.32 
N-AcCVWM 1.2 3.7 
CVFM 0 0.06* 
N-CholylCVFM 0.85 0.21 
N-AcCVFM 1.1 0.25 
N-Octanoyl-CVFM 
2.1 0.35 
N-AcHCVFM 0.96 0.24 
N-AcCCVFM 1.2 0.11 
N-AcDCVFM 1.3 1.9 
N-AcECVFM 1.4 2.4 
N-AcPCVFM 1.4 0.20 
N-AcGCVFM 1.9 0.43 
N-AcSCVFM 2.0 0.23 
N-AcMCVFM 2.2 1.4 
N-AcKCVFM 2.8 0.30 
KCVFM 0.77 1.1 
CCVFM 1.5 0.44 
______________________________________ 
Each peptide was incubated at a concentration of 3.6 .mu.M with purified 
farnesyltransferase, and the incorporated radioactivity was determined as 
described above. For purposes of standardization, the data are expressed 
as the ratio of incorporation of [.sup.3 H]farnesyl from [.sup.3 
H]farnesyl pyrophosphate into each tetrapeptides divided by the 
incorporation into CVIM, which was measured in each experiment. #The 
values for 50% inhibition of the farnesylation of p21.sup.Hras were 
obtained from experiments in which each peptide was tested at six 
concentration ranging from 0.03 to 10 .mu.M. 
*Mean of 9 consecutive experiments over a 3month period in which the 50% 
inhibition values ranged from 0.032 to 0.09 .mu.M. 
FIG. 25 shows that a phenylalanine-containing tetrapeptide with a free 
NH.sub.2 -group (CVFM) inhibited the farnesylation of an N-substituted 
peptide (N-OctanoylCVFM), further confirming the effect of N-modification 
on the farnesylation reaction. The result also suggests that CVFM and its 
N-substituted derivative interact with the same binding site on the 
enzyme. 
3. Discussion 
The foregoing examples demonstrate that peptides that contain aromatic 
residues in the A.sub.2 position of the CA.sub.1 A.sub.2 X sequence 
inhibit farnesylation of p21.sup.H-ras without themselves becoming 
farnesylated by the enzyme. In the present example it is shown that the 
resistance of these peptides to farnesylation also depends upon the 
presence of a free NH.sub.2 -terminus on cysteine. Substitution of this 
NH.sub.2 -group with acyl (acetyl or octanoyl) or amino acid residues 
allows the peptide to become a substrate for farnesylation. 
The farnesyltransferase enzyme displays two rather remarkable 
specificities. First, the wide variation in inhibitory activity over the 
range of sequences tested denotes a precise peptide recognition domain. 
Second, the A.sub.2 aromatic group and the NH.sub.2 -terminus must act in 
concert to disrupt farnesyl transfer. Separately, peptides with either of 
these moieties are well tolerated as substrates. Further modification at 
these two sites of the peptide may allow one to separately probe the 
structural requirements for enzyme binding and farnesyl transfer. 
It is likely that the resistance of the aromatic-containing peptides to 
farnesylation requires a positive charge at the NH.sub.2 -terminus. All of 
the modifications that restore farnesylation also remove the positive 
charge on this nitrogen. This interpretation is also consistent with a 
previous result in which a phenylalanine-containing peptide that lacks a 
primary NH.sub.2 -group (mercaptopropionic acid-VFM) was farnesylated. 
The mechanism whereby the positively-charged phenylalanine-containing 
peptides resist farnesylation is not known. One possibility is that the 
binding of a peptide with aromatic residues in the A.sub.2 position causes 
a conformational change in the enzyme that places the charged NH.sub.2 
-terminus in position to disrupt transfer of the farnesyl residue. 
Alternatively, it is possible that the peptides are farnesylated, but that 
they dissociate slowly from the enzyme, thereby preventing repeated cycles 
of farnesylation. These possibilities should be distinguishable by careful 
kinetic studies with the purified farnesyltransferase. 
In a practical sense the current findings raise questions regarding the use 
of aromatic-containing peptides such as CVFM to inhibit 
farnesyltransferase in intact cells. Non-substrate peptide inhibitors may 
be preferable for delivery into cells, and the requirement for a charged 
NH.sub.2 -terminus may retard passage of the peptide through the cell 
membrane. It may be necessary to mask this charge with a substituent that 
will be cleaved within the cell. Considering the ubiquity of esterases and 
amidases, this goal should be attainable. 
While the compositions and methods of this invention have been described in 
terms of preferred embodiments, it will be apparent to those of skill in 
the art that variations may be applied to the composition, methods and in 
the steps or in the sequence of steps of the method described herein 
without departing from the concept, spirit and scope of the invention. 
More specifically, it will be apparent that certain agents which are both 
chemically and physiologically related may be substituted for the agents 
described herein while the same or similar results would be achieved. All 
such similar substitutes and modifications apparent to those skilled in 
the art are deemed to be within the spirit, scope and concept of the 
invention as defined by the appended claims. 
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Goldstein, J. L., and Brown, M. S. (1991), J. Biol. Chem., 266, 
10672-10677. 
51. He, B., Chen, P., Chen, S. Y., Vancura, K. L., Michaelis, S., and 
Powers, S. (1991), Proc. Natl. Acad. Sci. USA, 88, 11373-11377. 
52. Kohl, N. E., Diehl, R. E., Schaber, M. D., Rands, E., Soderman, D. D., 
He, B., Moores, S. L., Pompliano, D. L., Ferro-Novick, S., Powers, S., 
Thomas, K. A., Gibbs, J. B. (1991), J. Biol. Chem., 266, 18884-18888. 
53. Goldstein, J. L., Brown, M. S., Stradley, S. J., Reiss, Y., and 
Gierasch, L. M. (1991), J. Biol Chem., 266, 15575-15578. 
54. Lipscomb, W. N. (1974), Tetrahedron, 30, 1725-1732. 
55. Petrillo, E. W. Jr., Ondetti, M. A. (1982), Medicinal Res. Rev., 2, 
1-41. 
56. Goldstein, J. L. and Brown, M. S. (1990), Nature, 343, 425-430. 
57. Farnsworth, C. C., Gelb, M. H., Glomset, J. A. (1990), Science, 247, 
320-322. 
58. Rilling, H. C., Breunger, E., Epstein, W. W., and Crain, P. F. (1990), 
Science, 247, 318-320. 
59. King, H. L. and Rilling, H. C. (1977), Biochemistry, 16, 3815-3819. 
60. Rilling, H. C. (1985), Meth. Enzymol., 110, 145-152. 
61. Barany, G. & Merrifield, R. B. (1980) in The Peptides, eds. Gross, E. & 
Meienhofer, J. (Academic Press, New York), pp. 1-284. 
62. Reiss, Y., Goldstein, J. L., Seabra, M. C., Casey, P. J. & Brown, M. S. 
(1990) Cell 62, 81-88. 
63. Reiss, Y., Seabra, M. C., Goldstein, J. L. & Brown, M. S. (1990) 
Methods: A Companion to Methods in Enzymology 1, 241-245. 
64. Nagasawa, H. T., Goon, D. J., Muldoon, W. P., and Zera, R. T. (1984) J. 
Med. Chem. 27, 591-596; Nagasawa, H. T., Goon, D. J., and Zera, R. T. 
(1982) J. Med. Chem. 25, 489-491. 
65. Hazelton, G. A., Hjelle, J. J., and Klaassen, C. D. (1986) J. Pharm. 
Exp. Therapeutics 237, 341-349. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 8 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
# 377 amino acidsH: 
# amino acidPE: 
(C) STRANDEDNESS: sing - #le 
# linear (D) TOPOLOGY: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- Met Ala Ala Thr Glu Gly Val Gly Glu Ser Al - #a Pro Gly Gly Glu Pro 
# 15 
- Gly Gln Pro Glu Gln Pro Pro Pro Pro Pro Pr - #o Pro Pro Pro Ala Gln 
# 30 
- Gln Pro Gln Glu Glu Glu Met Ala Ala Glu Al - #a Gly Glu Ala Ala Ala 
# 45 
- Ser Pro Met Asp Asp Gly Phe Leu Ser Leu As - #p Ser Pro Thr Tyr Val 
# 60 
- Leu Tyr Arg Asp Arg Ala Glu Trp Ala Asp Il - #e Asp Pro Val Pro Gln 
# 80 
- Asn Asp Gly Pro Ser Pro Val Val Gln Ile Il - #e Tyr Ser Glu Lys Phe 
# 95 
- Arg Asp Val Tyr Asp Tyr Phe Arg Ala Val Le - #u Gln Arg Asp Glu Arg 
# 110 
- Ser Glu Arg Ala Phe Lys Leu Thr Arg Asp Al - #a Ile Glu Leu Asn Ala 
# 125 
- Ala Asn Tyr Thr Val Trp His Phe Arg Arg Va - #l Leu Leu Arg Ser Leu 
# 140 
- Gln Lys Asp Leu Gln Glu Glu Met Asn Tyr Il - #e Ile Ala Ile Ile Glu 
145 1 - #50 1 - #55 1 - 
#60 
- Glu Gln Pro Lys Asn Tyr Gln Val Trp His Hi - #s Arg Arg Val Leu Val 
# 175 
- Glu Trp Leu Lys Asp Pro Ser Gln Glu Leu Gl - #u Phe Ile Ala Asp Ile 
# 190 
- Leu Asn Gln Asp Ala Lys Asn Tyr His Ala Tr - #p Gln His Arg Gln Trp 
# 205 
- Val Ile Gln Glu Phe Arg Leu Trp Asp Asn Gl - #u Leu Gln Tyr Val Asp 
# 220 
- Gln Leu Leu Lys Glu Asp Val Arg Asn Asn Se - #r Val Trp Asn Gln Arg 
225 2 - #30 2 - #35 2 - 
#40 
- His Phe Val Ile Ser Asn Thr Thr Gly Tyr Se - #r Asp Arg Ala Val Leu 
# 255 
- Glu Arg Glu Val Gln Tyr Thr Leu Glu Met Il - #e Lys Leu Val Pro His 
# 270 
- Asn Glu Ser Ala Trp Asn Tyr Leu Lys Gly Il - #e Leu Gln Asp Arg Gly 
# 285 
- Leu Ser Arg Tyr Pro Asn Leu Leu Asn Gln Le - #u Leu Asp Leu Gln Pro 
# 300 
- Ser His Ser Ser Pro Tyr Leu Ile Ala Phe Le - #u Val Asp Ile Tyr Glu 
305 3 - #10 3 - #15 3 - 
#20 
- Asp Met Leu Glu Asn Gln Cys Asp Asn Lys Gl - #u Asp Ile Leu Asn Lys 
# 335 
- Ala Leu Glu Leu Cys Glu Ile Leu Ala Lys Gl - #u Lys Asp Thr Ile Arg 
# 350 
- Lys Glu Tyr Trp Arg Tyr Ile Gly Arg Ser Le - #u Gln Ser Lys His Ser 
# 365 
- Arg Glu Ser Asp Ile Pro Ala Ser Val 
# 375 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
# 1701 base pairsH: 
# nucleic acid: 
(C) STRANDEDNESS: sing - #le 
# linear (D) TOPOLOGY: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2: 
- GCGGGCCGCG GAGGGGGCGG GGCTCCACCA CCACCTCAGC TGCGGACGGA GG - #CGAGATGG 
60 
- CGGCCACTGA GGGGGTCGGG GAATCTGCGC CAGGCGGTGA GCCGGGACAG CC - #AGAGCAGC 
120 
- CGCCGCCCCC GCCTCCTCCG CCGCCAGCAC AGCAGCCGCA GGAAGAAGAG AT - #GGCGGCCG 
180 
- AGGCCGGGGA AGCAGCGGCG TCCCCTATGG ACGACGGGTT TCTGAGCCTG GA - #CTCGCCCA 
240 
- CCTATGTCTT GTACAGGGAC AGGGCAGAGT GGGCTGACAT AGACCCAGTG CC - #CCAGAATG 
300 
- ATGGCCCCAG TCCAGTGGTC CAGATCATCT ACAGTGAAAA GTTTAGAGAC GT - #CTATGATT 
360 
- ACTTCCGAGC TGTTCTGCAG CGCGATGAAA GAAGCGAACG AGCCTTTAAG CT - #CACTCGAG 
420 
- ATGCTATTGA GTTAAACGCA GCCAACTATA CGGTGTGGCA TTTTCGGAGA GT - #TCTCTTGA 
480 
- GGTCGCTTCA GAAGGATCTG CAAGAAGAAA TGAACTACAT CATCGCAATA AT - #TGAGGAAC 
540 
- AGCCCAAAAA CTATCAAGTT TGGCACCATA GGAGAGTATT AGTGGAGTGG CT - #GAAAGATC 
600 
- CTTCTCAAGA GCTCGAGTTC ATCGCCGATA TCCTTAATCA GGATGCAAAG AA - #TTACCATG 
660 
- CCTGGCAGCA TCGACAGTGG GTCATTCAGG AGTTTCGACT TTGGGATAAT GA - #GCTGCAGT 
720 
- ATGTGGACCA GCTTCTCAAA GAGGATGTGA GAAATAACTC TGTGTGGAAC CA - #AAGACACT 
780 
- TCGTCATTTC TAATACCACT GGCTACAGTG ATCGCGCTGT GTTGGAGAGA GA - #AGTCCAAT 
840 
- ATACTCTGGA AATGATCAAA TTAGTGCCAC ACAATGAGAG TGCGTGGAAC TA - #CTTGAAAG 
900 
- GGATTTTGCA GGACCGTGGT CTTTCCAGAT ACCCTAATCT ATTAAACCAG TT - #GCTTGATT 
960 
- TACAACCAAG TCACAGCTCC CCCTACCTAA TTGCCTTTCT TGTGGATATC TA - #TGAAGACA 
1020 
- TGCTGGAAAA CCAGTGTGAC AACAAGGAGG ACATTCTTAA TAAAGCACTA GA - #GTTATGTG 
1080 
- AGATTCTAGC TAAAGAAAAG GACACTATAA GAAAGGAATA TTGGAGATAT AT - #TGGACGGT 
1140 
- CCCTCCAGAG TAAACACAGC AGAGAAAGTG ACATACCGGC GAGTGTATAG CA - #GCAAGAGC 
1200 
- GGCTGGAAGA AGTGGACAAT GCTTTCTAAG GCCTCTTATT CGGGAGTGTA GA - #GCGGTTAG 
1260 
- AGCGGTCATC TCATGCCTGT GAGCTAACGT TGTCCAGGTG CTGTTTCTAA CA - #AGAACTAA 
1320 
- GGATGACTCC TGTGTCTGAC GCTGTTCAGA CTAGCTAAGA GTCGATTTCC TA - #AAGCAAGG 
1380 
- TCATTGGAGG GGAGGGTGAA GAAAACTTTC CCGTAAAGGA ACTACTGCTT TT - #GTAGTCTT 
1440 
- CCCAACATTT AATCCAATCC TGTAGAATCA GCATCTCCTG AAAGACATGG TG - #CACGGCTG 
1500 
- TGTGCTGGGC GTGGCTAAGG GCAGCTGTGT CATGGGTTTG CAGTCATGGG AA - #CCTCGGAG 
1560 
- CGCTGCCCGG GACTGCATTG ATGATTAGGG CTGCTGGCCT CACCCACAGG AT - #CTTGCTAT 
1620 
- CACTGTAACC AACTAATGCC AAAAGAAAGG TTTTATAATA AAATCACATT AT - #CTACAAAC 
1680 
# 1701AA A 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
# 437 amino acid residues 
# amino acidPE: 
(C) STRANDEDNESS: sing - #le 
# linear (D) TOPOLOGY: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3: 
- Met Ala Ser Ser Ser Ser Phe Thr Tyr Tyr Cy - #s Pro Pro Ser Ser Ser 
# 15 
- Pro Val Trp Ser Glu Pro Leu Tyr Ser Leu Ar - #g Pro Glu His Ala Arg 
# 30 
- Glu Arg Leu Gln Asp Asp Ser Val Glu Thr Va - #l Thr Ser Ile Glu Gln 
# 45 
- Ala Lys Val Glu Glu Lys Ile Gln Glu Val Ph - #e Ser Ser Tyr Lys Phe 
# 60 
- Asn His Leu Val Pro Arg Leu Val Leu Gln Ar - #g Glu Lys His Phe His 
# 80 
- Tyr Leu Lys Arg Gly Leu Arg Gln Leu Thr As - #p Ala Tyr Glu Cys Leu 
# 95 
- Asp Ala Ser Arg Pro Trp Leu Cys Tyr Trp Il - #e Leu His Ser Leu Glu 
# 110 
- Leu Leu Asp Glu Pro Ile Pro Gln Ile Val Al - #a Thr Asp Val Cys Gln 
# 125 
- Phe Leu Glu Leu Cys Gln Ser Pro Asp Gly Gl - #y Phe Gly Gly Gly Pro 
# 140 
- Gly Gln Tyr Pro His Leu Ala Pro Thr Tyr Al - #a Ala Val Asn Ala Leu 
145 1 - #50 1 - #55 1 - 
#60 
- Cys Ile Ile Gly Thr Glu Glu Ala Tyr Asn Va - #l Ile Asn Arg Glu Lys 
# 175 
- Leu Leu Gln Tyr Leu Tyr Ser Leu Lys Gln Pr - #o Asp Gly Ser Phe Leu 
# 190 
- Met His Val Gly Gly Glu Val Asp Val Arg Se - #r Ala Tyr Cys Ala Ala 
# 205 
- Ser Val Ala Ser Leu Thr Asn Ile Ile Thr Pr - #o Asp Leu Phe Glu Gly 
# 220 
- Thr Ala Glu Trp Ile Ala Arg Cys Gln Asn Tr - #p Glu Gly Gly Ile Gly 
225 2 - #30 2 - #35 2 - 
#40 
- Gly Val Pro Gly Met Glu Ala His Gly Gly Ty - #r Thr Phe Cys Gly Leu 
# 255 
- Ala Ala Leu Val Ile Leu Lys Lys Glu Arg Se - #r Leu Asn Leu Lys Ser 
# 270 
- Leu Leu Gln Trp Val Thr Ser Arg Gln Met Ar - #g Phe Glu Gly Gly Phe 
# 285 
- Gln Gly Arg Cys Asn Lys Leu Val Asp Gly Cy - #s Tyr Ser Phe Trp Gln 
# 300 
- Ala Gly Leu Leu Pro Leu Leu His Arg Ala Le - #u His Ala Gln Gly Asp 
305 3 - #10 3 - #15 3 - 
#20 
- Pro Ala Leu Ser Met Ser His Trp Met Phe Hi - #s Gln Gln Ala Leu Gln 
# 335 
- Glu Tyr Ile Leu Met Cys Cys Gln Cys Pro Al - #a Gly Gly Leu Leu Asp 
# 350 
- Lys Pro Gly Lys Ser Arg Asp Phe Tyr His Th - #r Cys Tyr Cys Leu Ser 
# 365 
- Gly Leu Ser Ile Ala Gln His Phe Gly Ser Gl - #y Ala Met Leu His Asp 
# 380 
- Val Val Met Gly Val Pro Glu Asn Val Leu Gl - #n Pro Thr His Pro Val 
385 3 - #90 3 - #95 4 - 
#00 
- Tyr Asn Ile Gly Pro Asp Lys Val Ile Gln Al - #a Thr Thr His Phe Leu 
# 415 
- Gln Lys Pro Val Pro Gly Phe Glu Glu Cys Gl - #u Asp Ala Val Thr Ser 
# 430 
- Asp Pro Ala Thr Asp 
435 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
# 2464 base pairsH: 
# nucleic acid: 
(C) STRANDEDNESS: sing - #le 
# linear (D) TOPOLOGY: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4: 
- CGGGCGCGTT GTTGCTGGAC GAAGCTGAGT CCTATACAGC GCTCGCAGCT CT - #CCCGATCA 
60 
- TGGCTTCTTC GAGTTCCTTC ACCTATTATT GTCCTCCATC TTCTTCCCCT GT - #TTGGTCAG 
120 
- AACCGCTGTA TAGTCTGAGA CCTGAGCACG CGCGGGAGCG GTTGCAAGAC GA - #CTCAGTGG 
180 
- AAACAGTCAC GTCCATAGAA CAGGCCAAAG TAGAAGAAAA GATCCAGGAG GT - #CTTCAGTT 
240 
- CTTACAAGTT TAACCACCTC GTACCAAGGC TCGTTCTGCA GAGGGAGAAG CA - #CTTCCATT 
300 
- ATCTGAAAAG AGGCCTTCGA CAACTGACAG ATGCCTATGA GTGTCTGGAT GC - #CAGCCGCC 
360 
- CCTGGCTCTG CTACTGGATC CTGCACAGCT TGGAGCTCCT CGACGAACCC AT - #CCCCCAAA 
420 
- TAGTGGCTAC AGATGTGTGT CAGTTCTTGG AGCTGTGTCA GAGTCCAGAC GG - #TGGCTTTG 
480 
- GAGGGGGCCC TGGTCAGTAC CCACACCTCG CTCCCACGTA TGCAGCTGTC AA - #CGCGCTAT 
540 
- GCATCATTGG CACGGAGGAA GCCTACAACG TCATTAACAG AGAGAAGCTC CT - #TCAGTACT 
600 
- TGTACTCCCT AAAGCAACCG GATGGCTCTT TTCTCATGCA CGTCGGAGGA GA - #GGTGGATG 
660 
- TAAGAAGTGC GTACTGTGCT GCCTCAGTAG CCTCTCTCAC CAACATCATC AC - #TCCTGACC 
720 
- TCTTCGAAGG CACTGCTGAA TGGATAGCAA GGTGCCAGAA CTGGGAAGGC GG - #CATTGGCG 
780 
- GGGTGCCAGG GATGGAAGCC CACGGTGGCT ACACCTTCTG TGGCTTGGCT GC - #GCTGGTGA 
840 
- TCCTCAAGAA GGAACGTTCT TTGAACCTGA AGAGCTTGCT ACAATGGGTG AC - #AAGCCGGC 
900 
- AGATGCGGTT CGAAGGAGGA TTTCAGGGCC GCTGCAACAA GCTGGTGGAC GG - #CTGCTACT 
960 
- CCTTCTGGCA GGCAGGACTT CTGCCCCTGT TGCACCGGGC ACTCCACGCT CA - #AGGTGACC 
1020 
- CTGCCCTCAG CATGAGCCAC TGGATGTTCC ATCAGCAGGC GCTGCAGGAG TA - #CATCCTCA 
1080 
- TGTGCTGCCA GTGTCCGGCT GGGGGTCTCC TGGACAAACC TGGCAAGTCA CG - #TGACTTCT 
1140 
- ACCATACTTG CTACTGCCTG AGCGGCCTGT CCATTGCCCA GCATTTTGGA AG - #TGGAGCCA 
1200 
- TGCTGCACGA TGTGGTCATG GGTGTGCCTG AAAATGTTCT GCAGCCCACT CA - #CCCTGTGT 
1260 
- ACAACATCGG ACCTGATAAG GTGATCCAGG CCACCACACA CTTTCTGCAG AA - #GCCGGTCC 
1320 
- CAGGCTTTGA GGAATGCGAA GATGCGGTGA CCTCAGATCC TGCCACTGAC TA - #GAGGACCC 
1380 
- CATGGCTCCC CCAAATCCCC CGTCAGACAA GGTTTCTCCG TTTGGGTACA TA - #GCACAGTC 
1440 
- CGTGCTACTT GAGCCTTGGC CACTGTGGAG TTGTGGTTTC TTTGTCCTTT CC - #TGTCAAAC 
1500 
- AAAACAAAGC CATCAGCTCT GGGTTGGAAT ACACAATGGT GTGATTTTTA AA - #ATTATTTT 
1560 
- CATACCTGTC AAACCAAAAC TCTGGGAGCC GATGTAGTAA GCAGGGTTGG AG - #AGCAATGC 
1620 
- ATGCTGGGAA GCAGCAGCCT CCTCCAGCAG CCAGGCCCAC AATGCTGAAA TG - #GAAGGTGT 
1680 
- CTGTGAGTAT CTCCACATCA CAGCCACTGC TGTGCCTCCC ACCTACACAC CA - #TTCAGTCA 
1740 
- GCAGATGGGC TCCTCTCTGG TATAAATGTC AGCTCTGTGC AAGGGCGGCG CT - #GTGGGTCC 
1800 
- AGCCAATACA CGCTCTCTGG AAAACAGCAC TGGGCTCCAG TGGGCATATT CA - #TACTTGTC 
1860 
- TCTTTTACCC CAGTCATTTG CGAAGGACAG GGGCCAGGAA TGAAGAAGGG TC - #TTAGATTG 
1920 
- AGCCCTCCCC ACAACCTGAG GAGAACTGAT CTCATATTTC TCCAAGGCCA TG - #TTTGTATG 
1980 
- AGCAAGACTT GTTTTGCCCT AAGTATGACG ACTAGACCCA GGTAATCAAT TA - #TGAGTGGA 
2040 
- AAATCAACCT CTAGGTGAAC TCTGTGCCAG AGGAAGCAGC CTCCCCAGTG TC - #CAGCCCCC 
2100 
- GCCTCTCCCC ATCATGTACC AGGAGAGGCC CTCCTCACGG CAGTGCTGCA GC - #CCAGGCTC 
2160 
- CTTCTAGTCC TTTCTCCCCA CCCACCCTCC AAGACAGTGC TCTTTTCTCA TC - #CAGGGTGT 
2220 
- TAACACTACT AAGGCTTCAC CGTAATCGAT CACTCAGGAT TTACTCCTGC CC - #TGCCCACT 
2280 
- CCAGGCTCTC TGAAACACAG TCAAGTGCTA GGCAAGCTAG CTGCTGCTGG GA - #CAGTGACC 
2340 
- AGCAGGAAGG CAAGTGCTGT CCGTGTGCTG AATTCTGGAA CTGCCTCTGC AC - #CGGCTGAG 
2400 
- TTTGCTCACC TATCCACTGC TACAGTCATA GCAAGCTCAT GCCGCTGTCC CA - #GCCTGTGC 
2460 
# 2464 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
# 379 amino acid residues 
# amino acidPE: 
(C) STRANDEDNESS: sing - #le 
# linear (D) TOPOLOGY: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5: 
- Met Ala Ala Thr Glu Gly Val Gly Glu Ala Al - #a Gln Gly Gly Glu Pro 
# 15 
- Gly Gln Pro Ala Gln Pro Pro Pro Gln Pro Hi - #s Pro Pro Pro Pro Gln 
# 30 
- Gln Gln His Lys Glu Glu Met Ala Ala Glu Al - #a Gly Glu Ala Val Ala 
# 45 
- Ser Pro Met Asp Asp Gly Phe Val Ser Leu As - #p Ser Pro Ser Tyr Val 
# 60 
- Leu Tyr Arg Asp Arg Ala Glu Trp Ala Asp Il - #e Asp Pro Val Pro Gln 
#80 
- Asn Asp Gly Pro Asn Pro Val Val Gln Ile Il - #e Tyr Ser Asp Lys Phe 
# 95 
- Arg Asp Val Tyr Asp Tyr Phe Arg Ala Val Le - #u Gln Arg Asp Glu Arg 
# 110 
- Ser Glu Arg Ala Phe Lys Leu Thr Arg Asp Al - #a Ile Glu Leu Asn Ala 
# 125 
- Ala Asn Tyr Thr Val Trp His Phe Arg Arg Va - #l Leu Leu Lys Ser Leu 
# 140 
- Gln Lys Asp Leu His Glu Glu Met Asn Tyr Il - #e Thr Ala Ile Ile Glu 
145 1 - #50 1 - #55 1 - 
#60 
- Glu Gln Pro Lys Asn Tyr Gln Val Trp His Hi - #s Arg Arg Val Leu Val 
# 175 
- Glu Trp Leu Arg Asp Pro Ser Gln Glu Leu Gl - #u Phe Ile Ala Asp Ile 
# 190 
- Leu Asn Gln Asp Ala Lys Asn Tyr His Ala Tr - #p Gln His Arg Gln Trp 
# 205 
- Val Ile Gln Glu Phe Lys Leu Trp Asp Asn Gl - #u Leu Gln Tyr Val Asp 
# 220 
- Gln Leu Leu Lys Glu Asp Val Arg Asn Asn Se - #r Val Trp Asn Gln Arg 
225 2 - #30 2 - #35 2 - 
#40 
- Tyr Phe Val Ile Ser Asn Thr Thr Gly Tyr As - #n Asp Arg Ala Val Leu 
# 255 
- Glu Arg Glu Val Gln Tyr Thr Leu Glu Met Il - #e Lys Leu Val Pro His 
# 270 
- Asn Glu Ser Ala Trp Asn Tyr Leu Lys Gly Il - #e Leu Gln Asp Arg Gly 
# 285 
- Leu Ser Lys Tyr Pro Asn Leu Leu Asn Gln Le - #u Leu Asp Leu Gln Pro 
# 300 
- Ser His Ser Ser Pro Tyr Leu Ile Ala Phe Le - #u Val Asp Ile Tyr Glu 
305 3 - #10 3 - #15 3 - 
#20 
- Asp Met Leu Glu Asn Gln Cys Asp Asn Lys Gl - #u Asp Ile Leu Asn Lys 
# 335 
- Ala Leu Glu Leu Cys Glu Ile Leu Ala Lys Gl - #u Lys Asp Thr Ile Arg 
# 350 
- Lys Glu Tyr Trp Arg Tyr Ile Gly Arg Ser Le - #u Gln Ser Lys His Ser 
# 365 
- Thr Glu Asn Asp Ser Pro Thr Asn Val Gln Gl - #n 
# 375 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
# 1664 base pairsH: 
# nucleic acid: 
(C) STRANDEDNESS: sing - #le 
# linear (D) TOPOLOGY: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6: 
- ATGGCGGCCA CCGAGGGGGT CGGGGAGGCT GCGCAAGGGG GCGAGCCCGG GC - #AGCCGGCG 
60 
- CAACCCCCGC CCCAGCCGCA CCCACCGCCG CCCCAGCAGC AGCACAAGGA AG - #AGATGGCG 
120 
- GCCGAGGCTG GGGAAGCCGT GGCGTCCCCC ATGGACGACG GGTTTGTGAG CC - #TGGACTCG 
180 
- CCCTCCTATG TCCTGTACAG GGACAGAGCA GAATGGGCTG ATATAGATCC GG - #TGCCGCAG 
240 
- AATGATGGCC CCAATCCCGT GGTCCAGATC ATTTATAGTG ACAAATTTAG AG - #ATGTTTAT 
300 
- GATTACTTCC GAGCTGTCCT GCAGCGTGAT GAAAGAAGTG AACGAGCTTT TA - #AGCTAACC 
360 
- CGGGATGCTA TTGAGTTAAA TGCAGCCAAT TATACAGTGT GGCATTTCCG GA - #GAGTTCTT 
420 
- TTGAAGTCAC TTCAGAAGGA TCTACATGAG GAAATGAACT ACATCACTGC AA - #TAATTGAG 
480 
- GAGCAGCCCA AAAACTATCA AGTTTGGCAT CATAGGCGAG TATTAGTGGA AT - #GGCTAAGA 
540 
- GATCCATCTC AGGAGCTTGA ATTTATTGCT GATATTCTTA ATCAGGATGC AA - #AGAATTAT 
600 
- CATGCCTGGC AGCATCGACA ATGGGTTATT CAGGAATTTA AACTTTGGGA TA - #ATGAGCTG 
660 
- CAGTATGTGG ACCAACTTCT GAAAGAGGAT GTGAGAAATA ACTCTGTCTG GA - #ACCAAAGA 
720 
- TACTTCGTTA TTTCTAACAC CACTGGCTAC AATGATCGTG CTGTATTGGA GA - #GAGAAGTC 
780 
- CAATACACTC TGGAAATGAT TAAACTAGTA CCACATAATG AAAGTGCATG GA - #ACTATTTG 
840 
- AAAGGGATTT TGCAGGATCG TGGTCTTTCC AAATATCCTA ATCTGTTAAA TC - #AATTACTT 
900 
- GATTTACAAC CAAGTCATAG TTCCCCCTAC CTAATTGCCT TTCTTGTGGA TA - #TCTATGAA 
960 
- GACATGCTAG AAAATCAGTG TGACAATAAG GAAGACATTC TTAATAAAGC AT - #TAGAGTTA 
1020 
- TGTGAAATCC TAGCTAAAGA AAAGGACACT ATAAGAAAGG AATATTGGAG AT - #ACATTGGA 
1080 
- AGATCCCTTC AAAGCAAACA CAGCACAGAA AATGACTCAC CAACAAATGT AC - #AGCAATAA 
1140 
- CACCATCCAG AAGAACTTGA TGGAATGCTT TTATTTTTTA TTAAGGGACC CT - #GCAGGAGT 
1200 
- TTCACACGAG AGTGGTCCTT CCCTTTGCCT GTGGTGTAAA AGTGCATCAC AC - #AGGTATTG 
1260 
- CTTTTTAACA AGAACTGATG CTCCTTGGGT GCTGCTGCTA CTCAGACTAG CT - #CTAAGTAA 
1320 
- TGTGATTCTT CTAAAGCAAA GTCATTGGAT GGGAGGAGGA AGAAAAAGTC CC - #ATAAAGGA 
1380 
- ACTTTTGTAG TCTTATCAAC ATATAATCTA ATCCCTTAGC ATCAGCTCCT CC - #CTCAGTGG 
1440 
- TACATGCGTC AAGATTTGTA GCAGTAATAA CTGCAGGTCA CTTGTATGTA AT - #GGATGTGA 
1500 
- GGTAGCCGAA GTTTGGTTCA GTAAGCAGGG AATACAGTCG TTCCATCAGA GC - #TGGTCTGC 
1560 
- ACACTCACAT TATCTTGCTA TCACTGTAAC CAACTAATGC CAAAAGAACG GT - #TTTGTAAT 
1620 
# 166 - #4AAAAAAAA AAAAAAAAAA AAAA 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
# 444 amino acid residues 
# amino acidPE: 
(C) STRANDEDNESS: sing - #le 
# linear (D) TOPOLOGY: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7: 
- Met Ala Ser Ser Ser Ser Phe Thr Tyr Tyr Cy - #s Pro Pro Ser Ser Ser 
# 15 
- Pro Val Trp Ser Glu Pro Leu Tyr Ser Leu Ar - #g Pro Glu His Ala Arg 
# 30 
- Glu Arg Leu Gln Asp Asp Ser Val Glu Thr Va - #l Thr Ser Ile Glu Gln 
# 45 
- Ala Lys Val Glu Glu Lys Ile Gln Glu Val Ph - #e Ser Ser Tyr Lys Phe 
# 60 
- Asn His Leu Val Pro Arg Leu Val Leu Gln Ar - #g Glu Lys His Phe His 
# 80 
- Tyr Leu Lys Arg Gly Leu Arg Gln Leu Thr As - #p Ala Tyr Glu Cys Leu 
# 95 
- Asp Ala Ser Arg Pro Trp Leu Cys Tyr Trp Il - #e Leu His Ser Leu Glu 
# 110 
- Leu Leu Asp Glu Pro Ile Pro Gln Ile Val Al - #a Thr Asp Val Cys Gln 
# 125 
- Phe Leu Glu Leu Cys Gln Ser Pro Glu Gly Gl - #y Phe Gly Gly Gly Pro 
# 140 
- Gly Gln Tyr Pro His Leu Ala Pro Thr Tyr Al - #a Ala Val Asn Ala Leu 
145 1 - #50 1 - #55 1 - 
#60 
- Cys Ile Ile Gly Thr Glu Glu Ala Tyr Asp Il - #e Ile Asn Arg Glu Glu 
# 175 
- Leu Leu Gln Tyr Leu Tyr Ser Leu Lys Gln Pr - #o Asp Gly Ser Phe Leu 
# 190 
- Met His Val Gly Gly Glu Val Asp Val Arg Se - #r Ala Tyr Cys Ala Ala 
# 205 
- Ser Val Ala Ser Leu Thr Asn Ile Ile Thr Pr - #o Asp Leu Phe Glu Gly 
# 220 
- Thr Ala Glu Trp Ile Ala Arg Cys Gln Asn Tr - #p Glu Gly Gly Ile Gly 
225 2 - #30 2 - #35 2 - 
#40 
- Gly Val Pro Gly Met Glu Ala His Gly Gly Ty - #r Thr Phe Cys Gly Leu 
# 255 
- Ala Ala Leu Val Ile Leu Lys Arg Glu Arg Se - #r Leu Asn Leu Lys Ser 
# 270 
- Leu Leu Gln Trp Val Thr Ser Arg Gln Met Ar - #g Phe Glu Gly Gly Phe 
# 285 
- Gln Gly Arg Cys Asn Lys Leu Val Asp Gly Cy - #s Tyr Ser Phe Trp Gln 
# 300 
- Ala Gly Leu Leu Pro Leu Leu His Arg Ala Le - #u His Ala Gln Gly Asp 
305 3 - #10 3 - #15 3 - 
#20 
- Pro Ala Leu Ser Met Ser His Trp Met Phe Hi - #s Gln Gln Ala Leu Gln 
# 335 
- Glu Tyr Ile Leu Met Cys Cys His Cys Pro Al - #a Gly Gly Leu Leu Asp 
# 350 
- Lys Pro Gly Lys Ser Arg Asp Phe Tyr His Th - #r Cys Tyr Cys Leu Ser 
# 365 
- Gly Leu Ser Ile Ala Gln His Phe Gly Ser Gl - #y Ala Met Leu His Asp 
# 380 
- Val Val Leu Gly Val Pro Glu Asn Ala Leu Gl - #n Pro Thr His Pro Val 
385 3 - #90 3 - #95 4 - 
#00 
- Tyr Asn Ile Gly Pro Asp Lys Val Ile Gln Va - #l Thr Thr Tyr Phe Leu 
# 415 
- Gln Lys Pro Val Pro Gly Phe Glu Glu Cys Gl - #u Asp Ala Val Thr Ser 
# 430 
- His Pro Ala Thr Asp Leu Arg Ser His Pro Gl - #u Ile 
# 440 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
# 1413 base pairsH: 
# nucleic acid: 
(C) STRANDEDNESS: sing - #le 
# linear (D) TOPOLOGY: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8: 
- TCGACCCACG CGTCCGGCTT AACGAAGCAG AGTCCTACCA CTGCTGCTCT CC - #TGATCATG 
60 
- GCTTCTTCGA GTTCTTTCAC CTACTATTGC CCTCCATCTT CCTCCCCCGT CT - #GGTCAGAG 
120 
- CCGCTGTACA GTCTGAGGCC CGAGCACGCG CGAGAGCGGT TGCAGGACGA CT - #CGGTGGAA 
180 
- ACAGTCACGT CCATAGAACA GGCAAAAGTA GAAGAAAAGA TCCAAGAGGT CT - #TCAGTTCT 
240 
- TACAAGTTCA ACCACCTTGT ACCAAGGCTC GTTTTGCAGA GGGAGAAGCA CT - #TCCATTAT 
300 
- CTGAAAAGAG GCCTTCGACA ACTGACAGAT GCCTATGAGT GTCTGGATGC CA - #GCCGCCCA 
360 
- TGGCTCTGCT ATTGGATCCT GCACAGCTTG GAACTGCTAG ATGAACCCAT CC - #CCCAGATA 
420 
- GTGGCTACAG ATGTGTGTCA GTTCTTGGAG CTGTGTCAGA GCCCAGAAGG TG - #GCTTTGGA 
480 
- GGAGGACCCG GTCAGTATCC ACACCTTGCA CCCACATATG CAGCAGTCAA TG - #CATTGTGC 
540 
- ATCATTGGCA CCGAGGAGGC CTATGACATC ATTAACAGAG AAGAGCTTCT TC - #AGTATTTG 
600 
- TACTCCCTGA AGCAACCTGA CGGCTCCTTT CTCATGCATG TCGGAGGTGA GG - #TGGATGTG 
660 
- AGAAGCGCAT ACTGTGCTGC CTCGGTAGCC TCGCTGACCA ACATCATCAC TC - #CAGACCTC 
720 
- TTTGAGGGCA CTGCTGAATG GATAGCAAGG TGTCAGAACT GGGAAGGTGG CA - #TTGGCGGG 
780 
- GTACCAGGGA TGGAAGCCCA TGGTGGCTAT ACCTTCTGTG GCCTGGCCGC GC - #TGGTAATC 
840 
- CTCAAGAGGG AACGTTCCTT GAACTTGAAG AGCTTATTAC AATGGGTGAC AA - #GCCGGCAG 
900 
- ATGCGATTTG AAGGAGGATT TCAGGGCCGC TGCAACAAGC TGGTGGATGG CT - #GCTACTCC 
960 
- TTCTGGCAGG CGGGGCTCCT GCCCCTGCTC CACCGCGCAC TGCACGCCCA AG - #GTGACCCT 
1020 
- GCCCTTAGCA TGAGCCACTG GATGTTCCAT CAGCAGGCCC TGCAGGAGTA CA - #TCCTGATG 
1080 
- TGCTGCCACT GCCCTGCGGG GGGGCTCCTG GATAAACCTG GCAAGTCGCG TG - #ATTTCTAC 
1140 
- CACACCTGCT ACTGTCTGAG CGGCCTGTCC ATAGCCCAGC ACTTCGGCAG CG - #GAGCCATG 
1200 
- TTGCATGATG TGGTCCTGGG TGTGCCCGAA AACGCTCTGC AGCCCACTCA CC - #CAGTGTAC 
1260 
- AACATTGGAC CAGACAAGGT GATCCAGGTC ACTACATACT TTCTACAGAA GC - #CAGTCCCA 
1320 
- GGTTTTGAGG AATGCGAAGA TGCGGTGACC TCACATCCTG CAACTGACTT AA - #GGAGCCAC 
1380 
# 1413 GCCC CCGTTAAAAC GGA 
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