Process and composition for increasing squalene and sterol accumulation in higher plants

A process of increasing squalene and sterol accumulation in a transgenic plant by increasing the amount of a gene encoding a polypeptide having HMG-CoA reductase activity is disclosed. The amount is preferably increased by transforming plant cells with a recombinant DNA molecule comprising a vector operatively linked to an exogenous DNA segment that encodes a polypeptide having HMG-CoA reductase activity, and a promoter suitable for driving the expression of said polypeptide to form a transformed plant cell and regenerating a transgenic plant from that transformed cell. Also disclosed are a process of increasing pest resistance in a transgenic plant, transgenic plants and transgenic seeds capable of germinating into transgenic plants.

TECHNICAL FIELD 
The present invention relates to processes and compositions for increasing 
the accumulation of squalene and sterols in higher plants, and more 
particularly to increasing squalene and non-delta-5 sterol accumulation by 
increasing the amount of a gene encoding a polypeptide having HMG-CoA 
reductase activity. 
BACKGROUND OF THE INVENTION 
Acetate is the metabolic precursor of a vast array of compounds vital for 
cell and organism viability. Acetyl coenzyme A (CoA) reacts with 
acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA). HMG-CoA 
is reduced to mevalonate in an irreversible reaction catalyzed by the 
enzyme HMG-CoA reductase. Mevalonate is phosphorylated and decarboxylated 
to isopentenyl-pyrophosphate (IPP). Through the sequential steps of 
isomerization, condensation and dehydrogenation, IPP is converted to 
geranyl pyrophosphate (GPP). GPP combines with IPP to form farnesyl 
pyrophosphate (FPP), two molecules of which are reductively condensed to 
form squalene, a 30-carbon precursor of sterols. 
Sterols are derivatives of a fused, reduced ring system, 
cyclopenta-[.alpha.]-phenanthrene, comprising three fused cyclohexane 
rings (A, B and C) in a phenanthrene arrangement, and a terminal 
cyclopentane ring (D) having the formula and carbon atom position 
numbering shown below: 
##STR1## 
where R is an 8 to 10 carbon-atom side chain. 
In plants, squalene is converted to squalene epoxide, which is then 
cyclized to form cycloartenol (4,4,14.alpha.-trimethyl-9.beta.,19 
cyclo-5.alpha.-cholest-24-en-3.beta.-ol). Cycloartenol has two methyl 
groups at position 4, a methyl group at position 14, a methylene bridge 
between the carbon atoms at positions 9 and 19 that forms a disubstituted 
cyclopropyl group at those positions, and includes an 8 carbon sidechain 
of the formula: CH.sub.3 CH(CH.sub.2).sub.2 CH.dbd.C(CH.sub.3).sub.2. 
Cycloartenol is formed in an early stage in the biosynthetic pathway of 
sterol production in higher plants. Cycloartenol is formed from squalene 
epoxide, which is formed from squalene, a derivative of mevalonic acid 
(mevalonate). Squalene epoxide can alternatively be converted into 
pentacyclic sterols, containing five instead of four rings. Exemplary 
pentacyclic sterols include the phytoalexins and saponins. 
Being one of the first sterols in the higher plant biosynthetic pathway, 
cycloartenol serves as a precursor for the production of numerous other 
sterols. In normal plants, cycloartenol is converted to predominantly 
24-methylene cycloartenol (4,4,14.alpha.-trimethyl-9.beta.,19 
cyclo-22,23-dihydro-ergosta-24(28)-en-3-.beta. -ol), cycloeucalenol 
(4,14.alpha.-dimethyl-9.beta.,19 
cyclo-5.alpha.-ergost-24(28)-en-3.beta.-ol), obtusifoliol 
(4,14.alpha.-dimethyl-5.alpha.-ergosta-8,24(28)-dien-3.beta.-ol), 
isofucosterol (5.alpha.-stigmasta-5-Z-24(28)-dien-3.beta.-ol), sitosterol 
(5.alpha.-stigmasta-5-en-3.beta.-ol), 
stigmasterol-(stigmasta-5,E-22-dien-3.beta.-ol), campesterol 
(5.alpha.-ergosta-5-en-3.beta.-ol), and cholesterol 
(5.alpha.-cholesta-5-en-3.beta.-ol). 
Although sterols produced by plants, and particularly higher (vascular) 
plants, can be grouped by the presence or absence of one or more of 
several functionalities, plant sterols are classified into two general 
groups herein; i.e., those containing a double bond between the carbon 
atoms at positions 5 and 6 (delta-5 or .DELTA.5 sterols) and those not 
containing a double bond between the carbon atoms at positions 5 and 6 
(non-delta-5 sterols). 
Exemplary naturally occurring delta-5 plant sterols isofucosterol, 
sitosterol, stigmasterol, campesterol, cholesterol, and 
dihydrobrassicasterol. Exemplary naturally occurring non-delta-5 plant 
sterols are cycloartenol, 24-methylene cycloartenol, cycloeucalenol, and 
obtusifoliol. 
The most abundant sterols of vascular plants are campesterol, sitosterol 
and stigmasterol, all of which contain a double bond between the carbon 
atoms at positions 5 and 6 and are classified as delta-5 sterols. 
The ratio of delta-5 to non-delta-5 sterols in plants can be an important 
factor relating to insect pest resistance. Insect pests are unable to 
synthesize de novo the steroid nucleus and depend upon external sources of 
sterols in their food source for production of necessary steroid 
compounds. In particular, insect pests require an external source of 
delta-5 sterols. By way of example, externally provided delta-5 sterols 
are necessary for the production of ecdysteroids, hormones that control 
reproduction and development. See, e.g., Costet et al., Proc. Natl. Acad. 
Sci. USA, 84:643 (1987) and Corio-Costet et al., Archives of Insect 
Biochem. Physiol., 11:47 (1989). 
Treatment of wheat with the fungicide fenpropimorph reduced delta-5 sterol 
content from about 93 percent of total sterol to about 1 percent of total 
sterol and increased non-delta-5 sterol content from about 7 percent of 
total sterol to about 99 percent of total sterol. Where the phytophagous 
grasshopper Locusta migratoria was reared feeding on wheat seedlings 
treated with fenpropimorph, the concentration of ecdysteroids in eggs was 
reduced by 80 percent. Those eggs either did not develop (meiosis is 
inhibited) or they developed with complex abnormalities and malfunctions. 
Costet et al., Proc. Natl. Acad. Sci. USA, 84:643 (1987); Corio-Costet et 
al., Archives of Insect Biochem. Physiol., 11:47 (1989). 
Because insects can use delta-5 sterols for steroid production, those 
delta-5 sterols are referred to herein as "utilizable" sterols. 
Non-delta-5 sterols are referred to herein as "non-utilizable" sterols. 
Naturally occurring higher plants typically contain an excess of utilizable 
over non-utilizable sterols. Costet et al., Proc. Natl. Acad. Sci. USA, 
84:643 (1987); Corio-Costet et al., Archives of Insect Biochem. Physiol., 
11:47 (1989). Such plants thus can provide an appropriate food supply for 
insect pests. 
Plants having an abundance of non-utilizable sterols have also been 
produced by treatment with inhibitors of sterol biosynthesis such as the 
fungicides triarimol, tridemorph, and triparanol. Hosokawa et al., Lipids, 
19(6):449 (1984). The use of fungicides, however, is undesirable in light 
of the adverse environmental effects attendant with the use of such 
chemicals. 
All of the fungicides discussed above are known to inhibit sterol 
biosynthesis subsequent to the formation of cycloartenol. 
As set forth above, cycloartenol is a metabolic derivative of mevalonate, 
which is formed from the reduction of 3-hydroxy-3-methylglutaryl coenzyme 
A (HMG-CoA). The reduction of HMG-CoA to mevalonate is catalyzed by the 
enzyme HMG-CoA reductase. 
The HMG-CoA reductase enzymes of animals and yeasts are integral membrane 
glycoproteins of the endoplasmic reticulum. The intact enzyme comprises 
three regions: a catalytic region, containing the active site of the 
enzyme; a membrane binding region, anchoring the enzyme to the endoplasmic 
reticulum; and a linker region, joining the catalytic and membrane binding 
regions of the enzymes. The membrane binding region occupies the NH.sub.2 
-terminal portion of the intact protein, whereas the catalytic region 
occupies the COOH-terminal portion of the protein, with the linker region 
constituting the remaining portion. Basson, M. E. et al., Mol. Cell Biol., 
8(9):3797-3808 (1988). At present, the sub-cellular localization of 
HMG-CoA reductase in plants is not known. Russell, D. W. et al., Current 
Topics in Plant Biochemistry, Vol. 4, ed. by D. D. Randall et al., Univ. 
of Missouri (1985). 
The activity of HMG-CoA reductase in animals and yeasts is known to be 
subject to feedback inhibition by sterols. Such feedback inhibition 
requires the presence of the membrane binding region of the enzyme. See, 
e.g., Gil, G. et al., Cell, 41: 249-258(1985); Bard, M. and Downing, J. F. 
Journal of General Microbiology, 125:415-420(1981). 
Given that mevalonate is the precursor for sterols and other isoprenoids, 
it might be expected that increases in the amount or activity of HMG-CoA 
reductase would lead to increases in the accumulation of both sterols and 
other isoprenoids. In yeasts and non-photosynthetic microorganisms, 
increases in HMG-CoA reductase activity are not associated with 
predictable increases in the production of sterols or other isoprenoids. 
In mutant strains of the yeast Saccharomyces cerevisiae (S. cerevisiae) 
having abnormally high levels of HMG-CoA reductase activity, the 
production of two sterols, 4,14-dimethylzymosterol and 
14-methylfecosterol, is markedly increased above normal. Downing et al., 
Biochemical and Biophysical Research Communications, 94(3): 974-979(1980). 
When HMG-CoA reductase activity was increased by illumination in 
non-photosynthetic microorganisms, isoprenoid (carotenoid), but not sterol 
(ergosterol), synthesis was enhanced. Tada et al., Plant and Cell 
Physiology, 23(4): 615-621(1982). There are no studies reporting the 
effects of such increases in HMG-CoA reductase activity in plants. 
SUMMARY OF THE INVENTION 
The present invention provides a process of increasing sterol accumulation 
in a transgenic plant comprising: 
(a) transforming a plant cell with a recombinant DNA molecule comprising a 
vector operatively linked to a DNA segment that encodes a polypeptide 
having HMG-CoA reductase activity and a promoter suitable for driving the 
expression of said polypeptide in said plant cell to form a transformed 
plant cell; and 
(b) regenerating the transformed plant cell into the transgenic plant. 
A polypeptide having HMG-CoA reductase activity preferably comprises the 
catalytic region and at least a portion of the linker region but is free 
from the membrane binding region of a HMG-CoA reductase. In a preferred 
embodiment, the promoter is a promoter whose regulatory function is 
substantially unaffected by the level of sterol in said transgenic plant 
such as the CaMV 35S promoter. A preferred recombinant DNA molecule is 
plasmid HMGR.DELTA.227-pKYLX71. 
The plant cell is preferably obtained from plants of the group consisting 
of tobacco, cotton, soybean, tomato and alfalfa. A sterol whose 
accumulation is increased is preferably a non-delta-5 sterol and, more 
preferably cycloartenol. 
A similar process to that set forth above is used increase squalene 
accumulation and to increase pest resistance in a transgenic plant. 
The present invention further contemplates a transgenic plant produced in 
accordance with any of the above processes. 
Still further, the present invention contemplates a transgenic plant that 
(a) has an increased amount of a structural gene that encodes a 
polypeptide having HMG-CoA reductase activity and (b) over accumulates 
sterols or squalene relative to a native, non-transgenic plant of the same 
strain. 
The encoded polypeptide is preferably an intact HMG-CoA reductase enzyme or 
an active, truncated HMG-CoA reductase enzyme comprising the catalytic and 
at least a portion of the linker region that is free from the membrane 
binding region of a HMG-CoA reductase enzyme such as a hamster HMG-CoA 
reductase. 
Preferably, the transgenic plant is a transgenic tobacco, cotton, soybean, 
tomato or alfalfa plant. The present invention also contemplates a 
transgenic tobacco plant whose seeds have ATCC accession No. 40904 and 
mutants, recombinants, genetically engineered derivatives thereof and 
hybrids derived therefrom. 
In a still further aspect, the present invention contemplates a transgenic 
plant seed capable of germinating into a transgenic plant that over 
accumulates sterol or squalene relative to a native, non-transgenic plant 
of the same strain and mutants, recombinants, genetically engineered 
derivatives thereof and hybrids derived therefrom.

DETAILED DESCRIPTION OF THE INVENTION 
I. Definitions 
The following words and phrases have the meanings set forth below. 
Expression: The combination of intracellular processes, including 
transcription and translation undergone by a structural gene to produce a 
polypeptide. 
Plant integrating vector: A polynucleotide having a first portion 
containing a structural gene and control elements that direct and regulate 
expression of that structural gene when operatively linked to that gene 
and a second portion containing polynucleotide sequences that permit the 
first portion to be integrated into the chromosome of a plant cell. 
Operatively linked: A structural gene is covalently bonded in correct 
reading frame to another DNA (or RNA as appropriate) segment, such as to a 
plant integrating vector so that the structural gene is under the control 
of the plant integrating vector. 
Promoter: A recognition site on a DNA sequence or group of DNA sequences 
that provide an expression control element for a structural gene and to 
which RNA polymerase specifically binds and initiates RNA synthesis 
(transcription) of that gene. 
Recombinant DNA molecule: A hybrid DNA sequence comprising at least two 
nucleotide sequences not normally found together in nature. 
Regeneration: The process of growing a plant from a plant cell (e.g. plant 
protoplast or explant). 
Structural gene: A DNA sequence that is expressed as a polypeptide, i.e., 
an amino acid residue sequence. 
Transformation: A process of introducing an exogenous sequence (e.g. a 
vector, a recombinant DNA molecule) into a cell or protoplasts in which 
that exogenous DNA is incorporated into a chromosome. 
Transformed plant cell: A plant cell whose DNA has been altered by the 
introduction of an exogenous DNA molecule into that cell or protoplast. 
Transgenie plant cell: Any plant cell derived or regenerated from a 
transformed plant cell or protoplast or derived from a transgenic plant. 
Exemplary transgenic cells include plant calli derived from a transformed 
plant cell and particular cells such as leaf, root, stem, e.g. somatic 
cells, or reproductive (germ) cells obtained from a transgenic plant. 
Transgenic plant: A plant or progeny thereof derived from a transformed 
plant cell or protoplast, wherein the plant DNA contains an introduced 
exogenous DNA molecule not originally present in a native, non-transgenic 
plant of the same strain. The terms "transgenic plant" and "transformed 
plant" have sometimes been used in the art as synonymous terms to define a 
plant whose DNA contains an exogenous DNA molecule. However, it is thought 
more scientifically correct to refer to a regenerated plant or callus 
obtained from a transformed plant cell or protoplast as being a transgenic 
plant, and that usage will be followed herein. 
Vector: A DNA molecule capable of replication in a host cell and/or to 
which another DNA segment can be operatively linked so as to bring about 
replication of the attached segment. A plasmid is an exemplary vector. 
II. The Invention 
A. Processes for Increasing Sterol Accumulation 
In one aspect, the present invention relates to a process for increasing 
sterol accumulation in transgenic plants, as well as to the transgenic 
plants that exhibit increased sterol accumulation relative to a native 
variety of the plant. Preferably, the increase in sterol accumulation is 
the result of an increased accumulation of non-delta-5 sterols (i.e., 
sterols lacking a double bond between the carbon atoms at positions 5 and 
6 of the sterol nucleus). 
A plant contemplated by this invention is a vascular, multicellular higher 
plant. Such higher plants will hereinafter be usually referred to simply 
as "plants". Such "plants" include both complete entities having leaves, 
stems, seeds, roots and the like as well as callus and cell cultures that 
are monocotyledonous and dicotyledonous. Dicotyledonous plants are a 
preferred embodiment of the present invention. 
Preferred plants are members of the Solanaceae, Leguminosae, Ammiaceae, 
Brassicaceae, Gramineae, Carduaceae and Malvaceae families. Exemplary 
plant members of those families are tobacco, petunia and tomato 
(Solanaceae), soybean and alfalfa (Leguminosae), carrot (Ammiaceae), corn 
and barley (Gramineae, arabidopsis (Brassicaceae), guayule (Carduaceae), 
and cotton (Malvaceae). A preferred plant is tobacco of the strain 
Nicotiana tabacum (N. tabacum), cotton of the strain Coker line 312-5A, 
soybean of the strain Glycine max, alfalfa of the strain RYSI or tomato of 
the strain Lycopersicon esculentum. 
A transgenic plant contemplated by this invention is produced by 
transforming a plant cell or protoplast with an added, exogenous 
structural gene that encodes a polypeptide having HMG-CoA reductase 
activity to produce a transformed plant cell, and regenerating a 
transgenic plant from the transformed plant cell. The encoded polypeptide 
is expressed both in the transformed plant cell or protoplast and the 
resulting transgenic plant. (The phrase "plant cell" will hereinafter be 
used to include a plant protoplast, except where plant protoplasts are 
specifically discussed.) 
A non-transgenic plant that serves as the source of the plant cell that is 
transformed; i.e., the precursor cell, is referred to herein as a "native, 
non-transgenic" plant. The native, non-transgenic plant is of the same 
strain as the formed transgenic plant. 
Sterol production in a transgenic plant of the present invention is 
increased by increasing the activity of the enzyme HMG-CoA reductase, 
which enzyme catalyzes the conversion of 3-hydroxy-3-methylglutaryl 
Coenzyme A (HMG-CoA) to mevalonate. As used herein, "activity" means the 
total catalytic activity of HMG-CoA reductase in a plant cell. As used 
herein, the term "specific activity" means the activity normalized to 
cellular protein content. 
HMG-CoA reductase activity is increased by increasing the amount (copy 
number) of a gene encoding a polypeptide having HMG-CoA reductase 
catalytic activity. Expression of the increased amount of that encoded 
structural gene enhances the activity of that enzyme. 
The amount of the expressed gene is increased by transforming a plant cell 
with a recombinant DNA molecule comprising a vector operatively linked to 
a DNA segment that encodes a polypeptide having HMG-CoA reductase 
activity, and a promoter suitable for driving the expression of that 
polypeptide in that plant cell, and culturing the transformed plant cell 
into a transgenic plant. Such a polypeptide includes intact as well as 
catalytically active, truncated HMG-CoA reductase proteins. 
Thus, a transformed plant cell and a transgenic plant have one or more 
added, exogenous genes that encode a polypeptide having HMG-CoA reductase 
activity relative to a native, non-transgenic plant or untransformed plant 
cell of the same type. As such, a transformed plant cell or transgenic 
plant can be distinguished from an untransformed plant cell or native, 
non-transgenic plant by standard technology such as agarose separation of 
DNA fragments or mRNAs followed by transfer and appropriate blotting with 
DNA or RNA, e.g., Southern or Northern blotting, or by use of polymerase 
chain reaction technology, as are well known. Relative HMG-CoA reductase 
activity of the transformed cell or transgenic plant with untransformed 
cells and native, non-transgenic plants or cell cultures therefrom can 
also be compared, with a relative activity for that enzyme of about 1.5:1 
for transgenic (transformed):native (untransformed) showing 
transformation. Higher relative activity ratios such as about 15:1 have 
also been observed. 
Sterol accumulation can also be used to distinguish between native, 
non-transgenic and transgenic plants. A transgenic plant has at least 
about twice the total sterol content as a native, non-transgenic plant 
where a single added gene is present. Greater differences up to about 
forty-fold have also been observed. 
The increased accumulation of sterol is preferably the result of an 
increase in the accumulation of non-delta-5 sterols lacking a double bond 
between the carbon atoms at positions 5 and 6. 
By way of example, in transgenic tobacco made in accordance with a process 
of the present invention, the increase in sterol accumulation was found to 
be due predominantly to an increase in the accumulation of the non-delta-5 
sterol cycloartenol (See Example 3 hereinafter). Increases in non-delta-5 
sterols were also observed in transgenic cotton, soybean, tomato and 
alfalfa plant callus cultures (See Examples 6, 7, 8 and 9 hereinafter). 
B. Processes for Increasing Squalene Accumulation 
In another aspect, the present invention relates to processes for 
increasing squalene accumulation in transgenic plants, as well as to the 
transgenic plants that exhibit increased squalene accumulation relative to 
a native, non-transgenic plant of the same strain. 
Squalene production in a transgenic plant of the present invention is 
increased by increasing the activity of the enzyme HMG-CoA reductase. The 
increase in HMG-CoA reductase activity is carried out in accordance with 
the processes discussed above relating to a process for increasing sterol 
accumulation. 
Although similar processes are used to increase sterol and squalene 
accumulation in transgenic plants, there does not appear to be any causal 
or necessary relationship between the increased accumulation of those 
compounds. For example, observed increases in sterol accumulation of 
transgenic plants of the present invention do not correlate with increases 
in squalene accumulation in those same plants. Table 1, below, shows the 
increases in sterol and squalene accumulation in transgenic tobacco, 
cotton, soybean, tomato and alfalfa callus. The data in Table 1 are taken 
from the data in Tables 6, 7, 8, 9 and 10 hereinafter. The Delta values 
shown in Table 1 represent averages of the individual data in Tables 6-10. 
TABLE 1 
______________________________________ 
Delta.sup.2 
Plant Control Transgenic 
(Cont-Trans) 
______________________________________ 
Sterol Accumulation.sup.1 
Tobacco 0.21 0.78 0.57 
Cotton 0.16 0.60 0.44 
Soybean 0.37 0.85 0.48 
Tomato 0.04 0.99 0.95 
Alfalfa 0.24 1.26 1.02 
Squalene Accumulation.sup.1 
Tobacco &lt;0.010 0.126 0.126 
Cotton &lt;0.002 0.560 0.560 
Soybean 0.022 0.233 0.211 
Tomato &lt;0.002 0.090 0.090 
Alfalfa 0.002 0.052 0.050 
______________________________________ 
.sup.1 Sterol and squalene levels of Control and Transgenic callus are 
given as percentage of dry weight 
.sup.2 Delta values are calculated as control minus transgenic (ContTrans 
levels. Where the control value is trace (tr) or &lt;0.01, the delta value i 
calculated as the level in the transgenic callus. 
It can be seen that there is no correlation between increases in sterol and 
squalene accumulation. In tobacco, the increase in sterol accumulation 
(0.57) was associated with an increase in squalene accumulation of 0,126. 
In marked contrast, in alfalfa where the increase in sterol accumulation 
was twice that seen in tobacco (1.02 vs. 0.57), the accumulation of 
squalene was only one-twentieth that seen in tobacco (0.05 vs. 0.126). 
These data show the likely independent effects of transformation and 
formation of transgenic plants on sterol and squalene accumulation. 
Squalene accumulation can also be used to distinguish between transgenie 
and native, non-transgenic plants. Thus, a transgenic plant contemplated 
herein can accumulate about 5 to about 75 times the squalene of a native, 
non-transformed, plant. 
C. Structural Genes 
The present invention contemplates transforming a plant cell with a 
structural gene that encodes a polypeptide having HMG-CoA reductase 
activity. The HMG-CoA reductase enzymes of both animal and yeast cells 
comprise three distinct amino acid residue sequence regions, which regions 
are designated the catalytic region, the membrane binding region and the 
linker region. 
The catalytic region contains the active site of the HMG-CoA reductase 
enzyme and comprises about forty percent of the COOH-terminal portion of 
intact HMG-CoA reductase enzyme. 
The membrane binding region contains hydrophobic amino acid residues and 
comprises about fifty percent of the NH.sub.2 -terminal portion of intact 
HMG-CoA reductase enzyme. 
The linker region connects the catalytic and membrane binding regions, and 
constitutes the remaining about ten percent of the intact enzyme. 
As discussed in greater detail below, only the catalytic region of HMG-CoA 
reductase is needed herein to provide the desired enzyme activity. Thus, 
an exogenous structural gene that encodes a polypeptide corresponding to 
that catalytic region is the minimal gene required for transforming plant 
cells. The present invention therefore contemplates use of both intact and 
truncated structural genes that encode a polypeptide having HMG-CoA 
reductase activity. 
A structural gene encoding a polypeptide having HMG-CoA reductase activity 
can be obtained or constructed from a variety of sources and by a variety 
of methodologies. See, e.g., Carlson et al., Cell, 28:145 (1982); Rine et 
al., Proc. Nat. Acad. Sci. USA, 80:6750 (1983). Exemplary of such 
structural genes are the mammalian and yeast genes encoding HMG-CoA 
reductase or the catalytic region thereof. 
The mammalian genome contains a single gene encoding HMG-CoA reductase. The 
nucleotide base sequence of the hamster and human gene for HMG-CoA 
reductase have been described. A composite nucleotide sequence of cDNA 
corresponding to the mRNA (SEQ ID NO:1), as well as the derived amino acid 
residue sequence (SEQ ID NO:2), for hamster HMG-CoA reductase is provided 
in FIG. 2, reprinted from Chin et al., Nature, 308:613 (1984). The 
composite nucleotide sequence of FIG. 2 (SEQ ID NO:1), comprising about 
4768 base pairs, includes the nucleotide sequence encoding the intact 
hamster HMG-CoA reductase enzyme. 
Intact hamster HMG-CoA reductase comprises about 887 amino acid residues 
(SEQ ID NO:2). A structural gene encoding an intact hamster HMG-CoA 
reductase enzyme of 887 amino acid residues comprises base pairs from 
about nucleotide position 164 to about nucleotide position 2824 of FIG. 2 
(SEQ ID NO:1). 
A preferred structural gene is one that encodes a polypeptide corresponding 
to only the catalytic region of the enzyme. Two catalytically active 
segments of hamster HMG-CoA reductase have been defined. Liscum et al., J. 
Biol. Chem., 260(1):522 (1985). One segment containing a catalytic region 
has an apparent molecular weight of 62 kDa and comprises amino acid 
residues from about position 373 to about position 887. A second segment 
containing a catalytic region has an apparent molecular weight of 53 kDa 
segment and comprises amino acid residues from about position 460 to about 
position 887. The 62 kDa catalytically active segment is encoded by base 
pairs from about nucleotide position 1280 to about nucleotide position 
2824 of FIG. 2 (SEQ ID NO:1). The 53 kDa catalytically active segment is 
encoded by base pairs from about nucleotide position 1541 to about 
nucleotide position 2824 of FIG. 2 (SEQ ID NO:1). 
In a preferred embodiment, the utilized structural gene encodes the 
catalytic region and at least a portion of the linker region of HMG-CoA 
reductase. The linker region of hamster HMG-CoA reductase comprises amino 
acid residues from about position 340 to about position 373 or from about 
position 340 to about position 460, depending upon how the catalytic 
region is defined. These linker regions are encoded by base pairs from 
about nucleotide position 1180 to about nucleotide position 1283 or from 
about position 1180 to about position 1540 respectively of FIG. 2 (SEQ ID 
NO:1). The structural gene encoding the linker region is operatively 
linked to the structural gene encoding the catalytic region. 
In one particularly preferred embodiment, a structural gene encoding a 
catalytically active, truncated HMG-CoA reductase enzyme can optionally 
contain base pairs encoding a small portion of the membrane region of the 
enzyme. A truncated hamster HMG-CoA reductase gene, designated 
HMGR-.DELTA.227, comprising nucleotides 164-190 operatively linked to 
nucleotides 1187-2824 from FIG. 2 (SEQ ID NO:1), which encodes amino acid 
residues 1-9 (from the membrane binding region) and 342-887 has been used 
to transform plant cells. The schematic structure of the transforming 
plasmid (pRED-227.DELTA.) containing the truncated gene is reprinted in 
FIG. 4. A structural gene encoding a polypeptide comprising a 
catalytically active, truncated or intact HMG-CoA reductase enzyme from 
other organisms such as yeast can also be used in accordance with the 
present invention. 
Yeast cells contain two genes encoding HMG-CoA reductase. The two yeast 
genes, designated HMG1 and HMG2, encode two distinct forms of HMG-CoA 
reductase, designated HMG-CoA reductase 1 and HMG-CoA reductase 2. The 
nucleotide base sequence of HMG1 (SEQ ID NO:3) as well as the amino acid 
residue sequence of HMG-CoA reductase 1 (SEQ ID NO:4) are presented in 
FIG. 3, taken from Basson et al., Mol. Cell Biol., 8(9):3797 (1988). The 
nucleotide base sequences of HMG2 (SEQ ID NO:5) as well as the amino acid 
residue sequence of HMG-CoA reductase 2 (SEQ ID NO:6) are set forth 
hereinafter in the Sequence Listing. 
The entire HMG1 gene comprises about 3360 base pairs (SEQ ID NO:3). Intact 
HMG-CoA reductase 1 comprises an amino acid sequence of about 1054 amino 
acid residues (SEQ ID NO:4). Thus, the minimal portion of the HMG1 gene 
that encodes an intact enzyme comprises base pairs from about nucleotide 
position 121 to about position 3282 of FIG. 3 (SEQ ID NO:3). 
The entire HMG2 gene comprises about 3348 base pairs (SEQ ID NO:5). Intact 
HMG-CoA reductase 2 comprises about 1045 amino acid residues (SEQ ID 
NO:6). Thus, the minimal portion of HMG2 gene that encodes intact HMG-CoA 
reductase 2 comprises base pairs from about nucleotide position 121 to 
about position 3255 of FIG. 3 (SEQ ID NO:5). 
By analogy to the truncated hamster structural gene, structural genes 
encoding polypeptides comprising catalytically active, truncated HMG-CoA 
reductase enzymes from yeast can also be used in accordance with the 
present invention. 
The catalytic region of HMG-CoA reductase 1 comprises amino acid residues 
from about residue 618 to about residue 1054: i.e., the COOH-terminus. A 
structural gene that encodes the catalytic region comprises base pairs 
from about nucleotide position 1974 to about position 3282 of FIG. 3. 
The linker region of HMG-CoA reductase 1 comprises an amino acid sequence 
from about residue 525 to about residue 617. A structural gene that 
encodes the linker region comprises nucleotides from about position 1695 
to about position 1973 of FIG. 3. A structural gene encoding a polypeptide 
comprising the catalytic region and at least a portion of the linker 
region of yeast HMG-CoA reductase 1 preferably comprises the structural 
gene encoding the linker region of the enzyme operatively linked to the 
structural gene encoding the catalytic region of the enzyme. 
Also by analogy to the truncated hamster gene, a truncated HMG1 gene can 
optionally contain nucleotide base pair sequences encoding a small portion 
of the membrane binding region of the enzyme. Such a structural gene 
preferably comprises base pairs from about nucleotide position 121 to 
about position 147 and from about position 1695 to about position 3282 of 
FIG. 3. 
A construct similar to those above from an analogous portion of yeast 
HMG-CoA reductase 2 can also be utilized. 
It will be apparent to those of skill in the art that the nucleic acid 
sequences set forth herein, either explicitly, as in the case of the 
sequences set forth above, or implicitly with respect to nucleic acid 
sequences generally known and not presented herein, can be modified due to 
the built-in redundancy of the genetic code and non-critical areas of the 
polypeptide that are subject to modification and alteration. In this 
regard, the present invention contemplates allelic variants of structural 
genes encoding a polypeptide having HMG-CoA reductase activity. 
The previously described DNA segments are noted as having a minimal length, 
as well as total overall lengths. That minimal length defines the length 
of a DNA segment having a sequence that encodes a particular polypeptide 
having HMG-CoA reductase activity. As is well known in the art, so long as 
the required DNA sequence is present, (including start and stop signals), 
additional base pairs can be present at either end of the segment and that 
segment can still be utilized to express the protein. This, of course, 
presumes the absence in the segment of an operatively linked DNA sequence 
that represses expression, expresses a further product that consumes the 
enzyme desired to be expressed, expresses a product other than the desired 
enzyme or otherwise interferes with the structural gene of the DNA 
segment. 
Thus, so long as the DNA segment is free of such interfering DNA sequences, 
a DNA segment of the invention can be up to 15,000 base pairs in length. 
The maximum size of a recombinant DNA molecule, particularly a plant 
integrating vector, is governed mostly by convenience and the vector size 
that can be accommodated by a host cell, once all of the minimal DNA 
sequences required for replication and expression, when desired, are 
present. Minimal vector sizes are well known. 
D. Recombinant DNA Molecules 
A recombinant DNA molecule of the present invention can be produced by 
operatively linking a vector to a useful DNA segment discussed before to 
form a plasmid such as those discussed herein. A particularly preferred 
recombinant DNA molecule is discussed in detail in Example 1, hereafter. A 
vector capable of directing the expression of a polypeptide having HMG-CoA 
reductase activity is referred to herein as an HMG-CoA reductase "plant 
integrating vector". 
Such plant integrating vectors contain control elements that direct and 
regulate expression, including a promoter, a marker, a terminator and 
insertion sequences (see FIG. 5). The polypeptide coding genes are 
operatively linked to the plant integrating vector to allow the promoter 
sequence to direct RNA polymerase binding and expression of the desired 
polypeptide coding gene. 
Useful in expressing the polypeptide coding gene are promoters that are 
inducible, viral, synthetic, constitutive as described by Poszkowski et 
al., EMBO J., 3:2719 (1989) and Odell et al., Nature, 313:810 (1985), and 
temporally regulated, spatially regulated, and spatiotemporally regulated 
as given in Chau et al., Science, 244:174-181 (1989). The promoter 
preferably comprises a promoter sequence whose function in regulating 
expression of the structural gene is substantially unaffected by the 
amount of sterol or squalene in the cell. As used herein, the term 
"substantially unaffected" means that the promoter is not responsive to 
direct feedback control by the sterols or squalene accumulated in 
transformed cells or transgenic plants. 
A promoter is also selected for its ability to direct the transformed plant 
cell's or transgenic plant's transcriptional activity to the structural 
gene encoding a polypeptide having HMG-CoA reductase activity. Structural 
genes can be driven by a variety of promoters in plant tissues. Promoters 
can be near-constitutive, such as the CaMV 35S promoter, or tissue 
specific or developmentally specific promoters affecting dicots or 
monocots. 
As exemplified and discussed in detail hereinafter, where the 
near-constitutive promoter CaMV 35S is used to transform tobacco plants, 
increases in total sterol and squalene accumulation are found in a variety 
of transformed plant tissues (e.g. callus, leaf, seed and root). 
Alternatively, the effects of transformation (e.g. increased amount of a 
gene coding for HMG-CoA reductase, increased total sterol accumulation and 
increased squalene accumulation) can be directed to specific plant tissues 
by using plant integrating vectors containing a tissue-specific promoter. 
An exemplary tissue-specific promoter is the Lectin promoter, which is 
specific for seed tissue. The Lectin protein in soybean seeds is encoded 
by a single gene (Le1) that is only expressed during seed maturation and 
accounts for about 2 to about 5 percent of total seed mRNA. The Lectin 
gene and seed-specific promoter have been fully characterized and used to 
direct seed specific expression in transgenic tobacco plants. See, e,g., 
Vodkin et al., Cell, 34:1023 (1983) and Lindstrom et al., Developmental 
Genetics, 11:160 (1990). 
A plant integrating vector containing a structural gene coding for a 
polypeptide having HMG-CoA reductase activity is engineered to be under 
control of the Lectin promoter and that vector is introduced into soybean 
plants using a protoplast transformation method. Dhir et al., Plant Cell 
Reports, 10:97 (1991). The expression of the polypeptide having HMG-CoA 
reductase activity is directed specifically to the seeds of the transgenic 
plant. In this way, a transgenic soybean seed having increased squalene 
accumulation is produced. Such seeds can then be used to extract oil 
containing enhanced levels of squalene. As set forth hereinafter, such 
squalene-enhanced oil is characterized by a greater thermal stability when 
compared to non-squalene-enhanced oil. 
A transgenic plant of the present invention produced from a plant cell 
transformed with a tissue specific promoter can be crossed with a second 
transgenic plant developed from a plant cell transformed with a different 
tissue specific promoter to produce a hybrid transgenic plant that shows 
the effects of transformation in more than one specific tissue. 
Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yang et 
al. Proc. Natl. Acad. Sci. U.S.A., 87:4144-48 (1990)), corn alcohol 
dehydrogenase 1 (Vogel et al., J. Cell Biochem., (supplement 13D, 312) 
(1989)), corn zein 19KD gene (storage protein) (Boston et al., Plant 
Physiol., 83:742-46), corn light harvesting complex (Simpson, Science, 
233:34 (1986), corn heat shock protein (O'Dell et al., Nature, 313:810-12 
(1985), pea small subunit RuBP Carboxylase (Poulsen et al., Mol. Gen. 
Genet., 205:193-200 (1986); Cushmore et al., Gen. Eng. of Plants, Plenum 
Press, New York, 29-38 (1983), Ti plasmid mannopine synthase (Langridge et 
al., Proc. Natl. Acad. Sci. USA, 86:3219-3223 (1989), Ti plasmid nopaline 
synthase (Langridge et al., Proc. Natl. Acad. Sci. USA, 86:3219-3223 
(1989), petunia chalcone isomerase (Van Tunen et al., EMBO J., 7:1257 
(1988), bean glycine rich protein 1 (Keller et al., EMBO J., 8:1309-14 
(1989), CaMV 35s transcript (O'Dell et al., Nature, 313:810-12 (1985) and 
Potato patatin (Wenzler et al., Plant Mol. Biol., 12:41-50 (1989). 
Preferred promoters are the cauliflower mosaic virus (CaMV 35S) promoter 
and the S-E9 small subunit RuBP carboxylase promoter. 
The choice of which plant integrating vector and ultimately to which 
promoter a polypeptide coding gene is operatively linked depends directly 
on the functional properties desired, e.g. the location and timing of 
protein expression, and the host cell to be transformed. These are well 
known limitations inherent in the art of constructing recombinant DNA 
molecules. However, a vector useful in practicing the present invention is 
capable of directing the expression of the polypeptide coding gene, i.e., 
the gene encoding HMG-CoA reductase activity, included in the DNA segment 
to which it is operatively linked. 
Typical vectors useful for expression of genes in higher plants are well 
known in the art and include vectors derived from the tumor-inducing (Ti) 
plasmid of Agrobacterium tumefaciens described by Rogers et al., Meth. in 
Enzymol., 153:253-277 (1987). However, several other plant integrating 
vector systems are known to function in plants including pCaMVCN transfer 
control vector described by Fromm et al., Proc. Natl. Acad. Sci. USA, 
82:5824 (1985). Plasmid pCaMVCN (available from Pharmacia, Piscataway, 
N.J.) includes the cauliflower mosaic virus CaMV 35S promoter. 
The use of retroviral plant integrating vectors to form the recombinant 
DNAs of the present invention is also contemplated. As used herein, the 
term "retroviral plant integrating vector" refers to a DNA molecule that 
includes a promoter sequence derived from the long terminal repeat (LTR) 
region of a retrovirus genome. 
In preferred embodiments, the vector used to express the polypeptide coding 
gene includes a selection marker that is effective in a plant cell, 
preferably a drug resistance selection marker. One preferred drug 
resistance marker is the gene whose expression results in kanamycin 
resistance; i.e., the chimeric gene containing the nopaline synthase 
promoter, Tn5 neomycin phosphotransferase II and nopaline synthase 3' 
nontranslated region described by Rogers et al., in Methods For Plant 
Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press 
Inc., San Diego, Calif. (988). 
A variety of methods has been developed to operatively link DNA to vectors 
via complementary cohesive termini or blunt ends. For instance, 
complementary homopolymer tracts can be added to the DNA segment to be 
inserted and to the vector DNA. The vector and DNA segment are then joined 
by hydrogen bonding between the complementary homopolymeric tails to form 
recombinant DNA molecules. 
Alternatively, synthetic linkers containing one or more restriction 
endonuclease sites can be used to join the DNA segment to the plant 
integrating vector. The synthetic linkers are attached to blunt-ended DNA 
segments by incubating the blunt-ended DNA segments with a large excess of 
synthetic linker molecules in the presence of an enzyme that is able to 
catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage 
T4 DNA ligase. Thus, the products of the reaction are DNA segments 
carrying synthetic linker sequences at their ends. These DNA segments are 
then cleaved with the appropriate restriction endonuclease and ligated 
into a plant integrating vector that has been cleaved with an enzyme that 
produces termini compatible with those of the synthetic linker. Synthetic 
linkers containing a variety of restriction endonuclease sites are 
commercially available from a number of sources including New England 
BioLabs, Beverly, Mass. 
Also contemplated by the present invention are RNA equivalents of the above 
described recombinant DNA molecules. 
A preferred recombinant DNA molecule utilized in accordance with the 
present invention is plasmid HMGR.DELTA.227-pKYLX71. 
E. Transformed Plant Cells, Transgenic Plants, Processes of Transformation 
and Processes of Regeneration 
The amount of a gene coding for a polypeptide having HMG-CoA reductase 
activity is increased by transforming a desired plant cell with a suitable 
vector that contains that added exogenous structural gene. Expression of 
that gene in the transformed plant cell and transgenic plants developed 
from that transformed plant cell enhances the activity of HMG-CoA 
reductase. 
Methods for transforming polypeptide-coding genes into plant cells include 
Agrobacterium-mediated plant transformation, protoplast transformation, 
gene transfer into pollen, injection into reproductive organs and 
injection into immature embryos. Each of these methods has distinct 
advantages and disadvantages. Thus, one particular method of introducing 
genes into a particular plant strain may not necessarily be the most 
effective for another plant strain, but it is well known which methods are 
useful for a particular plant strain. 
Agrobacterium-mediated transfer is a widely applicable system for 
introducing genes into plant cells because the DNA can be introduced into 
whole plant tissues, thereby bypassing the need for regeneration of an 
intact plant from a protoplast. The use of Agrobacterium-mediated plant 
integrating vectors to introduce DNA into plant cells is well known in the 
art. See, for example, the methods described by Fraley et al., 
Biotechnology, 3:629 (1985) and Rogers et al., Methods in Enzymology, 
153:253-277 (1987). Further, the integration of the Ti-DNA is a relatively 
precise process resulting in few rearrangements. The region of DNA to be 
transferred is defined by the border sequences, and intervening DNA is 
usually inserted into the plant genome as described by Spielmann et al., 
Mol. Gen. Genet., 205:34 (1986) and Jorgensen et al., Mol. Gen. Genet., 
207:471 (1987). 
Modern Agrobacterium transformation vectors are capable of replication in 
E. coli as well as Agrobacterium, allowing for convenient manipulations as 
described by Klee et al., in Plant DNA Infectious Agents, T. Hohn and J. 
Schell, eds., Springer-Verlag, New York (1985) pp. 179-203. 
Moreover, recent technological advances in vectors for 
Agrobacterium-mediated gene transfer have improved the arrangement of 
genes and restriction sites in the vectors to facilitate construction of 
vectors capable of expressing various polypeptide coding genes. The 
vectors described by Rogers et al., Methods in Enzymology, 153:253 (1987), 
have convenient multi-linker regions flanked by a promoter and a 
polyadenylation site for direct expression of inserted polypeptide coding 
genes and are suitable for present purposes. 
In addition, Agrobacteria containing both armed and disarmed Ti genes can 
be used for the transformations. Both types of transforming systems are 
illustrated herein. Transformants from the former system result in callus 
from which the desired squalene or sterol can be obtained, whereas 
transformants obtained from the latter, disarmed Ti genes can be 
regenerated into complete transgenic plants from whose tissues, e.g. leaf, 
seed and root, the desired chemicals can be obtained. 
In those plant strains where Agrobacterium-mediated transformation is 
efficient, it is the method of choice because of the facile and defined 
nature of the gene transfer. 
Agrobacterium-mediated transformation of leaf disks and other tissues such 
as cotyledons and hypocotyls appears to be limited to plant strains that 
Agrobacterium naturally infects. Agrobacterium-mediated transformation is 
most efficient in dicotyledonous plants. Few monocots appear to be natural 
hosts for Agrobacterium, although transgenic plants have been produced in 
asparagus using Agrobacterium vectors as described by Bytebier et al., 
Proc. Natl. Acad. Sci. USA, 84:5345 (1987). Therefore, commercially 
important cereal grains such as rice, corn, and wheat must usually be 
transformed using alternative methods. However, as mentioned above, the 
transformation of asparagus using Agrobacterium can also be achieved. See, 
for example, Bytebier, et al., Proc. Natl. Acad. Sci. USA, 84:5345 (1987). 
A transgenic plant formed using Agrobacterium transformation methods 
typically contains a single gene on one chromosome. Such transgenic plants 
can be referred to as being heterozygous for the added gene. However, 
inasmuch as use of the word "heterozygous" usually implies the presence of 
a complementary gene at the same locus of the second chromosome of a pair 
of chromosomes, and there is no such gene in a plant containing one added 
gene as here, it is believed that a more accurate name for such a plant is 
an independent segregant, because the added, exogenous gene segregates 
independently during mitosis and meiosis. A transgenic plant containing a 
single structural gene that encodes a polypeptide having HMG-CoA reductase 
activity; i.e., an independent segregant, is a preferred transgenic plant. 
More preferred is a transgenic plant that is homozygous for the added 
structural gene; i.e., a transgenic plant that contains two added genes, 
one gene at the same locus on each chromosome of a chromosome pair. A 
homozygous transgenic plant can be obtained by sexually mating (selfing) 
an independent segregant transgenic plant that contains a single added 
gene, germinating some of the seed produced and analyzing the resulting 
plants produced for enhanced HMG-CoA reductase activity, sterol 
accumulation, or squalene accumulation or all three, relative to a control 
(native, non-transgenic) or an independent segregant transgenic plant. A 
homozygous transgenic plant exhibits enhanced HMG-CoA reductase activity, 
sterol and squalene accumulation as compared to both a native, 
non-transgenic plant and an independent segregant transgenic plant. 
It is to be understood that two different transgenic plants can also be 
mated to produce offspring that contain two independently segregating 
added, exogenous genes. Selfing of appropriate progeny can produce plants 
that are homozygous for both added, exogenous genes that encode a 
polypeptide having HMG-CoA activity. Back-crossing to a parental plant and 
out-crossing with a non-transgenic plant are also contemplated. 
Transformation of plant protoplasts can be achieved using methods based on 
calcium phosphate precipitation, polyethylene glycol treatment, 
electroporation, and combinations of these treatments. See, for example, 
Potrykus et al., Mol. Gen. Genet., 199:183 (1985); Lorz et al., Mol. Gen. 
Genet., 199:178 (1985); Fromm et al., Nature, 319:791 (1986); Uchimiya et 
al., Mol. Gen, Genet., 204:204 (1986); Callis et al., Genes and 
Development, 1:1183 (1987); and Marcotte et al., Nature, 335:454 (1988). 
Application of these systems to different plant strains depends upon the 
ability to regenerate that particular plant strain from protoplasts. 
Illustrative methods for the regeneration of cereals from protoplasts are 
described in Fujimura et al., Plant Tissue Culture Letters, 2:74 (1985); 
Toriyama et al., Theor Appl. Genet., 73:16 (1986); Yamada et al., Plant 
Cell Rep., 4:85 (1986); Abdullah et al., Biotechnology, 4:1087 (1986). 
To transform plant strains that cannot be successfully regenerated from 
protoplasts, other ways to introduce DNA into intact cells or tissues can 
be utilized. For example, regeneration of cereals from immature embryos or 
explants can be effected as described by Vasil, Biotechnology, 6:397 
(1988). In addition, "particle gun" or high-velocity microprojectile 
technology can be utilized. 
Using that latter technology, DNA is carried through the cell wall and into 
the cytoplasm on the surface of small metal particles as described in 
Klein et al., Nature, 327:70 (1987); Klein et al., Proc. Natl. Acad. Sci. 
U.S.A., 85:8502 (1988); and McCabe et al., Biotechnology, 6:923 (1988). 
The metal particles penetrate through several layers of cells and thus 
allow the transformation of cells within tissue explants. 
Metal particles have been used to successfully transform corn cells and to 
produce fertile, stable transgenic tobacco plants as described by 
Gordon-Kamm, W. J. et al., The Plant Cell, 2:603-618 (1990); Klein, T. M. 
et al., Plant Physiol., 91:440-444 (1989); Klein, T. M. et al., Proc. 
Natl. Acad. Sci. USA, 85:8502-8505 (1988); and Tomes, D. T. et al., Plant 
Mol. Biol., 14:261-268 (1990). Transformation of tissue explants 
eliminates the need for passage through a protoplast stage and thus speeds 
the production of transgenic plants. 
Thus, the amount of a gene coding for a polypeptide having HMG-CoA 
reductase activity can be increased in monocotyledonous plants such as 
corn by transforming those plants using particle bombardment methods. 
Maddock et al., Third International Congress of Plant Molecular Biology, 
Abstract 372 (1991). By way of example, a plant integrating vector 
containing a structural gene for HMG-CoA reductase and an appropriate 
selectable marker is transformed into a suspension of embryonic maize 
(corn) cells using a particle gun to deliver the DNA coated on 
microprojectiles. Transgenic plants are regenerated from transformed 
embryonic calli that express HMG-CoA reductase. 
DNA can also be introduced into plants by direct DNA transfer into pollen 
as described by Zhou et al., Methods in Enzymology, 101:433 (1983); D. 
Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., Plant Mol. Biol,. 
Reporter, 6:165 (1988). Expression of polypeptide coding genes can be 
obtained by injection of the DNA into reproductive organs of a plant as 
described by Pena et al., Nature, 325:274 (1987). DNA can also be injected 
directly into the cells of immature embryos and the rehydration of 
desiccated embryos as described by Neuhaus et al., Theor. Appl. Genet., 
75:30 (1987); and Benbrook et al., in Proceedings Bio Expo 1986, 
Butterworth, Stoneham, Mass., pp. 27-54 (1986). 
The development or regeneration of plants from either single plant 
protoplasts or various explants is well known in the art. See, for 
example, Methods for Plant Molecular Biology, A. Weissbach and H. 
Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). This 
regeneration and growth process typically includes the steps of selection 
of transformed cells, culturing those individualized cells through the 
usual stages of embryonic development through the rooted plantlet stage. 
Transgenic embryos and seeds are similarly regenerated. The resulting 
transgenic rooted shoots are thereafter planted in an appropriate plant 
growth medium such as soil. 
The development or regeneration of plants containing the foreign, exogenous 
gene that encodes a polypeptide having HMG-CoA activity introduced by 
Agrobacterium from leaf explants can be achieved by methods well known in 
the art such as described by Horsch et al., Science, 227:1229-1231 (1985). 
In this procedure, transformants are cultured in the presence of a 
selection agent and in a medium that induces the regeneration of shoots in 
the plant strain being transformed as described by Fraley et al., Proc. 
Natl. Acad. Sci. U.S.A., 80:4803 (1983). 
This procedure typically produces shoots within two to four months and 
those shoots are then transferred to an appropriate root-inducing medium 
containing the selective agent and an antibiotic to prevent bacterial 
growth. Shoots that rooted in the presence of the selective agent to form 
plantlets are then transplanted to soil or other media to allow the 
production of roots. These procedures vary depending upon the particular 
plant strain employed, such variations being well known in the art. 
Mature regenerated transgenic plants are obtained that exhibit increased 
sterol or squalene accumulation due to expression of the HMG-CoA reductase 
polypeptide gene. Preferably, the regenerated plants are self-pollinated 
to provide homozygous transgenic plants, as discussed before. Otherwise, 
pollen obtained from the regenerated plants is crossed to seed-grown 
plants of agronomically important, preferably inbred lines. Conversely, 
pollen from plants of those important lines is used to pollinate 
regenerated plants. The presence of the added gene in the progeny is 
assessed as discussed hereinafter. 
A transgenic plant of the present invention containing a desired HMG-CoA 
reductase polypeptide is cultivated using methods well known to one 
skilled in the art. Any of the transgenic plants of the present invention 
can be cultivated to isolate the desired sterol or squalene products they 
contain. 
A transgenic plant of this invention thus has an increased amount of a 
structural gene that encodes a polypeptide having HMG-CoA reductase 
activity. A preferred transgenic plant is an independent segregant for the 
added HMG-CoA reductase structural gene and can transmit that gene and its 
activity to its progeny. A more preferred transgenic plant is homozygous 
for that gene, and transmits that gene to all of its offspring on sexual 
mating. 
A transgenic plant of the invention accumulates sterols and, particularly 
non-delta-5 sterols relative to a native plant. A transgenic plant of the 
invention also accumulates squalene relative to a native, non-transgenic 
plant. A transgenic plant also exhibits resistance to pests such as the 
hornworms and budworms as is discussed hereinafter. 
F. Development of Commercial Hybrid Seed 
Seed from a transgenic plant is grown in the field or greenhouse, and 
resulting sexually mature transgenic plants are self-pollinated to 
generate true breeding plants. The progeny from these plants become true 
breeding lines that are evaluated for sterol or squalene accumulation, 
preferably in the field, under a range of environmental conditions. 
The commercial value of a transgenic plant with increased sterol or 
squalene accumulation is enhanced if many different hybrid combinations 
are available for sale. The user typically grows more than one kind of 
hybrid based on such differences as time to maturity, standability or 
other agronomic traits. Additionally, hybrids adapted to one part of a 
country are not necessarily adapted to another part because of differences 
in such traits as maturity, disease and herbicide resistance. Because of 
this, sterol or squalene accumulation is preferably bred into a large 
number of parental lines so that many hybrid combinations can be produced. 
Adding an enhanced sterol or squalene accumulation trait to an 
agronomically elite line is accomplished by a variety of techniques well 
known to those of skill in the art. For example, parent transgenic plants 
that are either homozygous or contain a single independent segregatable 
gene that encodes a polypeptide having HMG-CoA activity and thus for 
enhanced sterol or squalene accumulation are crossed with lines having 
other desirable traits, such as herbicide resistance (U.S. Pat. No. 
4,761,373) produce hybrids. Preferably, transgenic plants homozygous for 
enhanced sterol or squalene accumulation are used to generate hybrids. 
For example, a transgenic plant homozygous for enhanced sterol accumulation 
is crossed with a parent plant having other desired traits. The progeny, 
which are heterozygous or independently segregatable for enhanced sterol 
accumulation, are backcrossed with the parent to obtain transgenic plants 
having enhanced sterol accumulation and the other desired traits. The 
backcrossing of progeny with the parent may have to be repeated more than 
once to obtain a transgenic plant that possesses all desirable traits. 
Alternatively, transgenic plants with an enhanced sterol or squalene 
accumulation trait are made multiply transgenic by introducing into such 
plants other genes that encode and express other desirable traits, or are 
mutated as with radiation, e.g. X-rays or gamma rays, as in U.S. Pat. No. 
4,616,099, whose disclosures are incorporates by reference. Thus, the 
present invention also contemplates mutants and genetically engineered 
derivatives of transgenic plants having enhanced sterol or squalene 
accumulation. 
G. Accumulation of Sterols in Transgenic Plants 
The present invention provides processes for increasing the accumulation of 
sterols, particularly non-delta-5 sterols, in transgenic plants. This is 
accomplished by increasing the amount of a gene encoding for a polypeptide 
having HMG-CoA reductase activity and subsequent expression of that 
encoded polypeptide. 
In native, non-transgenic plants sterol accumulation is usually equal to 
about 0.3 weight percent of the dry weight on the plant. The predominant 
sterols accumulated by such normal plants are campesterol, sitosterol and 
stigmasterol. These sterols, .DELTA.5-derivatives of cycloartenol that 
have undergone desaturation of the 5(6) carbon-carbon bond of 
cycloartenol, comprise over 80 weight percent of total sterols in native 
plants. Cycloartenol normally comprises from about 3 to about 30 percent 
of the total sterols present in such a plant. 
Transgenic plants having an increased amount of a gene encoding a 
polypeptide having HMG-CoA reductase activity demonstrate a marked 
increase in total sterol accumulation when compared to a native, 
non-transgenic plant of the same strain. Further, the predominant sterol 
found in such transgenic plants is cycloartenol, which represents from 
about 60 to about 70 weight percent of total sterols of a transgenic 
plant. 
Thus, the present invention provides transgenic plants that overaccumulate 
sterols relative to a native, non-transgenic plant. Transgenic plants with 
a single added gene accumulate total sterol to a level about twice that 
found in native, non-transgenic plants. In particular, such transgenic 
plants accumulate non-delta-5 sterols (e.g. cycloartenol) to a level from 
about ten to about one hundred times greater than found in native, 
non-transgenic plants. 
These results are surprising and unexpected in light of studies relating 
HMG-CoA reductase activity and sterol accumulation in other organisms. 
In yeast, increases in HMG-CoA reductase activity are associated with 
increases in squalene, 4,14-dimethylzymosterol and 14-methylfecosterol. 
Downing et al., Biochemical and Biophysical Research Communications, 
94(3): 974-979 (1980). Increases in HMG-CoA reductase activity of yeast 
were not associated with increases in lanosterol, (a sterol of yeast 
analogous to cycloartenol). Benveniste, Ann. Rev. Plant Physiol., 
37:275-308 (1986). 
In non-photosynthetic microorganisms, light-induced increases in HMG-CoA 
reductase activity were not associated with increases in sterol 
accumulation. Tada et al., Plant and Cell Physiology, 23(4):615-621(1982). 
H. Increased Squalene Accumulation 
The present invention provides processes for increasing the accumulation of 
squalene in transgenic plants. This is accomplished by increasing the 
amount of a gene encoding for a polypeptide having HMG-CoA reductase 
activity and subsequent expression of that encoded polypeptide in the 
transgenic plant. 
Squalene has use as a bactericide, a pharmaceutical intermediate, and 
cosmetic ingredient. Further, enhanced squalene levels in or on rind can 
serve to protect citrus fruit against the harmful effects of chilling and 
freezing. 
There is an inverse relationship between squalene levels in the 
epicuticular wax of grapefruit and severity of chilling injury in that 
fruit. Norby and McDonald, Lipids, 25:807-810 (1990), J. Agric. Food 
Chem., 39:957-962 (1991), and U.S. Pat. No. 4,921,715. Further, where 
squalene was applied as a spray or dip to grapefruit, it prevented chill 
injury. Norby and McDonald, Hortscience, 25:94 (1990). 
In a preferred embodiment, the present invention provides processes for 
increasing the accumulation of squalene in a transgenic citrus plant. This 
is accomplished by increasing the amount of a gene encoding for 
a-polypeptide having HMG-CoA reductase activity and subsequent expression 
of that encoded polypeptide in the transgenic plant. 
The amount of a gene encoding for a polypeptide having HMG-CoA reductase 
activity is increased in a citrus plant by transforming a citrus plant 
cell in accordance with a process of the present invention. Means for 
transforming citrus plant cells using Agrobacterium-mediated 
transformation techniques are well known in the art. 
Still further, squalene is reported to improve the heat stability of 
vegetable oils. The addition of squalene to rapeseed oil was found to 
retard the formation of thermally unstable polar compounds in rapeseed oil 
heated to about 170.degree. C. for about 10 hours. Malecka, N., Die 
Nahrung, 35(5):541 (1991). 
Transgenic tobacco plant seeds of the present invention have an increased 
accumulation of squalene when compared to seeds of a native, 
non-transgenic seed (See Example 10 hereinafter). Thus, in another aspect, 
the present invention contemplates transgenic plant seeds whose oil 
contains an increased accumulation of squalene when compared to oil 
obtained from a native, non-transgenic seed. 
In native, non-transgenic plants squalene accumulation is less than about 
0.01 weight percent of the dry weight on the plant. In transgenic plants 
of the present invention squalene accumulation increases to a level of 
from about 0.115 weight percent of dry weight (tobacco and soybean) to 
0.56 weight percent (cotton) (See Examples 5-9 hereinafter). 
I. Harvesting of Sterols and Squalene 
If desired, after cultivation, the transgenic plant is harvested to recover 
the sterol or squalene product. This harvesting step can consist of 
harvesting a callus culture, the entire plant, or only the leaves, or 
roots of the plant. This step can either kill the plant or, if only a 
non-essential portion of the transgenic plant is harvested, can permit the 
remainder of the plant to continue to grow. 
In preferred embodiments, this harvesting step further comprises the steps 
of: 
(i) homogenizing at least a sterol-containing or a squalene-containing 
portion of the transgenic plant to produce a plant pulp and using the 
sterol- or squalene-containing pulp directly, as in dried pellets or 
tablets as where an animal food is contemplated; or 
(ii) extracting the squalene or sterol(s) from the plant pulp with an 
appropriate solvent such as an organic solvent or by supercritical 
extraction [Favati et al., J. Food Sci., 53:1532 (1988) and the citations 
therein] to produce a sterol- or squalene-containing liquid solution or 
suspension; and 
(iii) isolating the squalene or sterol(s) from the solution or suspension. 
At least a portion of the transgenic plant is homogenized to produce a 
plant pulp using methods well known to one skilled in the art. This 
homogenization can be done manually, by a machine, or by a chemical means 
as long as the transgenic plant portions are broken up into small pieces 
to produce a plant pulp. This plant pulp consists of a mixture of squalene 
or the sterol of interest, residual amounts of precursors, cellular 
particles and cytosol contents. This pulp can be dried and compressed into 
pellets or tablets and eaten or otherwise used to derive the benefits, or 
the pulp can be subjected to extraction procedures. 
The sterol or squalene can be extracted from the plant pulp produced above 
to form a sterol-or squalene-containing solution or suspension. Such 
extraction processes are common and well known to one skilled in this art. 
For example, the extracting step can consist of soaking or immersing the 
plant pulp in a suitable solvent. This suitable solvent is capable of 
dissolving or suspending the squalene or sterol present in the plant pulp 
to produce a sterol- or squalene-containing solution or suspension. 
Solvents useful for such an extraction process are well known to those 
skilled in the art and include several organic solvents and combinations 
thereof such as methanol, ethanol, isopropanol, acetone, acetonitrile, 
tetrahydrofuran (THF), hexane, and chloroform as well as water-organic 
solvent mixtures. A vegetable oil such as peanut, corn, soybean and 
similar oils can also be used for this extraction as can steam 
distillation. 
A whole plant or callus culture with an added, exogenous structural gene 
for a polypeptide having HMG-CoA reductase activity is grown under 
suitable conditions for a period of time sufficient for squalene or 
sterols to be synthesized and accumulated. The sterol- or 
squalene-containing plant cells, preferably in dried form, are then lysed 
chemically or mechanically, and the squalene or sterol is extracted from 
the lysed cells using a liquid organic solvent or steam distillation, as 
described before, to form a sterol- or squalene-containing liquid solution 
or suspension. The squalene or sterol is thereafter isolated from the 
liquid solution or suspension by usual means such as chromatography. 
The squalene or sterol is isolated from the solution or suspension produced 
above using methods that are well known to those skilled in the art of 
squalene and sterol isolation. These methods include, but are not limited 
to, purification procedures based on solubility in various liquid media, 
chromatographic techniques such as column chromatography and the like. 
J. Pest Resistance of Transgenic Plants 
Certain sterols accumulated by a transgenic plant of the present invention 
have use as systemic insecticidal or pesticidal agents. As set forth 
before, because insects are unable to synthesize de novo the steroid 
nucleus, they depend upon external, dietary sources of delta-5 sterols for 
production of necessary steroid compounds such as ecdysteroids. See, e.g., 
Costet et al., Proc. Natl. Acad. Sci. USA, 84:643 (1987) and Corio-Costet 
et al., Archives of Insect Biochem. Physiol., 11:47 (1989). 
This embodiment of the present invention relates to a process of increasing 
pest resistance of a transgenic plant comprising transforming a plant cell 
of a native, non-transgenic plant with a recombinant DNA molecule 
comprising a vector operatively linked to a DNA segment that encodes the 
catalytic region of HMG-CoA reductase, and a promoter suitable for driving 
the expression of said reductase in that plant, and regenerating a 
transgenic plant from the transformed plant cell. In preferred practice, 
the DNA segment also encodes at least a portion of the linker region but 
not the membrane binding region of HMG-CoA reductase. Use of the hamster 
gene is particularly preferred. The resulting transgenic plant exhibits 
enhanced resistance to insect pests. 
A transgenic plant is then preferably grown to sexual maturity and used to 
transmit its enhanced pest resistance to its offspring. Transgenic plants 
can also themselves be used agriculturally. 
Tobacco hornworm larvae grown on the leaves of transgenic plants 
regenerated from plant cells transformed with a truncated hamster HMG-CoA 
reductase gene, which transgenic plants have increased levels of 
non-delta-5 sterol and, particularly cycloartenol, demonstrated retarded 
development. Preliminary studies also indicate that tobacco bud worms 
(Heliothis virescens) fed on leaves of a similar transgenic plant 
exhibited retarded development under similar conditions. 
Other insects such as Locusta migratoria show marked developmental arrest 
and growth abnormalities when reared on plants deficient in delta-5 
sterols. Costet et al., Proc. Soc. Natl. Acad. Sci., USA, 84:643 (1987); 
Corro-Costet et al., Archives Insect Biochem. Physiol., 11:47 (1989). 
Further, initial feeding studies show that the growth and development at 
various stages of Heliothis and European corn borer, Ostrinia nubialis, 
are markedly inhibited by feeding those insect pests on artificial diets 
having reduced levels of delta-5 sterols and increased levels of 
non-delta-5 sterols. 
K. Harvesting of Transgenic Seed Oil 
Oil is extracted from transgenic plant seeds of the present invention by 
methods well known in the art. By way of example, oil can be extracted 
from plant seeds using extraction methods set forth above for harvesting 
sterols and squalene from transgenic plants. Alternatively, oil can be 
extracted from transgenic plant seeds by usually used methods for 
obtaining seed oils such as by crushing the seeds to produce a pulp and 
then pressing the pulp to obtain oil. The pulp can also be extracted with 
appropriate solvents (e.g. benzene) to obtain the oil. Industrial 
Chemistry: A Manual for the Student and Manufacturer, ed. by A. Rogers and 
A. B. Aubert, D. Van Nostrand Co., New York, pages 547-548 (1912). 
The following examples illustrate the best mode of carrying out the 
invention and are not to be construed as limiting of the specification and 
claims in any way. 
BEST MODE FOR CARRYING OUT THE INVENTION 
EXAMPLE 1 
Transformation of Plant Cells 
Plant cells were transformed in accordance with standard methods for 
expressing foreign genes in plants. Schardl et al., Gene, 61:1-11 (1987). 
A pKYLX series of vectors was used as the expression system. Preferred 
vectors are plasmids pKYLX6 and pKYLX7. Berger et al., Proc. Natl. Acad. 
Sci. USA, 86:8402-8406 (1989). 
Transformations were performed with a truncated Hamster HMG-CoA reductase 
gene (HMGR-.DELTA.227) obtained from the laboratories of Dr. J. L. 
Goldstein, See, e.g., Gil et al., Cell, 41:249-258(1985); Bard et al., 
Journal of General Microbiology, 125:415-420(1981). 
The HMGR-.DELTA.227 gene was incorporated into modified vectors pKYLX6 (an 
E. coli vector designed for intermediate constructs) and pKYLX7 (an A. 
tunefaciens vector designed for integration of cloned genes). Berger et 
al., Proc. Natl. Acad. Sci. USA, 86:8402-8406 (1989). The modified vectors 
pKYLX61 and pKYLX71 contained Hind III, Xho I, Bam HI, Pst I, and Sst I 
sites in place of the original Hind III Sst I fragment multiple cloning 
site region. 
The HMGR-.DELTA.227 gene was digested with Bam HI and Sst I, and the 
approximately, 2500 bp HMGR-.DELTA.227-Bam HI-Sst I fragment was inserted 
into plasmid pKYLX61. The resulting HMGR.DELTA.227-pKYLX61 construct was 
cleaved with Eco RI and Cla I, and an approximately 4000 bp fragment 
containing the promoter-gene-terminator portion was inserted into 
corresponding sites of pKYLX71 to generate plasmid HMGR.DELTA.227-pKYLX71 
(see FIG. 5). In plasmid HMGR.DELTA.227-KYLX71, the truncated 
HMGR-.DELTA.227 gene is under control of the strong, constitutive CaMV 35S 
promoter. 
The HMGR.DELTA.227-pKYLX71 plasmid was mobilized into Agrobacterium 
tumefaciens by a standard triparental mating between E. coli, harboring 
the HMGR.DELTA.227-pKYLX71 construct, Argrobacterium tumefaciens, 
harboring a disarmed Ti-plasmid, GV3850, and E. coli harboring the 
conjugation helper plasmid pRK2013. See, e.g., Schardl, et al., Gene, 
61:1-11 (1987); Ditta et al., Proc. Natl. Acad. Sci. USA 77:7347-7351 
(1980). As a result of the cross, Agrobacterium harboring the 
HMGR.DELTA.227-pKYLX71 construct, was selected for by resistance to 
rifampicin (encoded on the chromosome of Agrobacterium), and to 
tetracycline and kanamycin (encoded on the pKYLX71 vector). 
Alternatively, the HMGR .DELTA.-227-KYLX71 plasmid is mobilized into 
Agrobacterium tumefaciens strain 281, which contains a fully armed T-DNA 
plasmid to form a binary plasmid strain A281-.DELTA.227. See, e.g., 
Schardl et al. Gene, 61:1-11 (1987) and Montoya et al., J. Bacteriol., 
129:101 (1977) (See Example 7 hereinafter). 
Nicotiana tabacum L. cv. xanthii (N. tabacum) was transformed by the well 
known "leaf disk method". Horsch et al., Science 27:1229-1231 (1985). Leaf 
disks were incubated with Agrobacteria containing plasmid 
.DELTA.227-pKYLX71 for about 3 days. Transformed tissue was selected for 
by resistance to kanamycin (encoded by the pKYLX71 vector), cured of 
Agrobacteria using the antibiotic mefoxin, and regenerated into whole 
plants. Horsch et al., Science, 27:1229-1231 (1985). 
Transgenic plant tissue was checked for the presence of integrated copies 
of the HMGR .DELTA.227 gene sequences by the method of Mettler, Plant Mol. 
Biol. Reporter, 5:346-349 (1987). RNA transcription levels were determined 
by northern blotting or S-1 protection assays. Maniatis et al., Molecular 
Cloning: A Laboratory Manual, Cold Spring Harbour Lab., Cold Spring 
Harbour, N.Y. (1982). 
Transgenic plants exhibiting HMG-CoA reductase activity greater than 
native, non-transgenic (control) plants or transgenic plants regenerated 
from plant cells transformed without the HMGR-.DELTA.227-construct were 
sexually crossed with themselves, to generate progeny. 
EXAMPLE 2 
HMG-CoA Reductase Enzyme Activity in Transgenic Plants 
Transgenic plants were screened for expression of the truncated HMGR gene 
by examining HMG-CoA reductase activity in the 100,000xG supernatant of 
lysed cells using a standard assay, Chappell et al., Plant Physiol., 
85:469-473 (1987). 
Soluble HMG-CoA reductase enzyme activity was measured in callus cultures 
grown on selection (kanamycin) medium, seedlings germinated in the 
presence of kanamycin or on moistened filter paper, and leaves of various 
sizes from plants grown in the greenhouse. Examplary results of studies of 
HMG-CoA reductase activity in leaves from greenhouse-grown plants are also 
summarized in Table 2 below: 
TABLE 2 
______________________________________ 
Total HMG-CoA 
Plant Reductase Activity 
% of 
Sample No. (pmol/hr./leaf) 
Control 
______________________________________ 
Control 
30 258 100 
Transgenic 
5 860 300 
14 1,100 390 
15 633 220 
18 456 160 
23 713 250 
______________________________________ 
The control plant, 30, was transformed with a selection marker but not with 
the .DELTA.227 gene. Transgenic plants 5, 14, 15, 18 and 23 were 
regenerated from plant cells transformed with the HMGR-.DELTA.227 gene, as 
discussed above. 
Total HMG-CoA reductase activity was 1.6 to 3.9 times greater in transgenic 
plants harboring the .DELTA.227 gene as compared to the control plant. 
EXAMPLE 3 
Sterol Accumulation in Transgenic Plants 
Regenerated, transgenic N. tabacum, from cells transformed with the 
HMGR-.DELTA.227 gene according to the process of Example 1 were analyzed 
for total sterol content. The results are presented in Table 3. 
TABLE 3 
______________________________________ 
Plant HMG-CoA Reductase 
Total Sterols 
Sample (pmol mg dry wt.) 
(% of dry wt) 
______________________________________ 
Control 2.00 .+-. 0.19 0.27 .+-. 0.02 
Plants 
(n = 6) 
Transgenic 5.75 .+-. 1.55 0.89 .+-. 0.17 
Plants 
(n = 12) 
______________________________________ 
Transgenic plants had elevated HMG-CoA reductase activity and increased 
sterol content. 
In addition to determining total sterol content, transgenic N. tabacum were 
examined for the accumulation of squalene and specific sterols. The 
designated plant tissues were lyophilized and heated with agitation in an 
alcohol/water solution containing potassium hydroxide to effect extraction 
and saponification of sterols and sterol esters. Free sterols were then 
extracted into heptane and measured by gas chromatography using an 
internal standard. The results of such an analysis in a control (Cntrl) 
and a transgenic (Trg) plant are presented in Table 4. 
TABLE 4 
______________________________________ 
Percent Dry Weight of Squalene and Sterols 
Accumulated 
Callus Leaf Root 
Product Cntrl Trg Cntrl Trg Cntrl 
Trg 
______________________________________ 
Squalene tr 0.025 0.01 0.126 tr 0.019 
Sterols 
Campesterol 
0.009 0.021 0.057 0.056 0.058 
0.022 
Cholesterol 
0.004 tr tr tr tr tr 
Cycloartenol 
0.003 0.258 0.011 0.678 0.039 
0.642 
Sitosterol 
0.027 0.077 0.083 0.187 0.029 
0.194 
Stigmasterol 
0.003 0.012 0.132 0.078 tr 0.238 
______________________________________ 
tr = trace (&lt;0.001 percent dry wt.) 
In the control plant, cycloartenol represented from about 3(0.011/0.283) 
percent dry weight (leaf) to about 30(0.039/0.126) percent dry weight 
(root) of total sterol accumulation. The predominant sterols accumulated 
by control plants (i.e. sitosterol, campesterol) are .DELTA.5-sterol 
derivatives of cycloartenol that have undergone additional metabolic 
transformation. 
As a result of transformation with the HMGR-.DELTA.227 gene, the ratio of 
cycloartenol to its derivatives is reversed. In transgenic plants, 
cycloartenol accumulation represents from about 60 (root) to about 70 
(leaf) percent by weight of total sterol accumulation. 
These data show that transgenic plants of the present invention 
overaccumulate sterols relative to a native, non-transgenic plant. 
Transgenic, heterozygous plants overaccumulate total sterols to a level 
about twice that found in a native plant. The data further show that 
transgenic plants containing a single added, exogenous gene 
over-accumulate cycloartenol to a level about ten to about one hundred 
times greater than found in a native plant. 
EXAMPLE 4 
Insecticidal Effects of Transgenie Plants 
First instar larvae of the tobacco pests tobacco hornworm (Manduca sexta), 
were placed onto leaves of control or HMGR-.DELTA.227 transgenic N. 
tabacum on a moistened filtered paper in a petri dish. Additional leaf 
material, from control or transgenic plants, was added to each dish, and 
the larvae were grown for an additional 7 days. Larvae were then examined 
to determine growth and development. The results are presented in Table 5. 
TABLE 5 
______________________________________ 
Control 
Transgenic 
(n = 14) 
(n = 13) 
______________________________________ 
Development 
% of larvae in 28.6 100 
second instar 
% of larvae in 71.4 0 
premolt or third instar 
Growth 
Fresh Wet Weight (mg) 
42.8 24.4 
______________________________________ 
Tobacco hornworm (Manduca sexta) larvae grown on leaves from transgenic 
plants (from HMGR-.DELTA.227-transformed cells) demonstrated retarded 
development (no progression beyond the second instar stage) and inhibited 
growth (wet weight) as compared to controls. The cycloartenol levels of 
the control and transgenic plants used in this study were 0,017 and 1.02 
percent of dry leaf weight, respectively. This study thus illustrates both 
the process of increasing the accumulation of cycloartenol in a plant and 
of enhancing pest resistance in a plant. 
Preliminary studies with a member of the Heliothis group of insect pests, 
the tobacco bud worm (Heliothis virescens), indicate a slower growth rate 
for insects fed on leaves of transgenic plant 14 (Example 2) than on 
leaves of the native, non-transgenic, control plant 30 (Example 2). 
EXAMPLE 5 
Homozygous Transgenic Plants 
One of the previously described transformed plants, plant 14 of Example 2, 
was selfed; i.e., sexually mated with itself. 
Twelve seeds from that cross were germinated and raised into plants. The 
tissues of those siblings were then analyzed for HMG-CoA reductase 
activity, total squalene (squalene plus squalene monoepoxide), and total 
sterol content as described in Example 3. The specific activity of HMG-CoA 
reductase was also calculated. The results of that assay compared to 
similar data from siblings from a selfing control plant 30 (Example 2) are 
presented in Table 6, below. 
TABLE 6 
______________________________________ 
HMGR Specific 
Plant Activity.sup.1 
Protein.sup.2 
Activity.sup.3 
Sterols.sup.4 
Squalene.sup.5 
______________________________________ 
30-1 3.78 30.22 184 0.20 &lt;0.01 
30-2 2.20 30.00 146 0.25 &lt;0.01 
30-3 1.44 18.70 154 0.29 &lt;0.01 
30-4 2.13 23.67 180 0.31 &lt;0.01 
30-5 1.70 19.27 176 0.36 &lt;0.01 
30-6 1.77 19.32 183 0.22 &lt;0.01 
14-1 1.36 23.60 115 0.21 0.142 
14-2 2.07 26.55 156 0.17 0.127 
14-3 10.28 17.60 1168 1.10 0.101 
14-4 7.08 27.25 520 0.74 0.114 
14-5 4.13 20.92 394 1.59 0.107 
14-6 1.58 11.00 143 0.25 0.086 
14-7* 20.35 16.77 2426 2.05 0.119 
14-8 4.87 24.20 402 0.97 0.174 
14-9 2.37 12.95 366 0.19 0.126 
14-10 7.94 11.00 1444 1.02 0.075 
14-11 2.56 15.25 334 1.10 0.082 
14-12 4.39 21.10 416 1.29 0.130 
______________________________________ 
.sup.1 pmoles/0.5 hours. 
.sup.2 micrograms (.mu.g). 
.sup.3 pmoles of enzyme/hour/mg of total protein. 
.sup.4 percentage of dry weight. 
.sup.5 squalene plus squalene monoepoxide (percentage of dry weight 
*this plant died. 
The phenotype for altered sterol composition segregated in a standard 
Mendelian manner with a ratio of three plants containing the elevated 
HMG-CoA reductase activity to one plant lacking the elevated HMG-CoA 
reductase activity. 
On the basis of the above data, the plants were classified as (a) having no 
added HMG-CoA reductase gene, or (b) containing the added gene. 
Illustratively, plant 14-2 was thus determined to lack the added gene and 
plant 14-8 was determined to contain the added gene. 
Southern blot analyses were performed on the transformed plants and 
confirmed the presence of the integrated gene. 
These data show that seeds from a transformed plant are capable of 
expressing enhanced squalene and sterol accumulation. 
Taken together with the data of Example 3, these data show that the 
transgenic plants of the present invention overaccumulate squalene, total 
sterol, and particularly non-utilizable sterols relative to a native plant 
and that such plants are capable of producing seeds, which germinate into 
transgenic plants that overaccumulate squalene and those sterols. 
Seeds from a selfing of transgenic plant 14-8 were deposited pursuant to 
the Budapest Treaty requirements with the American Type Culture Collection 
(ATCC) at 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. on Sep. 28, 
1990, and were assigned accession number ATCC 40904. 
The above deposit is made for a term of at least thirty (30) years and at 
least five (5) years after the most recent request for the furnishing of a 
sample of the deposit was received by the depository. 
EXAMPLE 6 
Transformation of Cotton 
The cotton plant cell line Coker 312-5A was transformed with 
HMGR-.DELTA.227 gene incorporated into plasmid pKYLX6. Transformation by 
Agrobacterium-mediated gene transfer was accomplished using the method of 
Trolinder et al., Plant Cell Reports, 6:231-234 (1987). 
Total sterol, delta-5 sterol, non-delta-5 sterol and squalene levels were 
determined in control, non-transgenic and transgenic calli as described in 
Example 3. The results of those studies are presented below in Table 7. 
TABLE 7 
______________________________________ 
Total Non-Util Util 
Plant Sterol Sterol.sup.1 
Sterol.sup.2 
Squalene 
______________________________________ 
Control 0.16 0.03 0.13 &lt;0.002 
Tr-1 0.48 0.39 0.09 0.2 
Tr-2 0.53 0.44 0.09 0.39 
Tr-3 0.79 0.72 0.07 1.1 
______________________________________ 
*all data are expressed as percentage of dry weight. Tr1, Tr2, and Tr3 ar 
transgenic calli one, two and three 
.sup.1 nondelta-5 sterols 
.sup.2 delta5 sterols 
These data show that transgenic cotton calli of the present invention 
overaccumulate squalene, total sterol and non-utilizable sterols relative 
to native, non-transgenic calli. 
EXAMPLE 7 
Transformation of Soybean 
The HMGR 227-KYLX71 plasmid was mobilized into Agrobacterium tumefaciens 
strain 281. Strain 281 contains a fully armed T-DNA plasmid. Montoya et 
al., J. Bacteriol., 129:101-107 (1977). 
The resulting strain A281:227 is a binary plasmid strain. Upon 
transfection, transformed plant cells proliferate as an undifferentiated 
transgenic callus since the tumor inducing (Ti) genes are transferred with 
KYLX-227 as cointegrates. The advantage of this system is that no 
selection is needed for transformants because they are self-proliferating. 
This is particularly advantageous in strains that have low transformation 
frequency and would not hold up well under stringent selection pressure. 
This expands the possible host range, including even woody strains, and 
existing vectors can be used without further engineering. 
Sterilized cotyledons from soybean Glycine max cv Peking were inoculated 
with A281:227 by placing a small aliquot of the Agrobacterium culture in 
cuts made on the inner surface of the tissue. The infected cotyledons were 
then put on Gamborg's B5 media lacking hormones and cultured for two 
weeks. Gamborg et al., Exp. Cell Res., 50:1151-1158 (1968). Transgenic 
calli were then isolated and analyzed for total squalene, total sterol and 
total cyclopropyl-pentacyclic (non-utilizable sterol levels as described 
in Example 3. The results of those studies are presented below in Table 8. 
TABLE 8* 
______________________________________ 
Cyclopropyl/ 
Pentacyclic 
Sterol 
Plant Total Sterol Callus Squalene 
______________________________________ 
Control 0.37 0.002 0.022 
Transgenic 
0.85 0.12 0.233 
______________________________________ 
*Data are presented as a percentage of dry weight. Data for the control 
represent the average of five vector controls. 
Transgenic represents a single transformant designated D13. 
Pentacyclic triterpenoids are sterol compounds having a fifth ring formed 
from cyclization of the steroidal 17-position side chain. Examples of 
these compounds include alpha-amyrin, beta-amyrin and lupeol. Although 
these compounds are found in a wide variety of plants, they are usually 
present in only trace amounts. These compounds and their conjugates (e.g. 
saponins) are reported to have medicinal and insecticidal properties. 
The positive identification of pentacyclictype (non-utilizable) compounds 
in transgenic soybean callus was made by gas chromatography-mass 
spectroscopy (GC-MS). Quantification by GC analysis is difficult because 
these compounds coelute with cyclopropyl sterols. The results for soybean 
transformants given in Table 8, above, therefore give cyclopropyl and 
pentacyclic sterols as a combined quantity. 
EXAMPLE 8 
Transformation of Tomato 
Hypocotyls from tomato Lycopersicon esculentum cv. UC82B were transformed 
as described in accordance with the procedures of Example 7 for soybean. 
After two weeks the transgenic calli were isolated and analyzed for sterol 
and squalene levels as described in Example 3. 
The results of these studies are presented below in Table 9. 
TABLE 9 
______________________________________ 
Total Sterol 
Cyclopropyl 
Squalene 
______________________________________ 
CTRL 0.04 &lt;0.002 0.002 
TRNSG 
#10 1.42 0.84 0.142 
#19 0.56 0.21 0.039 
______________________________________ 
*Data are expressed as percentage of dry weight 
CTRL = nontransgenic control 
TRNSG = transgenic 
The data show that calli of tomato plant cells transformed by a process of 
the present invention results in increases in total sterol, cyclopropyl 
(non-delta-5) sterols and squalene accumulation. 
EXAMPLE 9 
Transformation of Alfalfa 
Hypocotyls from alfalfa strain RYSI were transformed in accordance with the 
procedures of Example 3 for tobacco. After three or four weeks transgenic 
calli were isolated and analyzed for sterol and squalene levels as 
described in Example 3. The results of those studies are shown below in 
Table 10. 
TABLE 10* 
______________________________________ 
Cyclopropyl/ 
Pentacyclic 
Total Sterol 
Sterol Squalene 
______________________________________ 
CTRL 0.24 &lt;0.002 0.002 
TRNSG 1.26 0.99 0.052 
______________________________________ 
*Data are expressed as percentage of dry weight 
CTRL = nontransgenic control 
TRNSG = transgenic 
The data from Table 10 show that transgenic alfalfa calli demonstrate large 
increases in total sterol, pentacyclic and cyclopropyl sterols, and 
squalene accumulation when compared to a native, non-transgenic calli. 
EXAMPLE 10 
Transgenic Tobacco Seeds 
Transgenic tobacco seeds produced in accordance with the procedures of 
Example 1 were assayed for total sterol and squalene accumulation as 
described in Example 3. The results of those studies are presented below 
in Table 11. 
TABLE 11* 
______________________________________ 
Total Sterol 
Cyclopropyl 
Squalene 
______________________________________ 
CTRL 0.286 0.031 0.011 
TRNSG 
#5 0.509 0.123 0.047 
#19 0.714 0.163 0.042 
______________________________________ 
*Data are expressed as percentage of dry weight 
CTRL = nontransgenic control 
TRNSG = transgenic 
The data in Table 11 show that transgenic tobacco seeds of the present 
invention have an increased accumulation of total sterol, cyclopropyl 
sterol (non-delta-5 sterol) and squalene when compared to a native, 
non-transgenic seed of the same strain. 
The present invention has been described with respect to preferred 
embodiments. It is readily apparent to those skilled in the art that 
modifications and/or variations of the disclosed subject matter can be 
made without departing from the scope of the invention set forth herein. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4768 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 164..2827 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TGTATGTCTTGTCTTTCTCCTAAGGGGCGTAGGCTCATTGATAACTCATGTCCTCACCTT60 
GCACTCCTTTTGGAATTATTTGGTTTGAGTGAAGAAGACCGGACCTTCGAGGTTCGCAAC120 
TTAAACAATAGACT TGTGAGGATCCAGGGACCGAGTGGCTACAATGTTGTCACGA175 
MetLeuSerArg 
CTTTTCCGTATGCATGG CCTCTTTGTGGCCTCCCATCCCTGGGAAGTT223 
LeuPheArgMetHisGlyLeuPheValAlaSerHisProTrpGluVal 
5101520 
ATTGTGGGGACGGT GACACTTACCATCTGTATGATGTCCATGAACATG271 
IleValGlyThrValThrLeuThrIleCysMetMetSerMetAsnMet 
253035 
TTCACTGGCAACAA CAAGATCTGTGGTTGGAATTACGAGTGCCCAAAA319 
PheThrGlyAsnAsnLysIleCysGlyTrpAsnTyrGluCysProLys 
404550 
TTTGAGGAGGATGTATT GAGCAGTGACATCATCATCCTCACCATAACA367 
PheGluGluAspValLeuSerSerAspIleIleIleLeuThrIleThr 
556065 
CGGTGCATCGCCATCCTGTACAT TTACTTCCAGTTCCAGAACTTACGT415 
ArgCysIleAlaIleLeuTyrIleTyrPheGlnPheGlnAsnLeuArg 
707580 
CAGCTTGGGTCGAAGTATATTTTAGGTATTGC TGGCCTGTTCACAATT463 
GlnLeuGlySerLysTyrIleLeuGlyIleAlaGlyLeuPheThrIle 
859095100 
TTCTCAAGTTTTGTCTTTAGTACAGTCGT CATTCACTTCTTAGACAAA511 
PheSerSerPheValPheSerThrValValIleHisPheLeuAspLys 
105110115 
GAACTGACGGGCTTAAATGAAGCTTTGCC CTTTTTCCTGCTTTTGATT559 
GluLeuThrGlyLeuAsnGluAlaLeuProPhePheLeuLeuLeuIle 
120125130 
GACCTTTCTAGAGCGAGTGCACTAGCAAAGTT TGCCCTAAGTTCAAAC607 
AspLeuSerArgAlaSerAlaLeuAlaLysPheAlaLeuSerSerAsn 
135140145 
TCTCAGGATGAAGTAAGGGAAAATATAGCTCGCGGAAT GGCAATTCTG655 
SerGlnAspGluValArgGluAsnIleAlaArgGlyMetAlaIleLeu 
150155160 
GGCCCCACATTCACCCTTGATGCTCTTGTGGAATGTCTTGTAATTGG A703 
GlyProThrPheThrLeuAspAlaLeuValGluCysLeuValIleGly 
165170175180 
GTTGGCACCATGTCAGGGGTGCGTCAGCTTGAAATCATGTGCTG CTTT751 
ValGlyThrMetSerGlyValArgGlnLeuGluIleMetCysCysPhe 
185190195 
GGCTGCATGTCTGTGCTTGCCAACTACTTCGTGTTCATGACATT TTTC799 
GlyCysMetSerValLeuAlaAsnTyrPheValPheMetThrPhePhe 
200205210 
CCAGCGTGTGTGTCCCTGGTCCTTGAGCTTTCTCGGGAAAGTCGAGA G847 
ProAlaCysValSerLeuValLeuGluLeuSerArgGluSerArgGlu 
215220225 
GGTCGTCCAATTTGGCAGCTTAGCCATTTTGCCCGAGTTTTGGAAGAA89 5 
GlyArgProIleTrpGlnLeuSerHisPheAlaArgValLeuGluGlu 
230235240 
GAAGAGAATAAACCAAACCCTGTAACCCAAAGGGTCAAGATGATTATG943 
GluGluA snLysProAsnProValThrGlnArgValLysMetIleMet 
245250255260 
TCTTTAGGTTTGGTTCTTGTTCATGCTCACAGTCGATGGATAGCTGAT991 
SerL euGlyLeuValLeuValHisAlaHisSerArgTrpIleAlaAsp 
265270275 
CCTTCCCCTCAGAATAGCACAACAGAACATTCTAAAGTCTCCTTGGGA1039 
Pro SerProGlnAsnSerThrThrGluHisSerLysValSerLeuGly 
280285290 
CTGGATGAAGATGTGTCCAAGAGAATTGAACCAAGTGTTTCTCTCTGG1087 
LeuAsp GluAspValSerLysArgIleGluProSerValSerLeuTrp 
295300305 
CAGTTTTATCTCTCCAAGATGATCAGCATGGACATTGAACAAGTGGTT1135 
GlnPheTyrLe uSerLysMetIleSerMetAspIleGluGlnValVal 
310315320 
ACCCTGAGCTTAGCTTTTCTGTTGGCTGTCAAGTACATTTTCTTTGAA1183 
ThrLeuSerLeuAlaPheL euLeuAlaValLysTyrIlePhePheGlu 
325330335340 
CAAGCAGAGACAGAGTCCACACTGTCTTTAAAAAATCCTATCACGTCT1231 
GlnAlaGluThrGlu SerThrLeuSerLeuLysAsnProIleThrSer 
345350355 
CCTGTCGTGACCCCAAAGAAAGCTCCAGACAACTGTTGTAGACGGGAG1279 
ProValValThrPro LysLysAlaProAspAsnCysCysArgArgGlu 
360365370 
CCTCTGCTTGTGAGAAGGAGCGAGAAGCTTTCATCGGTTGAGGAGGAG1327 
ProLeuLeuValArgAr gSerGluLysLeuSerSerValGluGluGlu 
375380385 
CCTGGGGTGAGCCAAGATAGAAAAGTTGAGGTTATAAAACCATTAGTG1375 
ProGlyValSerGlnAspArgL ysValGluValIleLysProLeuVal 
390395400 
GTGGAAACTGAGAGTGCAAGCAGAGCTACATTTGTGCTTGGCGCCTCT1423 
ValGluThrGluSerAlaSerArgAlaThr PheValLeuGlyAlaSer 
405410415420 
GGGACCAGCCCTCCAGTGGCAGCGAGGACACAGGAGCTTGAAATTGAA1471 
GlyThrSerProProValAlaAlaArg ThrGlnGluLeuGluIleGlu 
425430435 
CTCCCCAGTGAGCCTCGGCCTAATGAAGAATGTCTGCAGATACTGGAG1519 
LeuProSerGluProArgProAsnGl uGluCysLeuGlnIleLeuGlu 
440445450 
AGTGCCGAGAAAGGTGCAAAGTTCCTTAGCGATGCAGAGATCATCCAG1567 
SerAlaGluLysGlyAlaLysPheLeuS erAspAlaGluIleIleGln 
455460465 
TTGGTCAATGCCAAGCACATCCCAGCCTACAAATTGGAAACCTTAATG1615 
LeuValAsnAlaLysHisIleProAlaTyrLys LeuGluThrLeuMet 
470475480 
GAAACTCATGAACGTGGTGTATCTATTCGCCGGCAGCTCCTCTCCACA1663 
GluThrHisGluArgGlyValSerIleArgArgGlnLeuLeu SerThr 
485490495500 
AAGCTTCCAGAGCCTTCTTCTCTGCAGTACCTGCCTTACAGAGATTAT1711 
LysLeuProGluProSerSerLeuGlnTyrLeuProTy rArgAspTyr 
505510515 
AATTATTCCCTGGTGATGGGAGCTTGCTGTGAGAATGTGATCGGATAT1759 
AsnTyrSerLeuValMetGlyAlaCysCysGluAsnV alIleGlyTyr 
520525530 
ATGCCCATCCCTGTCGGAGTAGCAGGGCCTCTGTGCCTGGATGGTAAA1807 
MetProIleProValGlyValAlaGlyProLeuCysLeu AspGlyLys 
535540545 
GAGTACCAGGTTCCAATGGCAACAACGGAAGGCTGTCTGGTGGCCAGC1855 
GluTyrGlnValProMetAlaThrThrGluGlyCysLeuValAla Ser 
550555560 
ACCAACAGAGGCTGCAGGGCAATAGGTCTTGGTGGAGGTGCCAGCAGC1903 
ThrAsnArgGlyCysArgAlaIleGlyLeuGlyGlyGlyAlaSerSer 
565 570575580 
CGGGTCCTTGCAGATGGGATGACCCGGGGCCCAGTGGTGCGTCTTCCT1951 
ArgValLeuAlaAspGlyMetThrArgGlyProValValArgLeuPro 
585590595 
CGTGCTTGTGATTCTGCAGAAGTGAAGGCCTGGCTTGAAACACCCGAA1999 
ArgAlaCysAspSerAlaGluValLysAlaTrpLeuGluThrProGlu 
600605610 
GGGTTTGCGGTGATAAAGGACGCCTTCGATAGCACTAGCAGATTTGCA2047 
GlyPheAlaValIleLysAspAlaPheAspSerThrSerArgPheAla 
615620625 
CGTCTACAGAAGCTTCATGTGACCATGGCAGGGCGCAACCTGTACATC2095 
ArgLeuGlnLysLeuHisValThrMetAlaGlyArgAsnLeuTyrIle 
630 635640 
CGTTTCCAGTCCAAGACAGGGGATGCCATGGGGATGAACATGATTTCC2143 
ArgPheGlnSerLysThrGlyAspAlaMetGlyMetAsnMetIleSer 
645 650655660 
AAGGGCACTGAGAAAGCACTTCTGAAGCTTCAGGAGTTCTTTCCTGAA2191 
LysGlyThrGluLysAlaLeuLeuLysLeuGlnGluPhePheProGlu 
665670675 
ATGCAGATTCTGGCAGTTAGTGGTAACTACTGCACTGACAAGAAACCT2239 
MetGlnIleLeuAlaValSerGlyAsnTyrCysThrAspLysLysPro 
68 0685690 
GCCGCCATAAACTGGATCGAGGGAAGAGGAAAGACAGTTGTGTGTGAA2287 
AlaAlaIleAsnTrpIleGluGlyArgGlyLysThrValValCysGlu 
695 700705 
GCTGTTATTCCAGCCAAGGTGGTGAGAGAAGTATTAAAGACAACTACG2335 
AlaValIleProAlaLysValValArgGluValLeuLysThrThrThr 
710 715720 
GAAGCTATGATTGACGTAAACATTAACAAGAATCTTGTGGGTTCTGCC2383 
GluAlaMetIleAspValAsnIleAsnLysAsnLeuValGlySerAla 
725730 735740 
ATGGCTGGGAGCATAGGAGGCTACAATGCCCATGCAGCAAACATCGTC2431 
MetAlaGlySerIleGlyGlyTyrAsnAlaHisAlaAlaAsnIleVal 
745 750755 
ACTGCTATCTACATTGCATGTGGCCAGGATGCAGCACAGAATGTGGGG2479 
ThrAlaIleTyrIleAlaCysGlyGlnAspAlaAlaGlnAsnValGly 
760 765770 
AGTTCAAACTGTATTACTTTAATGGAAGCAAGTGGTCCCACGAATGAA2527 
SerSerAsnCysIleThrLeuMetGluAlaSerGlyProThrAsnGlu 
775780 785 
GACTTGTATATCAGCTGCACCATGCCATCTATAGAGATAGGAACTGTG2575 
AspLeuTyrIleSerCysThrMetProSerIleGluIleGlyThrVal 
790795 800 
GGTGGTGGGACCAACCTCCTACCACAGCAGGCCTGTCTGCAGATGCTA2623 
GlyGlyGlyThrAsnLeuLeuProGlnGlnAlaCysLeuGlnMetLeu 
805810815 820 
GGTGTTCAAGGAGCGTGCAAAGACAATCCTGGAGAAAATGCACGGCAA2671 
GlyValGlnGlyAlaCysLysAspAsnProGlyGluAsnAlaArgGln 
825830 835 
CTTGCCCGAATTGTGTGTGGTACTGTAATGGCTGGGGAGTTGTCCTTG2719 
LeuAlaArgIleValCysGlyThrValMetAlaGlyGluLeuSerLeu 
840845 850 
ATGGCAGCATTGGCAGCAGGACATCTTGTTAGAAGTCACATGGTTCAT2767 
MetAlaAlaLeuAlaAlaGlyHisLeuValArgSerHisMetValHis 
855860 865 
AACAGATCGAAGATAAATTTACAAGATCTGCAAGGAACGTGCACCAAG2815 
AsnArgSerLysIleAsnLeuGlnAspLeuGlnGlyThrCysThrLys 
870875880 
AAG TCAGCTTGAGCAGCCTGACAGTATTGAACTGAAACACGGGCATTGG2864 
LysSerAla 
885 
GTTCTCAAGGACTAACATGAAATCTGTGAATTAAAAATCTCAATGCAGTGTCTTGTGGAA2924 
GATGAATGAACGTGATCAGTGAGACGCCTGCTT GGTTTCTGGCTCTTTCAGAGACGTCTG2984 
AGGTCCTTTGCTCGGAGACTCCTCAGATCTGGAAACAGTGTGGTCCTTCCCATGCTGTAT3044 
TCTGAAAAGATCTCATATGGATGTTGTGCTCTGAGCACCACAGATGTGATCTGCAGCTCG3104 
TTTCTGAAAT GATGGAGTTCATGGTGATCAGTGTGAGACTGGCCTCTCCCAGCAGGTTAA3164 
AAATGGAGTTTTAAATTATACTGTAGCTGACAGTACTTCTGATTTTATATTTATTTAGTC3224 
TGAGTTGTAGAACTTTGCAATCTAAGTTTATTTTTTGTAACCTAATAATTCATTTG GTGC3284 
TGGTCTATTGATTTTTGGGGGTAAACAATATTATTCTTCAGAAGGGGACCTACTTCTTCA3344 
TGGGAAGAATTACTTTTATTCTCAAACTACAGAACAATGTGCTAAGCAGTGCTAAATTGT3404 
TCTCATGAAGAAAACAGTCACTGCATTTATCTC TGTAGGCCTTTTTTCAGAGAGGCCTTG3464 
TCTAGATTTTTGCCAGCTAGGCTACTGCATGTCTTAGTGTCAGGCCTTAGGAAAGTGCCA3524 
CGCTCTGCACTAAAGATATCAGAGCTCTTGGTGTTACTTAGACAAGAGTATGAGCAAGTC3584 
GGACCTCTCA GAGTGTGGGAACACAGTTTTGAAAGAAAAACCATTTCTCTAAGCCAATTT3644 
TCTTTAAAGACATTTTAACTTATTTAGCTGAGTTCTAGATTTTTCGGGTAAACTATCAAA3704 
TCTGTATATGTTGTAATAAAGTGTCTTATGCTAGGAGTTTATTCAAAGTGTTTAAG TAAT3764 
AAAAGGACTCAAATTTACACTGATAAAATACTCTAGCTTGGGCCAGAGAAGACAGTGCTC3824 
ATTAGCGTTGTCCAGGAAACCCTGCTTGCTTGCCAAGCCTAATGAAGGGAAAGTCAGCTT3884 
TCAGAGCCAATGATGGAGGCCACATGAATGGCC CTGGAGCTGTGTGCCTTGTTCTGTGGC3944 
CAGGAGCTTGGTGACTGAATCATTTACGGGCTCCTTTGATGGACCCATAAAAGCTCTTAG4004 
CTTCCTCAGGGGGTCAGCAGAGTTGTTGAATCTTAATTTTTTTTTTAATGTACCAGTTTT4064 
GTATAAATAA TAATAAAGAGCTCCTTATTTTGTATTCTATCTAATGCTTCGAGTTCAGTC4124 
TTGGGAAGCTGACATCTCATGTAGAAGATGGACTCTGAAAGACATTCCAAGAGTGCAGCG4184 
GCATCATGGGAGCCTCTTAGTGATTGTGTGTCAGTATTATTGTGGAAGATTGACTT TGCT4244 
TTTGTATGTGAAGTTTCAGATTGCTCCTCTTGTGACTTTTTAGCCAGTAACATTTTATTT4304 
ACCTGAGCTTGTCATGGAAGTGGCAGTGAAAAGTATTGAGTATTCATGCTGGTGACTGTA4364 
ACCAATGTCATCTTGCTAAAAACTCATGTTTTG TACAATTACTAAATTGTATACATTTTG4424 
TTATAGAATACTTTTTCCAGTTGAGTAAATTATGAAAGGAAGTTAACATTAACAGGTGTA4484 
AGCGGTGGCTTTTTTAAAATGAAGGATTAACCCTAAGCCCGAGACCCAGAAGCTAGCAAA4544 
GTCTGGCAGA GTGGTAAACTGTCCTGCTGGGGCCATCCAATCATCTCTCTCCATTACACT4604 
TTCTAACTTTGCAGCATTGGTGCTGGCCAGTGTATTGTTTCATTGATCTTCCTTACGCTT4664 
AGAGGGTTTGATTGGTTCAGATCTATAATCTCAGCCACATTGTCTTGGTATCAGCT GGAG4724 
AGAGTTAAGAGGAAGGGAAAATAAAGTTCAGATAGCCAAAACAC4768 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 887 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
Me tLeuSerArgLeuPheArgMetHisGlyLeuPheValAlaSerHis 
151015 
ProTrpGluValIleValGlyThrValThrLeuThrIleCysMetMet 
202530 
SerMetAsnMetPheThrGlyAsnAsnLysIleCysGlyTrpAsnTyr 
354045 
GluCysProLysPheGluGluAspV alLeuSerSerAspIleIleIle 
505560 
LeuThrIleThrArgCysIleAlaIleLeuTyrIleTyrPheGlnPhe 
657075 80 
GlnAsnLeuArgGlnLeuGlySerLysTyrIleLeuGlyIleAlaGly 
859095 
LeuPheThrIlePheSerSerPheValPheSerThrValValIle His 
100105110 
PheLeuAspLysGluLeuThrGlyLeuAsnGluAlaLeuProPhePhe 
115120125 
LeuLeuLeuIleAs pLeuSerArgAlaSerAlaLeuAlaLysPheAla 
130135140 
LeuSerSerAsnSerGlnAspGluValArgGluAsnIleAlaArgGly 
145150 155160 
MetAlaIleLeuGlyProThrPheThrLeuAspAlaLeuValGluCys 
165170175 
LeuValIleGlyValGlyThrMetSerGlyValA rgGlnLeuGluIle 
180185190 
MetCysCysPheGlyCysMetSerValLeuAlaAsnTyrPheValPhe 
195200205 
Met ThrPhePheProAlaCysValSerLeuValLeuGluLeuSerArg 
210215220 
GluSerArgGluGlyArgProIleTrpGlnLeuSerHisPheAlaArg 
225230 235240 
ValLeuGluGluGluGluAsnLysProAsnProValThrGlnArgVal 
245250255 
LysMetIleMetSerLeuGlyLe uValLeuValHisAlaHisSerArg 
260265270 
TrpIleAlaAspProSerProGlnAsnSerThrThrGluHisSerLys 
275280 285 
ValSerLeuGlyLeuAspGluAspValSerLysArgIleGluProSer 
290295300 
ValSerLeuTrpGlnPheTyrLeuSerLysMetIleSerMetAspIle 
305 310315320 
GluGlnValValThrLeuSerLeuAlaPheLeuLeuAlaValLysTyr 
325330335 
IlePhePheGlu GlnAlaGluThrGluSerThrLeuSerLeuLysAsn 
340345350 
ProIleThrSerProValValThrProLysLysAlaProAspAsnCys 
355 360365 
CysArgArgGluProLeuLeuValArgArgSerGluLysLeuSerSer 
370375380 
ValGluGluGluProGlyValSerGlnAspArgLysValGluVa lIle 
385390395400 
LysProLeuValValGluThrGluSerAlaSerArgAlaThrPheVal 
405410415 
LeuGlyAlaSerGlyThrSerProProValAlaAlaArgThrGlnGlu 
420425430 
LeuGluIleGluLeuProSerGluProArgProAsnGluGluCysLeu 
435 440445 
GlnIleLeuGluSerAlaGluLysGlyAlaLysPheLeuSerAspAla 
450455460 
GluIleIleGlnLeuValAsnAlaLysHisIle ProAlaTyrLysLeu 
465470475480 
GluThrLeuMetGluThrHisGluArgGlyValSerIleArgArgGln 
485490 495 
LeuLeuSerThrLysLeuProGluProSerSerLeuGlnTyrLeuPro 
500505510 
TyrArgAspTyrAsnTyrSerLeuValMetGlyAlaCysCysGluAs n 
515520525 
ValIleGlyTyrMetProIleProValGlyValAlaGlyProLeuCys 
530535540 
LeuAspGlyLysGluTyrGln ValProMetAlaThrThrGluGlyCys 
545550555560 
LeuValAlaSerThrAsnArgGlyCysArgAlaIleGlyLeuGlyGly 
565 570575 
GlyAlaSerSerArgValLeuAlaAspGlyMetThrArgGlyProVal 
580585590 
ValArgLeuProArgAlaCysAspSerAlaGluVal LysAlaTrpLeu 
595600605 
GluThrProGluGlyPheAlaValIleLysAspAlaPheAspSerThr 
610615620 
SerArgPheA laArgLeuGlnLysLeuHisValThrMetAlaGlyArg 
625630635640 
AsnLeuTyrIleArgPheGlnSerLysThrGlyAspAlaMetGlyMet 
6 45650655 
AsnMetIleSerLysGlyThrGluLysAlaLeuLeuLysLeuGlnGlu 
660665670 
PhePheProGluMetGlnIleLeu AlaValSerGlyAsnTyrCysThr 
675680685 
AspLysLysProAlaAlaIleAsnTrpIleGluGlyArgGlyLysThr 
690695700 
ValValCysGluAlaValIleProAlaLysValValArgGluValLeu 
705710715720 
LysThrThrThrGluAlaMetIleAspValAsnIleAsnLysAsnLeu 
725730735 
ValGlySerAlaMetAlaGlySerIleGlyGlyTyrAsnAlaHisAla 
740745750 
AlaAsnIleValT hrAlaIleTyrIleAlaCysGlyGlnAspAlaAla 
755760765 
GlnAsnValGlySerSerAsnCysIleThrLeuMetGluAlaSerGly 
770775 780 
ProThrAsnGluAspLeuTyrIleSerCysThrMetProSerIleGlu 
785790795800 
IleGlyThrValGlyGlyGlyThrAsnLeuLeuProGln GlnAlaCys 
805810815 
LeuGlnMetLeuGlyValGlnGlyAlaCysLysAspAsnProGlyGlu 
820825830 
As nAlaArgGlnLeuAlaArgIleValCysGlyThrValMetAlaGly 
835840845 
GluLeuSerLeuMetAlaAlaLeuAlaAlaGlyHisLeuValArgSer 
850 855860 
HisMetValHisAsnArgSerLysIleAsnLeuGlnAspLeuGlnGly 
865870875880 
ThrCysThrLysLysSerAla 
885 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3360 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 121..3282 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TTTATTAACTTA TTTTTTTCTTCTTTCTACCCAATTCTAGTCAGGAAAAGACTAAGGGCT60 
GGAACATAGTGTATCATTGTCTAATTGTTGATACAAAGTAGATAAATACATAAAACAAGC120 
ATGCCGCCGCTATTCAAGGGACTGAAACAGATGGCAAAGCCAATTGCC 168 
MetProProLeuPheLysGlyLeuLysGlnMetAlaLysProIleAla 
151015 
TATGTTTCAAGATTTTCGGCGAAACGACCAATTCATATAATACTTTTT 216 
TyrValSerArgPheSerAlaLysArgProIleHisIleIleLeuPhe 
202530 
TCTCTAATCATATCCGCATTCGCTTATCTATCCGTCATTCAGTATTAC26 4 
SerLeuIleIleSerAlaPheAlaTyrLeuSerValIleGlnTyrTyr 
354045 
TTCAATGGTTGGCAACTAGATTCAAATAGTGTTTTTGAAACTGCTCCA312 
PheA snGlyTrpGlnLeuAspSerAsnSerValPheGluThrAlaPro 
505560 
AATAAAGACTCCAACACTCTATTTCAAGAATGTTCCCATTACTACAGA360 
AsnLysAspSerA snThrLeuPheGlnGluCysSerHisTyrTyrArg 
65707580 
GATTCCTCTCTAGATGGTTGGGTATCAATCACCGCGCATGAAGCTAGT408 
AspSerSerL euAspGlyTrpValSerIleThrAlaHisGluAlaSer 
859095 
GAGTTACCAGCCCCACACCATTACTATCTATTAAACCTGAACTTCAAT456 
GluLeuProA laProHisHisTyrTyrLeuLeuAsnLeuAsnPheAsn 
100105110 
AGTCCTAATGAAACTGACTCCATTCCAGAACTAGCTAACACGGTTTTT504 
SerProAsnGluT hrAspSerIleProGluLeuAlaAsnThrValPhe 
115120125 
GAGAAAGATAATACAAAATATATTCTGCAAGAAGATCTCAGTGTTTCC552 
GluLysAspAsnThrLysT yrIleLeuGlnGluAspLeuSerValSer 
130135140 
AAAGAAATTTCTTCTACTGATGGAACGAAATGGAGGTTAAGAAGTGAC600 
LysGluIleSerSerThrAspGlyThrL ysTrpArgLeuArgSerAsp 
145150155160 
AGAAAAAGTCTTTTCGACGTAAAGACGTTAGCATATTCTCTCTACGAT648 
ArgLysSerLeuPheAspValLysT hrLeuAlaTyrSerLeuTyrAsp 
165170175 
GTATTTTCAGAAAATGTAACCCAAGCAGACCCGTTTGACGTCCTTATT696 
ValPheSerGluAsnValThrGlnA laAspProPheAspValLeuIle 
180185190 
ATGGTTACTGCCTACCTAATGATGTTCTACACCATATTCGGCCTCTTC744 
MetValThrAlaTyrLeuMetMetPheT yrThrIlePheGlyLeuPhe 
195200205 
AATGACATGAGGAAGACCGGGTCAAATTTTTGGTTGAGCGCCTCTACA792 
AsnAspMetArgLysThrGlySerAsnPheTrpL euSerAlaSerThr 
210215220 
GTGGTCAATTCTGCATCATCACTTTTCTTAGCATTGTATGTCACCCAA840 
ValValAsnSerAlaSerSerLeuPheLeuAlaLeuTyrValT hrGln 
225230235240 
TGTATTCTAGGCAAAGAAGTTTCCGCATTAACTCTTTTTGAAGGTTTG888 
CysIleLeuGlyLysGluValSerAlaLeuThrLeuPheG luGlyLeu 
245250255 
CCTTTCATTGTAGTTGTTGTTGGTTTCAAGCACAAAATCAAGATTGCC936 
ProPheIleValValValValGlyPheLysHisLysIleL ysIleAla 
260265270 
CAGTATGCCCTGGAGAAATTTGAAAGAGTCGGTTTATCTAAAAGGATT984 
GlnTyrAlaLeuGluLysPheGluArgValGlyLeuSerLysA rgIle 
275280285 
ACTACCGATGAAATCGTTTTTGAATCCGTGAGCGAAGAGGGTGGTCGT1032 
ThrThrAspGluIleValPheGluSerValSerGluGluGlyGlyArg 
290295300 
TTGATTCAAGACCATTTGCTTTGTATTTTTGCCTTTATCGGATGCTCT1080 
LeuIleGlnAspHisLeuLeuCysIlePheAlaPheIleGlyCysSer 
305 310315320 
ATGTATGCTCACCAATTGAAGACTTTGACAAACTTCTGCATATTATCA1128 
MetTyrAlaHisGlnLeuLysThrLeuThrAsnPheCysIleLeuSer 
325330335 
GCATTTATCCTAATTTTTGAATTGATTTTAACTCCTACATTTTATTCT1176 
AlaPheIleLeuIlePheGluLeuIleLeuThrProThrPheTyrSer 
340345350 
GCTATCTTAGCGCTTAGACTGGAAATGAATGTTATCCACAGATCTACT1224 
AlaIleLeuAlaLeuArgLeuGluMetAsnValIleHisArgSerThr 
355 360365 
ATTATCAAGCAAACATTAGAAGAAGACGGTGTTGTTCCATCTACAGCA1272 
IleIleLysGlnThrLeuGluGluAspGlyValValProSerThrAla 
370 375380 
AGAATCATTTCTAAAGCAGAAAAGAAATCCGTATCTTCTTTCTTAAAT1320 
ArgIleIleSerLysAlaGluLysLysSerValSerSerPheLeuAsn 
385390 395400 
CTCAGTGTGGTTGTCATTATCATGAAACTCTCTGTCATACTGTTGTTT1368 
LeuSerValValValIleIleMetLysLeuSerValIleLeuLeuPhe 
405 410415 
GTTTTCATCAACTTTTATAACTTTGGTGCAAATTGGGTCAATGATGCC1416 
ValPheIleAsnPheTyrAsnPheGlyAlaAsnTrpValAsnAspAla 
420 425430 
TTCAATTCATTGTACTTCGATAAGGAACGTGTTTCTCTACCAGATTTT1464 
PheAsnSerLeuTyrPheAspLysGluArgValSerLeuProAspPhe 
435440 445 
ATTACCTCGAATGCCTCTGAAAACTTTAAAGAGCAAGCTATTGTTAGT1512 
IleThrSerAsnAlaSerGluAsnPheLysGluGlnAlaIleValSer 
450455 460 
GTCACCCCATTATTATATTACAAACCCATTAAGTCCTACCAACGCATT1560 
ValThrProLeuLeuTyrTyrLysProIleLysSerTyrGlnArgIle 
465470475 480 
GAGGATATGGTTCTTCTATTGCTTCGTAATGTCAGTGTTGCCATTCGT1608 
GluAspMetValLeuLeuLeuLeuArgAsnValSerValAlaIleArg 
485490 495 
GATAGGTTCGTCAGTAAATTAGTTCTTTCCGCCTTAGTATGCAGTGCT1656 
AspArgPheValSerLysLeuValLeuSerAlaLeuValCysSerAla 
500505 510 
GTCATCAATGTGTATTTATTGAATGCTGCTAGAATTCATACCAGTTAT1704 
ValIleAsnValTyrLeuLeuAsnAlaAlaArgIleHisThrSerTyr 
515520525 
ACTGCAGACCAATTGGTGAAAACTGAAGTCACCAAGAAGTCTTTTACT1752 
ThrAlaAspGlnLeuValLysThrGluValThrLysLysSerPheThr 
530535540 
GCTCCTGT ACAAAAGGCTTCTACACCAGTTTTAACCAATAAAACAGTC1800 
AlaProValGlnLysAlaSerThrProValLeuThrAsnLysThrVal 
545550555560 
ATTTC TGGATCGAAAGTCAAAAGTTTATCATCTGCGCAATCGAGCTCA1848 
IleSerGlySerLysValLysSerLeuSerSerAlaGlnSerSerSer 
565570575 
TCAGG ACCTTCATCATCTAGTGAGGAAGATGATTCCCGCGATATTGAA1896 
SerGlyProSerSerSerSerGluGluAspAspSerArgAspIleGlu 
580585590 
AGCTTGGA TAAGAAAATACGTCCTTTAGAAGAATTAGAAGCATTATTA1944 
SerLeuAspLysLysIleArgProLeuGluGluLeuGluAlaLeuLeu 
595600605 
AGTAGTGGAAATAC AAAACAATTGAAGAACAAAGAGGTCGCTGCCTTG1992 
SerSerGlyAsnThrLysGlnLeuLysAsnLysGluValAlaAlaLeu 
610615620 
GTTATTCACGGTAAGTTACCTTT GTACGCTTTGGAGAAAAAATTAGGT2040 
ValIleHisGlyLysLeuProLeuTyrAlaLeuGluLysLysLeuGly 
625630635640 
GATACTACGAGAGCGGTTGC GGTACGTAGGAAGGCTCTTTCAATTTTG2088 
AspThrThrArgAlaValAlaValArgArgLysAlaLeuSerIleLeu 
645650655 
GCAGAAGCTCCTGTATTAGC ATCTGATCGTTTACCATATAAAAATTAT2136 
AlaGluAlaProValLeuAlaSerAspArgLeuProTyrLysAsnTyr 
660665670 
GACTACGACCGCGTATTTGGCGC TTGTTGTGAAAATGTTATAGGTTAC2184 
AspTyrAspArgValPheGlyAlaCysCysGluAsnValIleGlyTyr 
675680685 
ATGCCTTTGCCCGTTGGTGTTATAGGCCC CTTGGTTATCGATGGTACA2232 
MetProLeuProValGlyValIleGlyProLeuValIleAspGlyThr 
690695700 
TCTTATCATATACCAATGGCAACTACAGAGGGTTGTTT GGTAGCTTCT2280 
SerTyrHisIleProMetAlaThrThrGluGlyCysLeuValAlaSer 
705710715720 
GCCATGCGTGGCTGTAAGGCAATCAATGCTGGCGG TGGTGCAACAACT2328 
AlaMetArgGlyCysLysAlaIleAsnAlaGlyGlyGlyAlaThrThr 
725730735 
GTTTTAACTAAGGATGGTATGACAAGAGGCCCAGT AGTCCGTTTCCCA2376 
ValLeuThrLysAspGlyMetThrArgGlyProValValArgPhePro 
740745750 
ACTTTGAAAAGATCTGGTGCCTGTAAGATATGGTTAGA CTCAGAAGAG2424 
ThrLeuLysArgSerGlyAlaCysLysIleTrpLeuAspSerGluGlu 
755760765 
GGACAAAACGCAATTAAAAAAGCTTTTAACTCTACATCAAGATT TGCA2472 
GlyGlnAsnAlaIleLysLysAlaPheAsnSerThrSerArgPheAla 
770775780 
CGTCTGCAACATATTCAAACTTGTCTAGCAGGAGATTTACTCTTCATG252 0 
ArgLeuGlnHisIleGlnThrCysLeuAlaGlyAspLeuLeuPheMet 
785790795800 
AGATTTAGAACAACTACTGGTGACGCAATGGGTATGAATATGATTTCT 2568 
ArgPheArgThrThrThrGlyAspAlaMetGlyMetAsnMetIleSer 
805810815 
AAAGGTGTCGAATACTCATTAAAGCAAATGGTAGAAGAGTATGGCTGG 2616 
LysGlyValGluTyrSerLeuLysGlnMetValGluGluTyrGlyTrp 
820825830 
GAAGATATGGAGGTTGTCTCCGTTTCTGGTAACTACTGTACCGACAAA266 4 
GluAspMetGluValValSerValSerGlyAsnTyrCysThrAspLys 
835840845 
AAACCAGCTGCCATCAACTGGATCGAAGGTCGTGGTAAGAGTGTCGTC2712 
LysP roAlaAlaIleAsnTrpIleGluGlyArgGlyLysSerValVal 
850855860 
GCAGAAGCTACTATTCCTGGTGATGTTGTCAGAAAAGTGTTAAAAAGT2760 
AlaGluAlaThrI leProGlyAspValValArgLysValLeuLysSer 
865870875880 
GATGTTTCCGCATTGGTTGAGTTGAACATTGCTAAGAATTTGGTTGGA2808 
AspValSerA laLeuValGluLeuAsnIleAlaLysAsnLeuValGly 
885890895 
TCTGCAATGGCTGGGTCTGTTGGTGGATTTAACGCACATGCAGCTAAT2856 
SerAlaMetA laGlySerValGlyGlyPheAsnAlaHisAlaAlaAsn 
900905910 
TTAGTGACAGCTGTTTTCTTGGCATTAGGACAAGATCCTGCACAAAAT2904 
LeuValThrAlaV alPheLeuAlaLeuGlyGlnAspProAlaGlnAsn 
915920925 
GTTGAAAGTTCCAACTGTATAACATTGATGAAAGAAGTGGACGGTGAT2952 
ValGluSerSerAsnCysI leThrLeuMetLysGluValAspGlyAsp 
930935940 
TTGAGAATTTCCGTATCCATGCCATCCATCGAAGTAGGTACCATCGGT3000 
LeuArgIleSerValSerMetProSerI leGluValGlyThrIleGly 
945950955960 
GGTGGTACTGTTCTAGAACCACAAGGTGCCATGTTGGACTTATTAGGT3048 
GlyGlyThrValLeuGluProGlnG lyAlaMetLeuAspLeuLeuGly 
965970975 
GTAAGAGGCCCGCATGCTACCGCTCCTGGTACCAACGCACGTCAATTA3096 
ValArgGlyProHisAlaThrAlaP roGlyThrAsnAlaArgGlnLeu 
980985990 
GCAAGAATAGTTGCCTGTGCCGTCTTGGCAGGTGAATTATCCTTATGT3144 
AlaArgIleValAlaCysAlaValLeuA laGlyGluLeuSerLeuCys 
99510001005 
GCTGCCCTAGCAGCCGGCCATTTGGTTCAAAGTCATATGACCCACAAC3192 
AlaAlaLeuAlaAlaGlyHisLeuValGlnSer HisMetThrHisAsn 
101010151020 
AGGAAACCTGCTGAACCAACAAAACCTAACAATTTGGACGCCACTGAT3240 
ArgLysProAlaGluProThrLysProAsnAsnLeuAspAla ThrAsp 
1025103010351040 
ATAAATCGTTTGAAAGATGGGTCCGTCACCTGCATTAAATCC3282 
IleAsnArgLeuLysAspGlySerValThrCysIleLy sSer 
10451050 
TAAACTTAGTCATACGTCATTGGTATTCTCTTGAAAAAGAAGCACAACAGCACCATGTGT3342 
TACGTAAAATATTTACTT3360 
( 2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1054 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetProProLeuPheLysGlyLeuLysGlnMetAlaLysProIleAla 
15 1015 
TyrValSerArgPheSerAlaLysArgProIleHisIleIleLeuPhe 
202530 
SerLeuIleIleSerAlaPheAlaTyrLeuSer ValIleGlnTyrTyr 
354045 
PheAsnGlyTrpGlnLeuAspSerAsnSerValPheGluThrAlaPro 
505560 
AsnLysA spSerAsnThrLeuPheGlnGluCysSerHisTyrTyrArg 
65707580 
AspSerSerLeuAspGlyTrpValSerIleThrAlaHisGluAlaSer 
859095 
GluLeuProAlaProHisHisTyrTyrLeuLeuAsnLeuAsnPheAsn 
100105110 
SerProAsnGluThrAspSer IleProGluLeuAlaAsnThrValPhe 
115120125 
GluLysAspAsnThrLysTyrIleLeuGlnGluAspLeuSerValSer 
130135 140 
LysGluIleSerSerThrAspGlyThrLysTrpArgLeuArgSerAsp 
145150155160 
ArgLysSerLeuPheAspValLysThrLeuAlaTyrSerLeuTyrAsp 
165170175 
ValPheSerGluAsnValThrGlnAlaAspProPheAspValLeuIle 
180185190 
MetValThrA laTyrLeuMetMetPheTyrThrIlePheGlyLeuPhe 
195200205 
AsnAspMetArgLysThrGlySerAsnPheTrpLeuSerAlaSerThr 
210215 220 
ValValAsnSerAlaSerSerLeuPheLeuAlaLeuTyrValThrGln 
225230235240 
CysIleLeuGlyLysGluValSerAlaLeuThrLeu PheGluGlyLeu 
245250255 
ProPheIleValValValValGlyPheLysHisLysIleLysIleAla 
260265270 
GlnTyrAlaLeuGluLysPheGluArgValGlyLeuSerLysArgIle 
275280285 
ThrThrAspGluIleValPheGluSerValSerGluGluGlyGlyArg 
290 295300 
LeuIleGlnAspHisLeuLeuCysIlePheAlaPheIleGlyCysSer 
305310315320 
MetTyrAlaHisGlnLeuLysThrL euThrAsnPheCysIleLeuSer 
325330335 
AlaPheIleLeuIlePheGluLeuIleLeuThrProThrPheTyrSer 
340345 350 
AlaIleLeuAlaLeuArgLeuGluMetAsnValIleHisArgSerThr 
355360365 
IleIleLysGlnThrLeuGluGluAspGlyValValProSerThrAla 
370375380 
ArgIleIleSerLysAlaGluLysLysSerValSerSerPheLeuAsn 
385390395400 
LeuSerValValVa lIleIleMetLysLeuSerValIleLeuLeuPhe 
405410415 
ValPheIleAsnPheTyrAsnPheGlyAlaAsnTrpValAsnAspAla 
420 425430 
PheAsnSerLeuTyrPheAspLysGluArgValSerLeuProAspPhe 
435440445 
IleThrSerAsnAlaSerGluAsnPheLysGluGlnA laIleValSer 
450455460 
ValThrProLeuLeuTyrTyrLysProIleLysSerTyrGlnArgIle 
465470475480 
Glu AspMetValLeuLeuLeuLeuArgAsnValSerValAlaIleArg 
485490495 
AspArgPheValSerLysLeuValLeuSerAlaLeuValCysSerAla 
500505510 
ValIleAsnValTyrLeuLeuAsnAlaAlaArgIleHisThrSerTyr 
515520525 
ThrAlaAspGlnLeuValLysThrGl uValThrLysLysSerPheThr 
530535540 
AlaProValGlnLysAlaSerThrProValLeuThrAsnLysThrVal 
545550555 560 
IleSerGlySerLysValLysSerLeuSerSerAlaGlnSerSerSer 
565570575 
SerGlyProSerSerSerSerGluGluAspAspSerArgAspIleG lu 
580585590 
SerLeuAspLysLysIleArgProLeuGluGluLeuGluAlaLeuLeu 
595600605 
SerSerGlyAsnThr LysGlnLeuLysAsnLysGluValAlaAlaLeu 
610615620 
ValIleHisGlyLysLeuProLeuTyrAlaLeuGluLysLysLeuGly 
625630 635640 
AspThrThrArgAlaValAlaValArgArgLysAlaLeuSerIleLeu 
645650655 
AlaGluAlaProValLeuAlaSerAspArgLeuPr oTyrLysAsnTyr 
660665670 
AspTyrAspArgValPheGlyAlaCysCysGluAsnValIleGlyTyr 
675680685 
Met ProLeuProValGlyValIleGlyProLeuValIleAspGlyThr 
690695700 
SerTyrHisIleProMetAlaThrThrGluGlyCysLeuValAlaSer 
705710 715720 
AlaMetArgGlyCysLysAlaIleAsnAlaGlyGlyGlyAlaThrThr 
725730735 
ValLeuThrLysAspGlyMetThr ArgGlyProValValArgPhePro 
740745750 
ThrLeuLysArgSerGlyAlaCysLysIleTrpLeuAspSerGluGlu 
755760 765 
GlyGlnAsnAlaIleLysLysAlaPheAsnSerThrSerArgPheAla 
770775780 
ArgLeuGlnHisIleGlnThrCysLeuAlaGlyAspLeuLeuPheMet 
785 790795800 
ArgPheArgThrThrThrGlyAspAlaMetGlyMetAsnMetIleSer 
805810815 
LysGlyValGlu TyrSerLeuLysGlnMetValGluGluTyrGlyTrp 
820825830 
GluAspMetGluValValSerValSerGlyAsnTyrCysThrAspLys 
835 840845 
LysProAlaAlaIleAsnTrpIleGluGlyArgGlyLysSerValVal 
850855860 
AlaGluAlaThrIleProGlyAspValValArgLysValLeuLys Ser 
865870875880 
AspValSerAlaLeuValGluLeuAsnIleAlaLysAsnLeuValGly 
885890895 
S erAlaMetAlaGlySerValGlyGlyPheAsnAlaHisAlaAlaAsn 
900905910 
LeuValThrAlaValPheLeuAlaLeuGlyGlnAspProAlaGlnAsn 
915 920925 
ValGluSerSerAsnCysIleThrLeuMetLysGluValAspGlyAsp 
930935940 
LeuArgIleSerValSerMetProSerIleGlu ValGlyThrIleGly 
945950955960 
GlyGlyThrValLeuGluProGlnGlyAlaMetLeuAspLeuLeuGly 
965970 975 
ValArgGlyProHisAlaThrAlaProGlyThrAsnAlaArgGlnLeu 
980985990 
AlaArgIleValAlaCysAlaValLeuAlaGlyGluLeuSerLeuCys 
99510001005 
AlaAlaLeuAlaAlaGlyHisLeuValGlnSerHisMetThrHisAsn 
101010151020 
ArgLysProAlaGluProThr LysProAsnAsnLeuAspAlaThrAsp 
1025103010351040 
IleAsnArgLeuLysAspGlySerValThrCysIleLysSer 
104510 50 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3348 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 121..3255 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GGAATATTTTGTACGAGCAA GTTATAGTAAGACACTTCAGTGAGAAATTAATCTGACTTA60 
CTTTTACTTAATTGTGTTCTTTCCAAATTAGTTCAACAAGGTTCCCACATACAACCTCAA120 
ATGTCACTTCCCTTAAAAACGATAGTACATTTGGTAAAGCCCTTTGCT168 
Me tSerLeuProLeuLysThrIleValHisLeuValLysProPheAla 
151015 
TGCACTGCTAGGTTTAGTGCGAGATACCCAATCCACGTCATTGTTGTT216 
Cy sThrAlaArgPheSerAlaArgTyrProIleHisValIleValVal 
202530 
GCTGTTTTATTGAGTGCCGCTGCTTATCTATCCGTGACACAATCTTAC264 
AlaVa lLeuLeuSerAlaAlaAlaTyrLeuSerValThrGlnSerTyr 
354045 
CTTAACGAATGGAAGCTGGACTCTAATCAGTATTCTACATACTTAAGC312 
LeuAsnGluTr pLysLeuAspSerAsnGlnTyrSerThrTyrLeuSer 
505560 
ATAAAGCCGGATGAGTTGTTTGAAAAATGCACACACTACTATAGGTCT360 
IleLysProAspGluLeuPh eGluLysCysThrHisTyrTyrArgSer 
65707580 
CCTGTGTCTGATACATGGAAGTTACTCAGCTCTAAAGAAGCCGCCGAT408 
ProValSerAspThrTr pLysLeuLeuSerSerLysGluAlaAlaAsp 
859095 
ATTTATACCCCTTTTCATTATTATTTGTCTACCATAAGTTTTCAAAGT456 
IleTyrThrProPheHi sTyrTyrLeuSerThrIleSerPheGlnSer 
100105110 
AAGGACAATTCAACGACTTTGCCTTCCCTTGATGACGTTATTTACAGT504 
LysAspAsnSerThrThrLe uProSerLeuAspAspValIleTyrSer 
115120125 
GTTGACCATACCAGGTACTTATTAAGTGAAGAGCCAAAGATACCAACT552 
ValAspHisThrArgTyrLeuLeuSe rGluGluProLysIleProThr 
130135140 
GAACTAGTGTCTGAAAACGGAACGAAATGGAGATTGAGAAACAACAGC600 
GluLeuValSerGluAsnGlyThrLysTrpArgLe uArgAsnAsnSer 
145150155160 
AATTTTATTTTGGACCTGCATAATATTTACCGAAATATGGTGAAGCAA648 
AsnPheIleLeuAspLeuHisAsnIleTyrAr gAsnMetValLysGln 
165170175 
TTTTCTAACAAAACGAGCGAATTTGATCAGTTCGATTTGTTTATCATC696 
PheSerAsnLysThrSerGluPheAspGlnPh eAspLeuPheIleIle 
180185190 
CTAGCTGCTTACCTTACTCTTTTTTATACTCTCTGTTGCCTGTTTAAT744 
LeuAlaAlaTyrLeuThrLeuPheTyrThrLeuCy sCysLeuPheAsn 
195200205 
GACATGAGGAAAATCGGATCAAAGTTTTGGTTAAGCTTTTCTGCTCTT792 
AspMetArgLysIleGlySerLysPheTrpLeuSerPheSe rAlaLeu 
210215220 
TCAAACTCTGCATGCGCATTATATTTATCGCTGTACACAACTCACAGT840 
SerAsnSerAlaCysAlaLeuTyrLeuSerLeuTyrThrThrHisSer 
2 25230235240 
TTATTGAAGAAACCGGCTTCCTTATTAAGTTTGGTCATTGGACTACCA888 
LeuLeuLysLysProAlaSerLeuLeuSerLeuValIleGlyLeuPr o 
245250255 
TTTATCGTAGTAATTATTGGCTTTAAGCATAAAGTTCGACTTGCGGCA936 
PheIleValValIleIleGlyPheLysHisLysValArgLeuAlaAl a 
260265270 
TTCTCGCTACAAAAATTCCACAGAATTAGTATTGACAAGAAAATAACG984 
PheSerLeuGlnLysPheHisArgIleSerIleAspLysLysIleThr 
275280285 
GTAAGCAACATTATTTATGAGGCTATGTTTCAAGAAGGTGCCTACTTA1032 
ValSerAsnIleIleTyrGluAlaMetPheGlnGluGlyAlaTyrLeu 
290 295300 
ATCCGCGACTACTTATTTTATATTAGCTCCTTCATTGGATGTGCTATT1080 
IleArgAspTyrLeuPheTyrIleSerSerPheIleGlyCysAlaIle 
3053 10315320 
TATGCTAGACATCTTCCCGGATTGGTCAATTTCTGTATTTTGTCTACA1128 
TyrAlaArgHisLeuProGlyLeuValAsnPheCysIleLeuSerThr 
3 25330335 
TTTATGCTAGTTTTCGACTTGCTTTTGTCTGCTACTTTTTATTCTGCC1176 
PheMetLeuValPheAspLeuLeuLeuSerAlaThrPheTyrSerAla 
340 345350 
ATTTTATCAATGAAGCTGGAAATTAACATCATTCACAGATCAACCGTC1224 
IleLeuSerMetLysLeuGluIleAsnIleIleHisArgSerThrVal 
355 360365 
ATCAGACAGACTTTGGAAGAGGACGGAGTTGTCCCAACTACAGCAGAT1272 
IleArgGlnThrLeuGluGluAspGlyValValProThrThrAlaAsp 
370375 380 
ATTATATATAAGGATGAAACTGCCTCAGAACCACATTTTTTGAGATCT1320 
IleIleTyrLysAspGluThrAlaSerGluProHisPheLeuArgSer 
3853903 95400 
AACGTGGCTATCATTCTGGGAAAAGCATCAGTTATTGGTCTTTTGCTT1368 
AsnValAlaIleIleLeuGlyLysAlaSerValIleGlyLeuLeuLeu 
4054 10415 
CTGATCAACCTTTATGTTTTCACAGATAAGTTAAATGCTACAATACTA1416 
LeuIleAsnLeuTyrValPheThrAspLysLeuAsnAlaThrIleLeu 
420425 430 
AACACGGTATATTTTGACTCTACAATTTACTCGTTACCAAATTTTATC1464 
AsnThrValTyrPheAspSerThrIleTyrSerLeuProAsnPheIle 
435440 445 
AATTATAAAGATATTGGCAATCTCAGCAATCAAGTGATCATTTCCGTG1512 
AsnTyrLysAspIleGlyAsnLeuSerAsnGlnValIleIleSerVal 
450455460 
TTGCCAAAGCAATATTATACTCCGCTGAAAAAATACCATCAGATCGAA1560 
LeuProLysGlnTyrTyrThrProLeuLysLysTyrHisGlnIleGlu 
4654704754 80 
GATTCTGTTCTACTTATCATTGATTCCGTTAGCAATGCTATTCGGGAC1608 
AspSerValLeuLeuIleIleAspSerValSerAsnAlaIleArgAsp 
4854904 95 
CAATTTATCAGCAAGTTACTTTTTTTTGCATTTGCAGTTAGTATTTCC1656 
GlnPheIleSerLysLeuLeuPhePheAlaPheAlaValSerIleSer 
500505510 
ATCAATGTCTACTTACTGAATGCTGCAAAAATTCACACAGGATACATG1704 
IleAsnValTyrLeuLeuAsnAlaAlaLysIleHisThrGlyTyrMet 
515520525 
AACTTC CAACCACAATCAAATAAGATCGATGATCTTGTTGTTCAGCAA1752 
AsnPheGlnProGlnSerAsnLysIleAspAspLeuValValGlnGln 
530535540 
AAATCGGCAACGATT GAGTTTTCAGAAACTCGAAGTATGCCTGCTTCT1800 
LysSerAlaThrIleGluPheSerGluThrArgSerMetProAlaSer 
545550555560 
TCTGGCCTAGAA ACTCCAGTGACCGCGAAAGATATAATTATCTCTGAA1848 
SerGlyLeuGluThrProValThrAlaLysAspIleIleIleSerGlu 
565570575 
GAAATCCAGAAT AACGAATGCGTCTATGCTTTGAGTTCCCAGGACGAG1896 
GluIleGlnAsnAsnGluCysValTyrAlaLeuSerSerGlnAspGlu 
580585590 
CCTATCCGTCCTTTA TCGAATTTAGTGGAACTTATGGAGAAAGAACAA1944 
ProIleArgProLeuSerAsnLeuValGluLeuMetGluLysGluGln 
595600605 
TTAAAGAACATGAATAATACT GAGGTTTCGAATCTTGTCGTCAACGGT1992 
LeuLysAsnMetAsnAsnThrGluValSerAsnLeuValValAsnGly 
610615620 
AAACTGCCATTATATTCCTTAGAGAAAAAA TTAGAGGACACAACTCGT2040 
LysLeuProLeuTyrSerLeuGluLysLysLeuGluAspThrThrArg 
625630635640 
GCGGTTTTAGTTAGGAGAAAGGCACTT TCAACTTTGGCTGAATCGCCA2088 
AlaValLeuValArgArgLysAlaLeuSerThrLeuAlaGluSerPro 
645650655 
ATTTTAGTTTCCGAAAAATTGCCCTTC AGAAATTATGATTATGATCGC2136 
IleLeuValSerGluLysLeuProPheArgAsnTyrAspTyrAspArg 
660665670 
GTTTTTGGAGCTTGCTGTGAAAATGTCATC GGCTATATGCCAATACCA2184 
ValPheGlyAlaCysCysGluAsnValIleGlyTyrMetProIlePro 
675680685 
GTTGGTGTAATTGGTCCATTAATTATTGATGGAACA TCTTATCACATA2232 
ValGlyValIleGlyProLeuIleIleAspGlyThrSerTyrHisIle 
690695700 
CCAATGGCAACCACGGAAGGTTGTTTAGTGGCTTCAGCTATGCGT GGT2280 
ProMetAlaThrThrGluGlyCysLeuValAlaSerAlaMetArgGly 
705710715720 
TGCAAAGCCATCAATGCTGGTGGTGGTGCAACAACTGTTTTA ACCAAA2328 
CysLysAlaIleAsnAlaGlyGlyGlyAlaThrThrValLeuThrLys 
725730735 
GATGGTATGACTAGAGGCCCAGTCGTTCGTTTCCCTACTTTA ATAAGA2376 
AspGlyMetThrArgGlyProValValArgPheProThrLeuIleArg 
740745750 
TCTGGTGCCTGCAAGATATGGTTAGACTCGGAAGAGGGACAAAAT TCA2424 
SerGlyAlaCysLysIleTrpLeuAspSerGluGluGlyGlnAsnSer 
755760765 
ATTAAAAAAGCTTTTAATTCTACATCAAGGTTTGCACGTTTGCAACAT 2472 
IleLysLysAlaPheAsnSerThrSerArgPheAlaArgLeuGlnHis 
770775780 
ATTCAAACCTGTCTAGCAGGCGATTTGCTTTTTATGAGATTTCGGACA2520 
IleGl nThrCysLeuAlaGlyAspLeuLeuPheMetArgPheArgThr 
785790795800 
ACTACCGGTGACGCAATGGGTATGAACATGATATCGAAAGGTGTCGAA2568 
Th rThrGlyAspAlaMetGlyMetAsnMetIleSerLysGlyValGlu 
805810815 
TACTCTTTGAAACAAATGGTAGAAGAATATGGTTGGGAAGATATGGAA2616 
Ty rSerLeuLysGlnMetValGluGluTyrGlyTrpGluAspMetGlu 
820825830 
GTTGTCTCCGTATCTGGTAACTATTGTACTGATAAGAAACCTGCCGCA2664 
ValVa lSerValSerGlyAsnTyrCysThrAspLysLysProAlaAla 
835840845 
ATCAATTGGATTGAAGGTCGTGGTAAAAGTGTCGTAGCTGAAGCTACT2712 
IleAsnTrpIl eGluGlyArgGlyLysSerValValAlaGluAlaThr 
850855860 
ATTCCTGGTGATGTCGTAAAAAGTGTTTTAAAGAGCGATGTTTCCGCT2760 
IleProGlyAspValValLy sSerValLeuLysSerAspValSerAla 
865870875880 
TTAGTTGAATTAAATATATCCAAGAACTTGGTTGGATCCGCAATGGCT2808 
LeuValGluLeuAsnIl eSerLysAsnLeuValGlySerAlaMetAla 
885890895 
GGATCTGTTGGTGGTTTCAACGCGCACGCAGCTAATTTGGTCACTGCA2856 
GlySerValGlyGlyPh eAsnAlaHisAlaAlaAsnLeuValThrAla 
900905910 
CTTTTCTTGGCATTAGGCCAAGATCCTGCGCAGAACGTCGAAAGTTCC2904 
LeuPheLeuAlaLeuGlyGl nAspProAlaGlnAsnValGluSerSer 
915920925 
AACTGTATAACTTTGATGAAGGAAGTTGATGGTGATTTAAGGATCTCT2952 
AsnCysIleThrLeuMetLysGluVa lAspGlyAspLeuArgIleSer 
930935940 
GTTTCCATGCCATCTATTGAAGTTGGTACGATTGGCGGGGGTACTGTT3000 
ValSerMetProSerIleGluValGlyThrIleGl yGlyGlyThrVal 
945950955960 
CTGGAGCCTCAGGGCGCCATGCTTGATCTTCTCGGCGTTCGTGGTCCT3048 
LeuGluProGlnGlyAlaMetLeuAspLeuLe uGlyValArgGlyPro 
965970975 
CACCCCACTGAACCTGGAGCAAATGCTAGGCAATTAGCTAGAATAATC3096 
HisProThrGluProGlyAlaAsnAlaArgGl nLeuAlaArgIleIle 
980985990 
GCGTGTGCTGTCTTGGCTGGTGAACTGTCTCTGTGCTCCGCACTTGCT3144 
AlaCysAlaValLeuAlaGlyGluLeuSerLeuCy sSerAlaLeuAla 
99510001005 
GCCGGTCACCTGGTACAAAGCCATATGACTCACAACCGTAAAACAAAC3192 
AlaGlyHisLeuValGlnSerHisMetThrHisAsnArgL ysThrAsn 
101010151020 
AAAGCCAATGAACTGCCACAACCAAGTAACAAAGGGCCCCCCTGTAAA3240 
LysAlaAsnGluLeuProGlnProSerAsnLysGlyProProCysLys 
1025103010351040 
ACCTCAGCATTATTATAACTCTTGTAGTTTACATGGTGATACTTTATATCTTTGT3295 
ThrSerAlaLeuLeu 
1045 
ATTGTCTAGCTATT CTAAATCATCTGCATGTAATAAGAAGTTGATCAAAATGA3348 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1045 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
MetSerLeuProLeuLysThrIle ValHisLeuValLysProPheAla 
151015 
CysThrAlaArgPheSerAlaArgTyrProIleHisValIleValVal 
2025 30 
AlaValLeuLeuSerAlaAlaAlaTyrLeuSerValThrGlnSerTyr 
354045 
LeuAsnGluTrpLysLeuAspSerAsnGlnTyrSerThrTyrLeuSer 
505560 
IleLysProAspGluLeuPheGluLysCysThrHisTyrTyrArgSer 
65707580 
ProValSerAspT hrTrpLysLeuLeuSerSerLysGluAlaAlaAsp 
859095 
IleTyrThrProPheHisTyrTyrLeuSerThrIleSerPheGlnSer 
100 105110 
LysAspAsnSerThrThrLeuProSerLeuAspAspValIleTyrSer 
115120125 
ValAspHisThrArgTyrLeuLeuSerGluGluPro LysIleProThr 
130135140 
GluLeuValSerGluAsnGlyThrLysTrpArgLeuArgAsnAsnSer 
145150155160 
As nPheIleLeuAspLeuHisAsnIleTyrArgAsnMetValLysGln 
165170175 
PheSerAsnLysThrSerGluPheAspGlnPheAspLeuPheIleIle 
180185190 
LeuAlaAlaTyrLeuThrLeuPheTyrThrLeuCysCysLeuPheAsn 
195200205 
AspMetArgLysIleGlySerLysP heTrpLeuSerPheSerAlaLeu 
210215220 
SerAsnSerAlaCysAlaLeuTyrLeuSerLeuTyrThrThrHisSer 
225230235 240 
LeuLeuLysLysProAlaSerLeuLeuSerLeuValIleGlyLeuPro 
245250255 
PheIleValValIleIleGlyPheLysHisLysValArgLeuAla Ala 
260265270 
PheSerLeuGlnLysPheHisArgIleSerIleAspLysLysIleThr 
275280285 
ValSerAsnIleIl eTyrGluAlaMetPheGlnGluGlyAlaTyrLeu 
290295300 
IleArgAspTyrLeuPheTyrIleSerSerPheIleGlyCysAlaIle 
305310 315320 
TyrAlaArgHisLeuProGlyLeuValAsnPheCysIleLeuSerThr 
325330335 
PheMetLeuValPheAspLeuLeuLeuSerAlaT hrPheTyrSerAla 
340345350 
IleLeuSerMetLysLeuGluIleAsnIleIleHisArgSerThrVal 
355360365 
Ile ArgGlnThrLeuGluGluAspGlyValValProThrThrAlaAsp 
370375380 
IleIleTyrLysAspGluThrAlaSerGluProHisPheLeuArgSer 
385390 395400 
AsnValAlaIleIleLeuGlyLysAlaSerValIleGlyLeuLeuLeu 
405410415 
LeuIleAsnLeuTyrValPheTh rAspLysLeuAsnAlaThrIleLeu 
420425430 
AsnThrValTyrPheAspSerThrIleTyrSerLeuProAsnPheIle 
435440 445 
AsnTyrLysAspIleGlyAsnLeuSerAsnGlnValIleIleSerVal 
450455460 
LeuProLysGlnTyrTyrThrProLeuLysLysTyrHisGlnIleGlu 
465 470475480 
AspSerValLeuLeuIleIleAspSerValSerAsnAlaIleArgAsp 
485490495 
GlnPheIleSer LysLeuLeuPhePheAlaPheAlaValSerIleSer 
500505510 
IleAsnValTyrLeuLeuAsnAlaAlaLysIleHisThrGlyTyrMet 
515 520525 
AsnPheGlnProGlnSerAsnLysIleAspAspLeuValValGlnGln 
530535540 
LysSerAlaThrIleGluPheSerGluThrArgSerMetProAl aSer 
545550555560 
SerGlyLeuGluThrProValThrAlaLysAspIleIleIleSerGlu 
565570575 
GluIleGlnAsnAsnGluCysValTyrAlaLeuSerSerGlnAspGlu 
580585590 
ProIleArgProLeuSerAsnLeuValGluLeuMetGluLysGluGln 
595 600605 
LeuLysAsnMetAsnAsnThrGluValSerAsnLeuValValAsnGly 
610615620 
LysLeuProLeuTyrSerLeuGluLysLysLeu GluAspThrThrArg 
625630635640 
AlaValLeuValArgArgLysAlaLeuSerThrLeuAlaGluSerPro 
645650 655 
IleLeuValSerGluLysLeuProPheArgAsnTyrAspTyrAspArg 
660665670 
ValPheGlyAlaCysCysGluAsnValIleGlyTyrMetProIlePr o 
675680685 
ValGlyValIleGlyProLeuIleIleAspGlyThrSerTyrHisIle 
690695700 
ProMetAlaThrThrGluGly CysLeuValAlaSerAlaMetArgGly 
705710715720 
CysLysAlaIleAsnAlaGlyGlyGlyAlaThrThrValLeuThrLys 
725 730735 
AspGlyMetThrArgGlyProValValArgPheProThrLeuIleArg 
740745750 
SerGlyAlaCysLysIleTrpLeuAspSerGluGlu GlyGlnAsnSer 
755760765 
IleLysLysAlaPheAsnSerThrSerArgPheAlaArgLeuGlnHis 
770775780 
IleGlnThrC ysLeuAlaGlyAspLeuLeuPheMetArgPheArgThr 
785790795800 
ThrThrGlyAspAlaMetGlyMetAsnMetIleSerLysGlyValGlu 
8 05810815 
TyrSerLeuLysGlnMetValGluGluTyrGlyTrpGluAspMetGlu 
820825830 
ValValSerValSerGlyAsnTyr CysThrAspLysLysProAlaAla 
835840845 
IleAsnTrpIleGluGlyArgGlyLysSerValValAlaGluAlaThr 
850855860 
IleProGlyAspValValLysSerValLeuLysSerAspValSerAla 
865870875880 
LeuValGluLeuAsnIleSerLysAsnLeuValGlySerAlaMetAla 
885890895 
GlySerValGlyGlyPheAsnAlaHisAlaAlaAsnLeuValThrAla 
900905910 
LeuPheLeuAlaL euGlyGlnAspProAlaGlnAsnValGluSerSer 
915920925 
AsnCysIleThrLeuMetLysGluValAspGlyAspLeuArgIleSer 
930935 940 
ValSerMetProSerIleGluValGlyThrIleGlyGlyGlyThrVal 
945950955960 
LeuGluProGlnGlyAlaMetLeuAspLeuLeuGlyVal ArgGlyPro 
965970975 
HisProThrGluProGlyAlaAsnAlaArgGlnLeuAlaArgIleIle 
980985990 
Al aCysAlaValLeuAlaGlyGluLeuSerLeuCysSerAlaLeuAla 
99510001005 
AlaGlyHisLeuValGlnSerHisMetThrHisAsnArgLysThrAsn 
1010 10151020 
LysAlaAsnGluLeuProGlnProSerAsnLysGlyProProCysLys 
1025103010351040 
ThrSerAlaLeuLeu 
1045