Nucleic acid molecules encoding cytochrome P450-type proteins involved in the brassinosteroid synthesis in plants

The invention describes nucleic acid molecules encoding cytochrome P450-t proteins involved in the brassionosteroid synthesis in plants, transgenic plant cells and plants containing such nucleic acid molecules as well as processes for the identification of other proteins involved in brassinosteroid synthesis and processes for the identification of substances acting as brassinosteroids or as brassinosteroid inhibitors in plants.

The present invention relates to nucleic acid molecules encoding cytochrome 
P450-type proteins involved in the brassionosteroid synthesis in plants, 
to transgenic plant cells and plants containing such nucleic acid 
molecules as well as to processes for the identification of other proteins 
involved in brassinosteroid synthesis and processes for the identification 
of substances acting as brassinosteroids or as brassinosteroid inhibitors 
in plants. 
In 1979 a novel plant growth-promoting factor, termed brassinolide, was 
isolated from the pollen of rape (Brassica napus) and identified as a 
novel type of steroid lactone. It was found that brassinolide-like steroid 
compounds (called brassinosteroids) occur in all plant species examined at 
very low concentrations (for review, see Mandava, Ann. Rev. Plant Physiol. 
Plant Mol. Biol. 39 (1988), 23-52). Initial studies of the physiological 
action of brassinolide showed that this particular factor (i) accelerated 
the germination and growth of plant seedlings at low temperatures, (ii) 
promoted the increase of cell size and elongation by induction of a 
longitudinal arrangement of cortical microtubuli and cellulose 
microfilaments on the surface of cells, (iii) promoted xylem 
differentiation by amplifying the tracheal elements, (iv) resulted in 
significant increase of dry weight of plants and their fruits, (v) 
promoted leaf unrolling and enlargement, (vi) induced H+ export and 
membrane hyperpolarization characteristic for auxin induced cell growth, 
(vii) inhibited the division of crown-gall tumour cells and radial growth 
of stems, (viii) repressed anthocyanin production in light-grown plants, 
(ix) inhibited the de-etiolation induced, e.g. by cytokinin in the dark, 
(x) promoted tissue senescence in the dark, but prolonged the life-span of 
plants in the light and (xi) induced plant pathogen resistance responses 
to numerous bacterial and fungal species (listed by Mandava (1988), loc. 
cit.). 
Following the initial isolation of and physiological studies with 
brassinolides, numerous brassinosteroid compounds, representing putative 
biosynthetic intermediates, were identified in different plant species. 
Because the in vivo concentration of these compounds was found to be 
extremely low, efforts had been made to develop methods for chemical 
synthesis of these compounds (for review, see: Adam and Marquardt, 
Phytochem. 25 (1986), 1787-1799). These compounds were tested in field 
experiments using soybean, maize, rice and other crops as well as trees in 
order to confirm the results of physiological studies. However, the field 
trials showed that due to poor uptake of steroids through the plant 
epidermis, the amount of steroids required for spraying or fertilization 
was considerable. Several methods for the chemical synthesis of 
brassinolides had been described since then, however, their practical use 
in agriculture is rather limited. Because the prize of brassinolide 
treatments is comparably high, their application cannot compete with the 
application of other known fertilizers and pesticides. Thus, up to know 
the practical application of these compounds has largely been abandoned, 
except for their occassional application as crop safeners. 
The interest in brassinosteroids as possible growth regulators has 
furthermore faded since plant physiologists claimed that physiological 
data did not indicate that these compounds were indeed functional growth 
factors because their concentrations in most plant species were very low 
in comparison to other growth factors, such as auxins, cytokinins, 
abscisic acid, ethylen and gibberellins. In addition, no plant mutant 
defective in brassinolide synthesis was available to demonstrate that 
these compounds are essential for plant growth and development. Therefore, 
brassinosteroids were classified as minor secondary plant metabolites with 
a questionable biological function. 
In order to be able to demonstrate that brassinosteroids can indeed be used 
as potential growth regulators of plants and to exploit the possible 
advantages and potentials of these substances, it would be necessary to 
identify plant mutants defective in brassinosteroid synthesis which would 
allow the characterization of genes involved in brassinosteroid synthesis. 
Thus, the problem underlying the present invention is to identify plant 
mutants defective in brassinosteroid synthesis and to identify nucleic 
acid molecules encoding proteins involved in the synthesis of 
brassinosteroids in plants. 
The problem is solved by the provision of the embodiments characterized in 
the claims. 
Thus, the invention relates to nucleic acid molecules encoding a protein 
having the biological, namely the enzymatic, activity of a cytochrome 
P450-type hydroxylase or encoding a biologically active fragment of such a 
protein. Such nucleic acid molecules encode preferably a protein that 
comprises the amino acid sequence shown in Seq ID No. 2 or a fragment 
thereof that has the biological activity of a cytochrome P450-type 
hydroxylase. More preferably such nucleic acid molecules comprise the 
nucleotide sequence shown in Seq ID No. 1, namely the indicated coding 
region, or a corresponding ribonucleotide sequence. 
The present invention also relates to nucleic acid molecules coding for a 
protein having the amino acid sequence as coded for by the exons of the 
nucleotide sequence given in SEQ ID NO:3 or coding for a fragment of such 
a protein, wherein the protein and the fragment have the biological 
activity of a cytochrome P450 hydroxylase. In particular, the present 
invention relates to nucleic acid molecules comprising the nucleotide 
sequence depicted in SEQ ID NO:3, namely the nucleotide sequence of the 
indicated exons, or a corresponding ribonucleotide sequence. Furthermore, 
the present invention relates to nucleic acid molecules which hybridize to 
any of the nucleic acid molecules as described above and which code for a 
protein having the biological activity of a cytochrome P450-type 
hydroxylase or for a biologically active fragment of such a protein as 
well as to nucleic acid molecules which are complementary to any of the 
nucleic acid molecules as described above. The present invention also 
relates to nucleic acid molecules encoding a cytochrome P450-type 
hydroxylase, or a biologically active fragment thereof, the sequence of 
which differs from the sequence of the above-described nucleic acid 
molecules due to the degeneracy of the genetic code. 
By "hybridizing" it is meant that such nucleic acid molecules hybridize 
under conventional hybridization conditions, preferably under stringent 
conditions such as described by, e.g, Sambrook et al. (Molecular Cloning; 
A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 
Cold Spring Harbor, N.Y. (1989)). Nucleic acid molecules hybridizing with 
the above-described nucleic acid molecules can in general be derived from 
any plant possessing such molecules, preferably from monocotyledonous or 
dicotyledonous plants, in particular from any plant of interest in 
agriculture, horticulture or wood culture, such as crop plants, namely 
those of the family Poaceae, any other starch producing plants, such as 
potato, maniok, leguminous plants, oil producing plants, such as oilseed 
rape, linenseed, etc., plants using protein as storage substances, such as 
soybean, plants using sucrose as storage substance, such as sugar beet or 
sugar cane, trees, ornamental plants etc. Preferably the nucleic acid 
molecules according to the invention are derived from plants belonging to 
the family Brassicaceae. Nucleic acid molecules hybridizing to the 
above-described nucleic acid molecules can be isolated, e.g., from 
libraries, such as cDNA or genomic libraries by techniques well known in 
the art. For example, hybridizing nucleic acid molecules can be identified 
and isolated by using the above-described nucleic acid molecules or 
fragments thereof or complements thereof as probes to screen libraries by 
hybridizing with said molecules according to standard techniques. Possible 
is also the isolation of such nucleic acid molecules by applying the 
polymerase chain reaction (PCR) using as primers oligonucleotides derived 
from the above-described nucleic acid molecules. 
The term "hybridizing nucleic acid molecules" also includes fragments, 
derivatives and allelic variants of the above-described nucleic acid 
molecules that code for a protein having the biological activity of a 
cytochrome P450-type hydroxylase or a biologically active fragment 
thereof. Fragments are understood to be parts of nucleic acid molecules 
long enough to code for the described protein or a biologically active 
fragment thereof. The term "derivative" means in this context that the 
nucleotide sequence of these nucleic acid molecules differs from the 
sequences of the above-described nucleic acid molecules in one or more 
positions and are highly homologous to said nucleic acid molecules. 
Homology is understood to refer to a sequence identity of at least 40%, 
particularly an identity of at least 60%, preferably more than 80% and 
still more preferably more than 90%. The deviations from the sequences of 
the nucleic acid molecules described above can, for example, be the result 
of substitutions, deletions, additions, insertions or recombination. 
Homology further means that the respective nucleic acid molecules or 
encoded proteins are functionally and/or structurally equivalent. The 
nucleic acid molecules that are homologous to the nucleic acid molecules 
described above and that are derivatives of said nucleic acid molecules 
are, for example, variations of said nucleic acid molecules which 
represent modifications having the same biological function, in particular 
encoding proteins with the same biological function. They may be naturally 
occuring variations, such as sequences from other plant varieties or 
species, or mutations. These mutations may occur naturally or may be 
obtained by mutagenesis techniques. Furthermore, these variations may be 
synthetically produced sequences. The allelic variations may be naturally 
occuring variants as well as synthetically produced or genetically 
engineered variants. 
The proteins encoded by the various derivatives and variants of the 
above-described nucleic acid molecules share specific common 
characteristics, such as enzymatic activity, molecular weight, 
immunological reactivity, conformation, etc., as well as physical 
properties, such as electrophoretic mobility, chromatographic behaviour, 
sedimentation coefficients, pH optimum, temperature optimum, stability, 
solubility, spectroscopic properties, etc. 
Cytochrome P450 proteins can be characterized by several features. For 
example, they are membrane-associated NAD(P)H dependent monooxygenases 
which normally form in vivo a complex with reductases. The CO-complex of 
these proteins shows an absorption maximum in the range of 450 nm. 
The proteins encoded by the nucleic acid molecules according to the 
invention comprise preferably domains characteristic for cytochrome P450 
proteins, especially those characteristic for microsomal cytochrom P450 
proteins, such as the conserved N-terminal membran-anchoring domain, the 
proline rich domain, the heme-binding domain and the oxygen-binding domain 
(see, for example, Nebert and Gonzalez, Ann. Rev. Biochem. 56 (1987), 
945-993). Furthermore, it is preferred that the proteins encoded by the 
nucleic acid molecules according to the invention contain domains 
characteristic for steroid hydroxylases, namely steroid binding domains. 
Preferably the proteins have the enzymatic activity of a steroid 
hydroxylase. 
In a preferred embodiment the nucleic acid molecules according to the 
invention encode a cytochrome P450-type protein with the enzymatic 
activity of a hydroxylase which is involved in the conversion of 
cathasterone to teasterone (see FIG. 1). This enzymatic activity may be 
determined by feeding experiments as described in the examples. 
The proteins encoded by the nucleic acid molecules according to the 
invention which, due to the presence of certain domains and due to their 
enzymatic activity can be classified as cytochrome P450 proteins, display 
overall a very low homology to known cytochrome P450s (less than 40%). 
Thus, these proteins are novel and constitute a new family of cytochrome 
P450 proteins with a novel substrate specificity. 
The nucleic acid molecules according to the invention are preferably RNA or 
DNA molecules, preferably cDNA or genomic DNA. 
The present invention is based on the finding that a particular Arabidopsis 
mutant generated by gene-tagging, which showed dwarfism and several other 
morphological and developmental abnormalities, can be restored to the 
wildtype phenotype by the addition of specific brassinosteroid compounds. 
Furthermore, the mutated gene and a corresponding cDNA had been isolated 
and characterized as encoding a cytochrome P450-type hydroxylase, the 
overexpression of which in the tagged mutant can also restore the wildtype 
phenotype. Moreover, it has been found that overexpression of the cDNA in 
transgenic plants leads to several physiological and phenotypic changes 
which might be useful for the engineering of improved plants for 
agriculture, wood culture or horticulture. 
The present invention provides evidence that the described nucleic acid 
molecules encode proteins with an enzymatic activity involved in 
brassinosteroid synthesis in plants. Furthermore, the present invention 
shows that a mutant defective in this enzyme activity shows severe 
physiological and phenotypic changes, for example, dwarfism, which can be 
reverted by addition of specific brassinosteroid compounds, and that 
plants overexpressing such an enzyme activity also show phenotypic 
changes, such as increased cell elongation. 
Thus, the present invention for the first time clearly establishes that 
brassinosteroids are indeed of central importance as plant growth 
regulators and, furthermore, provides extremely useful tools to 
(i) identify mutants deficient in brassinosteroid snythesis; 
(ii) identify and isolate genes encoding proteins involved in the 
brassionosteroid synthesis in plants or in its regulation; 
(iii) generate plants with modified brassinosteroid synthesis and 
consequently with modified physiological and/or phenotypic 
characteristics; and 
(iv) identify compounds which may act as potential brassionosteroids on 
plants. 
The different possible applications of the nucleic acid molecules according 
to the invention as well as molecules derived from them will be described 
in detail in the following. 
In one aspect the present invention relates to nucleic acid probes which 
specifically hybridize with a nucleic acid molecule as described above. 
This means that they hybridize, preferably under stringent conditions, 
only with the nucleic acid molecules as described above and show no or 
very little cross-hybridization with nucleic acid molecules coding for 
other proteins. The nucleic acid probes according to the invention 
comprise a nucleic acid molecule of at least 15 nucleotides. Nucleic acid 
probe technology is well known to those skilled in the art who will 
readily appreciate that such probes may vary in length. The nucleic acid 
probes are useful for various applications. On the one hand, they may be 
used as PCR primers for amplification of nucleic acid molecules according 
to the invention. On the other hand, they can be useful tools for the 
detection of the expression of molecules according to the invention in 
plants, for example, by in-situ hybridization or Northern-Blot 
hybridization. Other applications are the use as hybridization probe to 
identify nucleic acid molecules hybridizing to the nucleic acid molecules 
according to the invention by homology screening of genomic or cDNA 
libraries. Nucleic acid probes according to the invention which are 
complementary to an RNA molecule as described above may also be used for 
repression of expression of such an RNA due to an antisense effect or for 
the construction of appropriate ribozymes which specifically cleave such 
RNA molecules. Furthermore, the person skilled in the art is well aware 
that it is also possible to label such a nucleic acid probe with an 
appropriate marker for specific applications. 
The present invention also relates to vectors, particularly plasmids, 
cosmids, viruses, bacteriophages and other vectors used conventionally in 
genetic engineering that contain a nucleic acid molecule according to the 
invention. 
In a preferred embodiment the nucleic acid molecule present in the vector 
is linked to regulatory elements which allow the expression of the nucleic 
acid molecule in procaryotic or eucaryotic cells. Expression comprises 
transcription of the nucleic acid molecule into a translatable mRNA. 
Regulatory elements ensuring expression in procaryotic or eucaryotic cells 
are well known to those skilled in the art. In the case of eucaryotic 
cells they comprise normally promoters ensuring initiation of 
transcription and optionally poly-A signals ensuring termination of 
transcription and stabilization of the transcript. Additional regulatory 
elements may include transcriptional as well as translational enhancers. 
The present invention furthermore relates to host cells comprising a vector 
as described above or a nucleic acid molecule according to the invention 
wherein the nucleic acid molecule is foreign to the host cell. 
By "foreign" it is meant that the nucleic acid molecule is either 
heterologous with respect to the host cell, this means derived from a cell 
or organism with a different genomic background, or is homologous with 
respect to the host cell but located in a different genomic environment 
than the naturally occuring counterpart of said nucleic acid molecule. 
This means that, if the nucleic acid molecule is homologous with respect 
to the host cell, it is not located in its natural location in the genome 
of said host cell, in particular it is surrounded by different genes. In 
this case the nucleic acid molecule may be either under the control of its 
own promoter or under the control of a heterologous promoter. The vector 
or nucleic acid molecule according to the invention which is present in 
the host cell may either be integrated into the genome of the host cell or 
it may be maintained in some form extrachromosomally. 
The host cell can be any procaryotic or eucaryotic cell, such as bacterial, 
fungal, plant or animal cells. Preferred fungal cells are, for example, 
those of the genus Saccharomyces, in particular those of the species S. 
cerevisiae. 
The present invention furthermore relates to proteins encoded by the 
nucleic acid molecules according to the invention or to fragments of such 
proteins which have the biological activity of a cytochrome P450-type 
hydroxylase. 
Furthermore, the present invention relates to antibodies specifically 
recognizing proteins according to the invention or parts, i.e specific 
fragments or epitopes, of such proteins. Specific eptitopes or fragments 
may, for example, comprise amino acid sequences which constitute domains 
which are characteristic for the proteins according to the invention, such 
as the substrate binding domain or the like. These antibodies can be 
monoclonal antibodies, polyclonal antibodies or synthetic antibodies as 
well as fragments of antibodies, such as Fab fragments etc. These 
antibodies can be used, for example, for the immunoprecipitation and 
immunolocalization of proteins according to the invention as well as for 
the monitoring of the synthesis of such proteins, for example, in 
recombinant organisms, and for the identification of proteins interacting 
with the proteins according to the invention. 
Another subject of the invention is a process for the preparation of such 
proteins which comprises the cultivation of host cells according to the 
invention which, due to the presence of a vector or a nucleic acid 
molecule according to the invention, are able to express such a protein, 
under conditions which allow expression of the protein and recovering of 
the so-produced protein from the culture. Depending on the specific 
constructs and conditions used, the protein may be recovered from the 
cells, from the cultur medium or from both. For the person skilled in the 
art it is well known that it is not only possible to express a native 
protein but also to express the protein as fusion proteins or to add 
signal sequences directing the protein to specific compartments of the 
host cell, ensuring secretion of the protein into the culture medium, etc. 
The nucleic acid molecules according to the invention are in particular 
useful for the genetic manipulation of plant cells in order to modify the 
brassinosteroid synthesis and to obtain plants with modified, preferably 
with improved or useful phenotypes. Thus, the present invention relates 
also to transgenic plant cells which contain stably integrated into the 
genome a nucleic acid molecule according to the invention linked to 
regulatory elements which allow for expression of the nucleic acid 
molecule in plant cells and wherein the nucleic acid molecule is foreign 
to the transgenic plant cell. For the meaning of foreign, see supra. 
The presence and expression of the nucleic acid molecule in the transgenic 
plant cells leads to the synthesis of a protein with the biological 
activity of a cytochrome P450-type hydroxylase which has an influence on 
brassinosteroid synthesis in the plant cells and leads to physiological 
and phenotypic changes in plants containing such cells. 
Thus, the present invention also relates to transgenic plants comprising 
transgenic plant cells according to the invention. 
Due to the expression of a protein having the biological activity of a 
cytochrome P450-type hydroxylase this transgenic plants may show various 
physiological, developmental and/or morphological modifications in 
comparison to wildtype plants. For example, these transgenic plants may 
display an increased induction of pathogenesis related genes (see, for 
example, Uknes et al., Plant Cell 4 (1992), 645-656), modified morphology, 
namely a stimulation of growth, increased cell elongation and/or increased 
wood production due to stimulated xylem differentiation. Furthermore, 
these transgenic plants may show accelarated seed germination at low 
temperatures, an increase in dry weight, repressed anthocyanin production 
during growth in light and/or inhibited de-etiolation which is induced, 
e.g. by cytokinin, in the dark. 
The provision of the nucleic acid molecules according to the invention 
furthermore opens up the possibility to produce transgenic plant cells 
with a reduced level of the cytochrome P450-type hydroxylase as described 
above and, thus, with a defect in brassinosteroid synthesis. Techniques 
how to achieve this are well known to the person skilled in the art. These 
include, for example, the expression of antisense-RNA, ribozymes, of 
molecules which combine antisense and ribozyme functions and/or of 
molecules which provide for a cosupression effect. 
When using the antisense approach for reduction of the above described 
enzymatic activity in plant cells, the nucleic acid molecule encoding the 
antisense-RNA is preferably is of homologous origin with respect to the 
plant species used for transformation. However, it is also possible to use 
nucleic acid molecules which display a high degree of homology to 
endogenously occuring nucleic acid molecules encoding the respective 
enzyme activity. In this case the homology is preferably higher than 80%, 
particularly higher than 90% and still more preferably higher than 95%. 
The reduction of the synthesis of a protein according to the invention in 
the transgenic plant cells results in an alteration in the brassinosteroid 
synthesis and/or metabolism in the cells. In transgenic plants comprising 
such cells this can lead to various physiological, developmental and/or 
morphological changes. 
Thus, the present invention also relates to transgenic plants comprising 
the above-described transgenic plant cells. These may show, for example, 
morphological changes, such as dwarfism, and/or developmental changes in 
comparison to wildtype plants, such as a reduced elongation of the 
hypocotyl of seedlings germinating in the dark or male sterility. 
Furthermore, these plants may display physiological changes in comparison 
to wildtype plants, such as an altered stress tolerance. Preferably the 
transgenic plants according to the invention show at least one of the 
following features: 
the seedlings which result from germination in the dark have a short 
hypocotyl, no apical hook, open cotyledons and/or extended leaf primordia 
when compared to wildtype seedlings; 
the length of epidermal cell files in the hypocotyl is reduced about 5-fold 
when compared to wildtype plants; 
the length of epidermal cell files in the roots of the seedlings is 
decreased by about 20 to 50% when compared to wildtype plants; 
the epidermal cells of the hypocotyl show thick transverse files of 
cellulose fibers (see, for example, FIGS. 2D,E); 
the epidermal cells of the hypocotyl show perpendicular divisions leading 
to differentiation of stomata guard cells (FIG. 2B), 
the cotyledons show dense stomata and trichomes normally characteristic for 
leaves (FIG. 2C); 
derepression of photomorphogenesis and de-etiolation in the dark; 
a 20 to 30-fold reduction in size in comparison to wildtype plants when 
grown in soil under white light (dwarfism); 
a reduction of the number of longitudinal mesophyll cell files in leaves 
and a failure of palisade cells to elongate; 
an amplification and duplication of stomatal guard cells in the leaf 
epidermis (FIGS. 2F,G); 
unequal division of cambium in the stem; 
production of extranumerary phloem cell files at the expense of xylem 
cells; 
the failure of the pollen to elongate during germination thereby resulting 
in male sterility; 
a differential regulation of stress responsive genes. 
The present invention also relates to cultured plant tissues comprising 
transgenic plant cells as described above which either show overexpression 
of a protein according to the invention or a reduction in synthesis of 
such a protein. 
In yet another aspect the invention also relates to harvestable parts and 
to propagation material of the transgenic plants according to the 
invention which either contain transgenic plant cells expressing a nucleic 
acid molecule according to the invention or which contain cells which show 
a reduced activity of the described protein. Harvestable parts can be in 
principle any useful parts of a plant, for example, leaves, stems, fruit, 
seeds, roots etc. 
Propagation material includes, for example, seeds, fruits, cuttings, 
seedlings, tubers, rootstocks etc. 
For the expression of the nucleic acid molecules according to the invention 
in sense or antisense orientation in plant cells, the molecules are placed 
under the control of regulatory elements which ensure the expression in 
plant cells. These regulatory elements may be heterologous or homologous 
with respect to the nucleic acid molecule to be expressed as well with 
respect to the plant species to be transformed. In general, such 
regulatory elements comprise a promoter active in plant cells. To obtain 
expression in all tissues of a transgenic plant, preferably constitutive 
promoters are used, such as the 35 S promoter of CaMV (Odell et al., 
Nature 313 (1985), 810-812) or promoters of the polyubiquitin genes of 
maize (Christensen et al., Plant Mol. Biol. 18 (1982), 675-689). In order 
to achieve expression in specific tissues of a transgenic plant it is 
possible to use tissue specific promoters (see, e.g., Stockhaus et al., 
EMBO J. 8 (1989), 2245-2251). Known are also promoters which are 
specifically active in tubers of potatoes or in seeds of different plants 
species, such as maize, Vicia, wheat, barley etc. Inducible promoters may 
be used in order to be able to exactly control expression. An example for 
inducible promoters are the promoters of heat shock proteins. 
The regulatory elements may further comprise transcriptional and/or 
translational enhancers functional in plants cells. 
Furthermore, the regulatory elements may include transcription termination 
signals, such as a poly-A signal, which lead to the addition of a poly A 
tail to the transcript which may improve its stability. 
In the case that a nucleic acid molecule according to the invention is 
expressed in sense orientation it is in principle possible to modifiy the 
coding sequence in such a way that the protein is located in any desired 
compartment of the plant cell. These include the endoplasmatic reticulum, 
the vacuole, the mitochondria, the plastides, the apoplast, the cytoplasm 
etc. Methods how to carry out this modifications and signal sequences 
ensuring localization in a desired compartment are well known to the 
person skilled in the art. 
Methods for the introduction of foreign DNA into plants are also well known 
in the art. These include, for example, the transformation of plant cells 
or tissues with T-DNA using Agrobacterium tumefaciens or Agrobacterium 
rhizogenes (EP-A 120 516; EP-A 116 718; Hoekema in: The Binary Plant 
Vector System, Offsetdrukkerij Kanters BV, Alblasserdam (1985), Chapter V; 
Fraley et al., Crit. Rev. Plant Sci. 4, 1-46 und An et al., EMBO J. 4 
(1985), 277-287), the fusion of protoplasts, direct gene transfer (see, 
e.g., EP-A 164 575), injection, electroporation, biolistic methods like 
particle bombardment and other methods. 
The transformation of most dicotyledonous plants is possible with the 
methods described above. But also for the transformation of 
monocotyledonous plants several successful transformation techniques have 
been developed. These include the transformation using biolistic methods 
(Wan and Lemaux, Plant Physiol. 104 (1994), 37-48; Vasil et al., 
Bio/Technology 11 (1993), 1553-1558), protoplast transformation, 
electroporation of partially permeabilized cells, introduction of DNA 
using glas fibers, etc. 
In general, the plants which can be modified according to the invention and 
which either show overexpression of a protein according to the invention 
or a reduction of the synthesis of such a protein can be derived from any 
desired plant species. They can be monocotyledonous plants or 
dicotyledonous plants, preferably they belong to plant species of interest 
in agriculture, wood culture or horticulture interest, such as crop plants 
(e.g. maize, rice, barley, wheat, rye, oats etc.), potatoes, oil producing 
plants (e.g. oilseed rape, sunflower, pea nut, soy bean, etc.), cotton, 
sugar beet, sugar cane, leguminous plants (e.g. beans, peas etc.), wood 
producing plants, preferably trees, etc. 
The present invention furthermore provides a process for the identification 
and isolation of nucleic acid molecules encoding proteins which are 
involved in brassinosteroid synthesis in plants or in its regulation which 
comprises the steps of: 
(a) screening naturally occurring, artificially mutagenised or genetically 
engineered dwarf mutants for those whose seedlings upon germination in the 
dark display no or only little elongation of the hypocotyl; 
(b) identifying those dwarf mutants identified in step (a) in which 
elongation of the hypocotyl in the dark can be stimulated by adding 
different brassinosteroids or brassinosteroid-like compounds; 
(c) identification and isolation of the gene(s) which are capable of 
complementing those dwarf mutants identified in step (b); 
(d) characterization of the isolated gene(s) and its (their) encoded 
product(s). 
This process is based on the finding that mutants which are defective in 
brassinosteroid synthesis show the characteristic features of reduced 
growth (dwarfism) and a reduced elongation of the hypocotyl in seedlings 
grown in the dark when compared to wildtype plants. By these features it 
is possible to select plant mutants which may have a defect in 
brassinosteroid synthesis. This can be confirmed if it possible to 
complement the mutant phenotype by addition of brassinosteroid compounds 
(step (b)). Various brassinosteroid compounds are known to the person 
skilled in the art. They may be naturally occuring brassinosteroids or 
chemically synthesized analogs. The way of application of the 
brassinosteroid compounds to the plants is not critical. Spraying of 
solutions is preferred. From the identified mutants which are indeed 
defective in brassinosteroid synthesis, the mutated gene can be identified 
and isolated. 
One possibility is: (i) to create a plurality of mutants by gene-tagging 
(e.g. by T-DNA or transposons), (ii) identify according to step (a) and 
(b) the mutants which are defective in brassinosteroid synthesis, (iii) to 
prepare a genomic library of the and (iv) to isolate the mutated gene with 
the help of the DNA used for tagging of the genes. This leads to the 
identification of the tagged mutated gene. This can subsequently be used 
to isolate wildtype cDNA and genomic clones using standard techniques, for 
example, hybidization techniques or PCR. Such an approach is in principle 
described in the examples, see supra. 
Alternatively, the identification and isolation of the mutated gene can be 
carried out as follows: (i) precise genetic mapping of the mutation which 
allows then (ii) the isolation of yeast artificial chromosome (YAC) clones 
carrying the corresponding gene on a smaller chromosome fragment, (iii) 
using these YAC clones to isolate corresponding cosmid clones, (iv) using 
these cosmid clones for the genetic complementation of the mutation, (v) 
identifying those cosmid clones which can complement the mutation and (vi) 
isolating with the help of the cosmid clone the corresponding cDNA and/or 
genomic sequences. This procedure is generally known as genomic walking 
and is well known to the person skilled in the art. 
The identified and isolated genes and/or cDNAs can subsequently be 
characterized according to standard techniques, such as restriction 
mapping and sequencing. The biologigal activity of the encoded product may 
be determined by homology comparisons with known proteins, in vivo feeding 
assays of the mutant etc. 
In a preferred embodiment the above-described process is carried out with 
transgenic plants showing reduced activity of the enzyme according to the 
invention which had been generated with the above-described nucleic acid 
molecules, for example, by expressing an antisense-RNA. 
The described process, thus, allows to identify nucleic acid molecules 
which encode a protein having the same enzymatic properties as the protein 
according to the invention and which is, thus, able to complement a mutant 
defective in this protein, even though the nucleic acid molecule encoding 
the protein may not hybridize to the nucleic acid molecules described 
above. 
Thus, the present invention also relates to nucleic acid molecules 
obtainable by the above-described process. In principle, any nucleic acid 
molecule encoding a protein involved in brassinosteroid synthesis or in 
its regulation may be identified by this method as long as its mutation 
leads to dwarfism and reduction of hypocotyl elongation in the dark. 
Preferably such nucleic acid molecules encode proteins involved in one or 
more enzymatic step(s) of the brassinosteroid synthesis pathway, and more 
preferably proteins which show the same enzymatic properties as the 
proteins according to the invention. 
By the provision of the knowledge that plant mutants defective in 
brassinosteroid synthesis may be identified by the features that they 
display dwarfism and reduced elongation of the hypocotyl of seeds 
germinating in the dark, the present invention allows to establish a 
simple method for identifying chemical compounds which can act like 
brassinosteroids in plants and which therefore may constitute potential 
growth factors in plants. 
Thus, the present invention also provides a method for the identification 
of chemical compounds which can act as brassinosteroids in plants 
comprising the steps of: 
(a) contacting a transgenic plant according to the invention which show a 
reduced activity of the protein according to the invention, or a mutant as 
identified by steps (a) and (b) of the method described above, which show 
a defect in brassinosteroid synthesis, with a plurality of chemical 
compounds; and 
(b) determining those compounds which are capable of compensating in the 
plants or mutants as defined in (a) the effects that resulted from defects 
in the brassinosteroid synthesis. 
Plants used in step (a) of this method may be plants which show reduced 
activity of the proteins according to the invention and, thus, have a 
defect in brassinosteroid synthesis which leads to dwarfism and reduced 
elongation of the hypocotyl of seedlings germinating in the dark. 
Alternatively, other plant mutants may be used which had been identified 
as being defective in brassinosteroid synthesis since they display 
dwarfism and reduced elongation of the hypocotyl in the dark and can be 
restored to the wildtype phenotype by addition of specific 
brassinosteroids. 
Chemical compounds which can partly or fully restore the wildtype phenotype 
may constitute potential growth factors for plants. 
In another aspect the present invention also relates to a method for the 
identification of chemical compounds which can act as brassinosteroids in 
plants comprising the steps of: 
(a) contacting germinating seeds of a plant according to the invention 
which show a reduced activity of the protein according to the invention 
and thus a defect in brassinosteroid synthesis, or of a dwarf mutant the 
seedlings of which show reduced elongation of the hypocotyl in the dark 
and in which normal growth can be restored by addition of specific 
brassinosteroids, with a plurality of chemical compounds; and 
(b) determining those compounds which are capable of restoring normal 
growth of the hypocotyl and/or roots in the seedlings. 
Furthermore, the present invention relates to a method for the 
identification of chemical compounds which can act as inhibitors of 
brassinosteroids or can suppress the biological activities of 
brassinosteroids comprising the steps of: 
(a) contacting plant cells or plants overexpressing a nucleic acid molecule 
according to the invention and, thus, showing a modified brassinosteroid 
synthesis and the above-described physiological and/or phenotypic changes, 
with a plurality of chemical compounds; and 
(b) identifying those compounds which lead to a weakening of the effects 
which resulted from altered brassinosteroid synthesis in these cells or 
plants. 
The present invention also relates to a method for the identification of 
chemical compounds which can act as inhibitors of brassinosteroids or can 
suppress the biological activities of brassinosteroids comprising the 
steps of: 
(a) contacting germinating seedlings of a plant according to the invention 
which show reduced activity of the protein according to the invention and, 
thus, a defect in brassinosteroid synthesis, or of a dwarf mutant the 
seedlings of which show reduced elongation of the hypocotyl in the dark 
and in which normal growth can be restored by addition of specific 
brassinosteroids, with brassinosteroids which are capable of restoring 
normal elongation of the hypocotyl of the seedlings germinating in the 
dark and simultaneously with a plurality of chemical compounds; and 
(b) determining those compounds which compete with the brassinosteroids to 
restore normal elongation of the hypocotyl. 
Inhibitors identified by the two above-described methods may prove useful 
as herbicides, pesticides or safeners. 
Beside the above described possibilities to use the nucleic acid molecules 
according to the invention for the genetic engineering of plants with 
modified characteristics and their use to identify homologous molecules, 
the described nucleic acid molecules may also be used for several other 
applications, for example, for the identification of nucleic acid 
molecules which encode proteins which interact with the cytochrome 
P450-type hydroxylase described above. This can be achieved by assays well 
known in the art, for example, by use of the so-called yeast "two-hybrid 
system". In this system the protein encoded by the nucleic acid molecules 
according to the invention or a smaller part thereof is linked to the 
DNA-binding domain of the GAL4 transcription factor. A yeast strain 
expressing this fusion protein and comprising a lacZ reporter gene driven 
by an appropriate promoter, which is recognized by the GAL4 transcription 
factor, is transformed with a library of cDNAs which will express plant 
proteins fused to an activation domain. Thus, if a protein encoded by one 
of the cDNAs is able to interact with the fusion protein comprising the 
P450 protein, the complex is able to direct expression of the reporter 
gene. In this way the nucleic acid molecules according to the invention 
and the encoded cytochrome P450 can be used to identify proteins 
interacting with the cytochrome P450, such as protein kinases, protein 
phosphatases, NAD(P)H oxidoreductases and/or cytochrome b5 proteins which 
are known to interact in plants and animals with cytochrome P450 proteins. 
Other methods for identifying proteins which interact with the proteins 
according to the invention or nucleic acid molecules encoding such 
molecules are, for example, the in vitro screening with the phage display 
system as well as filter binding assays. 
Furthermore, is it possible to use the nucleic acid molecules according to 
the invention as molecular markers in plant breeding as well as for the 
generation of modified cytochrome P450 proteins, as, e.g., proteins with 
an altered substrate specifity. 
Moreover, the nucleic acid molecules and proteins according to the 
invention can be used for the production of teasterone in any desired 
recombinant organism such as bacteria, fungi, animals or plants. 
Furthermore, the overexpression of nucleic acid molecules according to the 
invention may be used for the alteration or modification of plant/insect 
or in general plant/pathogene interactions. The term pathogene includes, 
for example, bacteria and fungi as well as protozoa. 
The nucleic acid molecules according to the invention as well as the 
encoded proteins and the brassinosteroid compounds identified by a method 
according to the invention can also be used for the regulation of stem and 
leaf (as well as other plant organ) development, which includes the 
regulation of the proportion of phloem and xylem in all crops and trees, 
namely in those plants which are of interest in wood production. 
A further possible use of the nucleic acid molecules, proteins and 
brassionosteroid compounds identified by a method according to the 
invention is the regulation of the differentiation and of the number of 
stomatal guard cells which may be of interest in the breeding or genetic 
engineering of plants with better stress tolerance, including drought, 
osmotic and other stresses.

EXAMPLE 1 
Construction And Identification Of T-DNA Tagged Mutant Impaired In The 
Regulation Of Cell Elongation And Skotomorphogenic Development 
A genetic technology, using the transferred DNA (T-DNA) of Agrobacterium 
tumefaciens Ti plasmid as an insertional mutagen, was developed for 
induction of gene mutations by gene tagging in higher plants. Namely, 
tissue culture transformation of Arabidopsis thaliana was carried out with 
a modified Ti plasmid derived vector, i.e. pPCV5013Hyg, as described in 
Koncz et al. (Proc. Natl. Acad. Sci. USA 86 (1989), 8467-8471), Koncz et 
al. (Plant Mol. Biol. 20 (1992b), 963-976) and Koncz et al. (Specialized 
vectors for gene tagging and expression studies. In: Plant Molecular 
Biology Manual Vol 2, Gelvin and Schilperoort (Eds.), Dordrecht, The 
Netherlands: Kluwer Academic Publ. (1994), 1-22)). This gene tagging 
technology was applied, using the model plant Arabidopsis thaliana, for 
generation of a collection of T-DNA insertional mutants, in order to 
identify mutations and corresponding genes, controlling plant development, 
in particular cell growth in different plant organs. 
By screening for mutants defective in hypocotyl and/or root elongation 
during skotomorphogenesis, a recessive mutation causing constitutive 
photomorphogenesis and dwarfism (cpd) was identified. Unlike the wild 
type, the cpd mutant developed a short hypocotyl, no apical hook, open 
cotyledons, and extended leaf primordia in the dark (FIGS. 2A,B). As 
compared to wildtype, the length of epidermal cell files was reduced at 
least 5-fold in the hypocotyl, but decreased only by 20 to 50% in the root 
of mutant seedlings. Epidermal cells of the mutant hypocotyl were 
decorated by thick transverse files of cellulose microfibrils (FIGS. 2D,E) 
and showed perpendicular divisions leading to differentiation of stomatal 
guard cells (FIG. 2B). Dense stomata and trichomes characteristic for 
leaves were also observed on the epidermis of mutant cotyledons (FIG. 2C). 
During growth for 5 weeks in the dark the mutant developed numerous 
rosette leaves, while wild type seedlings opened their cotyledons without 
leaf expansion under these conditions (FIG. 3 A). These phenotypic traits 
indicated a derepression of photomorphogenesis and de-etiolation in the 
dark-grown cpd mutant. Hybridization of steady-state RNAs prepared from 
these seedlings, using an ubiquitin (UBI) gene probe as an internal 
control, confirmed that morphological signs of de-etiolation in the mutant 
were accompanied by an increase in the expression of light-regulated 
genes, coding for the small subunit of ribulose 1,5-bisphosphate 
carboxylase (RBCS) and the chlorophyll a/b-binding protein (CAB, FIG. 3A). 
When grown in soil under white light, the size of cpd mutant plants was 20 
to 30-fold smaller than that of the same age wild type plants. Exposure to 
light induced greening and chloroplast differentiation in the periderm of 
mutant roots (data not shown) and resulted in a further inhibition of cell 
elongation, leading to an overall reduction of the length of petioles, 
leaves, inflorescence-stems and flower organs (FIGS. 2H,I). Histological 
analysis showed that in the round-shape epinastic mutant leaves the number 
of longitudinal mesophyll cell files was reduced and the palisade cells 
failed to elongate (FIGS. 2J,K). The cell walls were straightened in the 
adaxial leaf epidermis of the mutant, which displayed an amplification and 
duplication of stomatal guard cells (FIGS. 2F,G). Stem cross sections 
showed an unequal division of cambium, producing extranumerary phloem cell 
files at the expense of xylem cells in the mutant (FIGS. 2L,M). The cpd 
mutant was viable in soil and produced eggs and pollen of wild type size. 
However, the mutant did not set seeds because its pollen failed to 
elongate during germination, resulting in male sterility. 
EXAMPLE 2 
Genetic Analysis Of The cpd Mutation 
For trisomic analysis and linkage mapping a cpd/+ line was crossed with the 
tester lines as described (Koncz et al. (1992b), loc. cit.) and hygromycin 
resistant F1 hybrids were selected by germinating seeds in MSAR medium 
(Koncz et al. (1994), loc. cit.). 
After outcrossing of the mutant with wild type, the cpd mutation 
co-segregated with a single T-DNA insertion, carrying a hygromycin 
resistance (hpt) marker gene from the Agrobacterium transformation vector 
pPCV5013Hyg (Koncz et al. (1989)`, loc. cit.). The cpd mutation and the 
T-DNA insertion were mapped to chromosome 5-14.3 (FIG. 4A), using trisomic 
testers and the ttg marker of chromosome 5 in repulsion as described in 
the following. 
After outcrossing of the cpd mutant with wild type, 8 F2 families yielded 
an offspring of 1297 wild type and 437 dwarf plants (2.97:1), fitting 
(c.sup.2 0.037, homogeneity: 2,599; P=0.85) the expected 3:1 ratio for 
monogenic segregation of the recessive cpd mutation. From these F2 
families, 5383 mutants were tested on hygromycin and all displayed 
resistance, indicating a tight linkage between the T-DNA insertion and the 
cpd mutation. 
In contrast to other trisomic hybrids, segregating the mutation at a ratio 
of 3:1, the chromosome 5 trisomic tester T31 produced an aberrant F2 ratio 
of 588 wild type (336 resistant and 252 sensitive to hygromycin) and 60 
cpd mutant (all hygromycin resistant) plants. The ratios of wild type to 
mutant (9.8:1) and hygromycin resistant to sensitive (1.57:1) progeny 
matched with the ratios expected for synteny (.gtoreq.8:1 and between 
1.25:1 and 2.41:1, respectively). 
The T-DNA insert and the cpd mutation were simultaneously mapped, using the 
ttg marker of chromosome 5 in repulsion. For determination of the cpd-ttg 
map distance, two mapping populations were raised, one including plants 
grown in soil and another using seedlings germinated in MSAR medium and 
tested in the presence of 15 .mu.g/ml hygromycin. The soil-grown 
population was scored for the hairless ttg and dwarf cpd phenotypes in F2 
and seeds from fertile plants were carried to full-F3 analysis. By 
labeling cpd as "a" and ttg as "b", the actual scores in the soil-grown 
population were 1054 AaBb, 685 aaB. (424 aaBB and 261 aaBb by 
extrapolation), 387 AAbb, 261 aAbb, 248 AaBB, 251 AABb, 21 AABB and 25 
aabb. Progeny analysis showed that the AaBb, aAbb, AaBB and aabb classes 
were hygromycin resistant, in contrast to the hygromycin sensitive classes 
AAbb, AABb and AABB. In the population scored on MSAR medium with 
controlled seed germination the data were 815 AaB., 512 aaB., 193 AAB., 
300 AAbb, 159 aAbb and 17 aabb. Both mapping populations yielded identical 
frequencies for the double recombinant fraction (cpd-ttg). The 
recombination frequencies and derived map distances were calculated by the 
maximum likelihood method as described (Koncz et al., Methods in 
Arabidopsis Research ; Singapore, World Scienticic, 1992a). From these 
data the smaller map distance, corrected for the error resulting from 
uneven seed germination in soil, was accepted, resulting in 21.18.+-.0.86 
cM for the cpd(5-14.3)-ttg(5-35.5) interval. By scoring 1520 recombinant 
chromosomes, no crossing-over between the T-DNA-encoded hygromycin 
resistance marker and the cpd mutation was found, indicating that the 
T-DNA insertion was located in the cpd locus. 
The physical map of the T-DNA-tagged locus was determined by DNA 
hybridization and showed that the cpd mutant contained a T-DNA insert of 
4.8 kb, which underwent internal rearrangements (FIG. 4A). 
EXAMPLE 3 
Isolation Of The T-DNA Tagged Locus As Well As Wildtype cDNAs And Genomic 
DNAs Of The Cpd Locus 
To isolate the T-DNA-tagged locus, a genomic DNA library was constructed by 
ligation of cpd DNA, digested partially by MboI, into the BamHI site of 
the .lambda.EMBL 3 vector (Sambrook et al. (1989), loc. cit.). The 
T-DNA-tagged locus was isolated by constructing a genomic DNA library from 
the cpd mutant and mapped by hybridization with T-DNA derived probes (FIG. 
4A). 
The T-DNA/plant DNA insert junctions were subcloned, sequenced and used as 
probes to determine precisely the genomic location of the T-DNA insertion 
by isolation of Arabidopsis YAC (yeast artificial chromosome) clones. The 
YAC clones (FIG. 4A) overlapped with the ASA1 (anthranylate synthase, 
chr5-14.7) and hy5 (long hypocotyl locus, chr5-14.8) region of chromosome 
5 (R. Schmidt, unpublished; Hauge et al., Plant J. 3 (1993), 745-754), 
thus matching the map position (chr5-14.3) determined for the T-DNA-tagged 
cpd mutation by genetic linkage analysis. 
Plant DNA sequences flanking the hpt-pBR segment of T-DNA (FIG. 4A) 
hybridized with a mRNA of 1.7 kb present in wildtype seedlings and cell 
suspension cultures, but failed to detect any transcript in the cpd mutant 
(FIG. 4B). 
Following the physical mapping of the .lambda.EMBL3 clones, the T-DNA-plant 
DNA juntion fragments (flanked by BamHI and HindIII sites in the plant 
DNA, FIG. 4A) were used as probes for the isolation of 4 genomic and 4 
cDNA clones from wildtype Arabidopsis .lambda.EMBL4 genomic and 
.lambda.gt10 cDNA libraries. To identify yeast artificial chromosome 
clones containing the CPD locus, wildtype Arabidopsis YAC libraries were 
screened by hybridization (Koncz et al. (1992b), loc. cit.), using the ocs 
T-DNA-plant DNA junction fragment (BamHI-EcoRI fragment in FIG. 4A) as a 
probe. These clones were mapped and their fragments were subcloned and 
sequenced, in order to characterize the CPD cDNA (EMBL data base: 
accession number X87367; Seq ID No. 1) and gene (EMBL data base: accession 
number X87368; Seq ID No. 3). The 5'-end of the CPD transcript of 1735 
bases was mapped 166 bp upstream of the ATG codon (data not shown), 
whereas the polyadenylation signal was located 104 nucleotides downstream 
of the stop codon in the 3'-UTR of 131 bases. 
In support of the RNA hybridization data, nucleotide sequence comparison of 
the T-DNA insert junctions with wildtype cDNA and genomic DNA sequences 
showed that the T-DNA was inserted 10 bp 3'-downstream of the ATG start 
codon of a gene, preventing the transcription of its coding region. 
DNA analyses and cloning, screening of lambda phage libraries, DNA and RNA 
filter hybridizations and sequencing of double-stranded DNA templates were 
performed using standard molecular techniques (Sambrook et al. (1989), 
loc. cit.). For hybridization of RNA blots, the following cDNA probes were 
used: RBCS (EST ATTS0402, GenBank (gb): X13611), CAB140 (Ohio Arabidopsis 
Stock Center (OASC) 38A1T7, gb A29280), alkaline peroxidase (EST ATTS0366, 
gb P24102), nonchloroplastic SOD (OASC 2G11T7P), GST2 (gb L11601), HSP70 
(gb M23108), lignin-forming peroxidase (EST ATTS0592, gb P11965), chalcone 
synthase (Trezzini et al., Plant Mol. Biol. 21 (1993), 385-389), 
lipoxygenase (Lox2, gb L23968), S-adenosyl-methionine synthase (OASC 
40G2T7, gb P23686), Hsp18.2 (gb X17295), ADH (gb M12196), PR1, PR2 and PR5 
(Uknes et al., Plant Cell 4 (1992), 645-656). 
The RNA blot shown in FIG. 4B was hybridized with plant DNA sequences 
flanking the hpt-pBR segment of the T-DNA (PstI-HindIII fragment in FIG. 
4A). 
EXAMPLE 4 
Analysis Of The cpd cDNAs And Genomic Clones 
The analysis of CPD DNAs and derived protein sequences was performed using 
the GCG and BLAST computer programs (Deveraux et al., Nucl. Acids Res. 12 
(1984), 387-395; Altschul et al., J. Mol. Biol. 215 (1990), 403-410), as 
well as P450 sequence compilations (Gotoh, J. Biol. Chem. 267 (1992), 
83-90; Nelson et al., DNA 12 (1993), 1-51; Frey et al., Mol. Gen. Genet. 
246(1995), 100-109). 
DNA sequence analysis revealed that the CPD gene (Seq ID No. 3) consists of 
8 exons (FIG. 4A) with consensus splice sites at the exon-intron 
boundaries. The CPD cDNA (Seq ID No. 1) showed over 90% homology with 
expressed sequence tags e.g. ESTs EMBL Z29017 and GenBank T43151! from 
several organ specific Arabidopsis cDNA libraries, indicating that the CPD 
transcript is ubiquitous. Hybridization analysis with the cDNA probe (FIG. 
4B) indeed showed that the levels of steady-state CPD mRNA were comparable 
in roots, leaves and flowers, but considerably lower in 
inflorescence-stems and green siliques (fruits). The expression of the CPD 
gene was found to be modulated by external signals, such as light, 
cytokinin growth factor and sucrose provided as carbon source. The levels 
of CPD mRNA were elevated in dark-grown wild type seedlings by either 
increasing the sucrose content of the media (from 3 mM to 90 mM) or by 
light at low concentrations of sucrose, but decreased by combined 
cytokinin and sucrose treatments, particularly in the light (FIG. 3B). 
Translation of the CPD cDNA defined a coding region of 472 codons (Seq ID 
No. 2) for a protein of 53,785 Da, in the following referred to as CYP90. 
The deduced amino acid sequence of this protein detected homology in the 
database with the conserved N-terminal membrane-anchoring, proline-rich, 
oxygen and heme binding domains of microsomal cytochrome P450s (FIG. 6); 
50 to 90% sequence identity with conserved P450 domains defined by Nebert 
and Gonzalez (Ann. Rev. Biochem. 56 (1987), 945-993). The CPD gene encoded 
protein thus appeared to possess all functionally important domains of 
P450 monooxygenases (Pan et al., J. Biol. Chem. 270 (1995), 8487-8494). In 
addition, the sequence comparison also indicated a homology between CYP90 
and specific domains of steroid hydroxylases. Members of the CYP2 family, 
including the rat testosterone-16a-hydroxylase (CYP2B1; 24% identity; 
Fujii-Kuriyama et al., Proc. Natl. Acad. Sci. 79 (1982), 2793-2797) showed 
thus sequence similarity with CYP90 in their central variable region 
(positions 135-249, FIG. 6), carrying the steroid substrate-binding 
domains SRS2 and SRS3 (Gotoh, (1992), loc. cit.). Moreover, in the CYP21 
family, represented by the human progesterone-21-hydroxylase (CYP21A2; 19% 
identity; White et al., Proc. Natl. Acad. Sci. 83 (1986), 5111-5115), the 
positions of introns 7 and 8 corresponded to those of introns 3 and 5 in 
the CPD gene, suggesting a significant evolutionary relationship (Nelson 
et al., (1993), loc. cit.). Nonetheless, because its overall sequence 
identity with other P450s was less than 40%, the CPD gene product was 
assigned to a novel P450 family, CYP90, clustering on the evolutionary 
tree with CYP85 from tomato, CYP87 from sunflower (both unpublished) and 
CYP88 from maize (Winkler and Helentjaris, Plant Cell 7 (1995), 1307-1317; 
P450 Nomenclature Committee, D. Nelson, personal comm.). 
EXAMPLE 5 
Complementation Of The cpd Mutation 
To demonstrate that the T-DNA insertion was indeed responsible for the cpd 
mutation, the coding region of the longest wildtype CPD cDNA (extending 47 
bp ustream of the ATG codon) was cloned in the BamHI-site of plant gene 
expression vector pPCV701, conjugated from E.coli to Agrobacterium, and 
transformed into the homozygous cpd mutant by Agrobacterium-mediated 
Arabidopsis transformation as described (Koncz et al. (1994), loc. cit.). 
The cDNA was expressed in the homozygous cpd mutant under the control of 
the auxin-regulated mannopine synthase (mas) 2' promoter (FIG. 5A; Koncz 
et al. (1994), loc. cit.). Transgenic plants, selected and regenerated 
with the aid of a kanamycin resistance gene carried by the pPCV701 vector, 
were all wildtype and fertile, demonstrating genetic complementation of 
the cpd mutation. Kanamycin resistant progeny of many complemented lines 
developed more expanded leaves and inflorescence branches than the wild 
type. One such complemented cpd line (FIG. 5C) contained at least 3 
independently segregating pPCV701 T-DNA insertions, since it yielded 268 
kanamycin resistant wildtype and 4 kanamycin sensitive cpd mutant progeny. 
DNA fingerprinting confirmed the presence of multiple pPCV701 T-DNA 
insertions in this complemented line which produced a considerably higher 
amount of CPD transcript from the mas 2' promoter driven cDNA copies than 
the wild type from the single copy CPD gene (FIG. 5B). 
EXAMPLE 6 
Effects Of Overexpression Of A cpd cDNA 
In contrast to the dark-grown cpd mutant (FIG. 3A), in light-grown plants 
neither the absence nor the overexpression of CPD transcript affected the 
level of steady-state RNAs of light-regulated RBCS and CAB genes (FIG. 
3B). The transcript levels of chalcone synthase (CHS), alcohol 
dehydrogenase (ADH), lipoxygenase (LOX2), S-adenosyl-methionine synthase 
(SAM) and heat shock 18.2 (Hsp18.2) genes were elevated in the cpd mutant, 
whereas the mRNA levels of other stress-regulated genes, such as alkaline 
peroxidase (APE), superoxide dismutase (SOD), glutathione-S-transferase 
(GST), heat shock 70 (HSP70) or lignin forming peroxidase (LPE), were 
comparable in the cpd mutant, wildtype and CPD overexpressing plants. The 
expression of the pathogenenesis related genes PR1, 2 and 5 were 
remarkably low in the cpd mutant. However, overexpression of the CPD cDNA 
resulted in a significant induction of these PR genes in the complemented 
lines overexpressing cpd. 
EXAMPLE 7 
Complementation Of cpd Mutants With Brassinosteroids And Other Plant Growth 
Factors 
The above described sequence homology data were not sufficient to predict 
unambiguously the substrate specificity of CYP90 (Nelson et al. (1993), 
loc. cit.). Therefore, the elongation response of the cpd mutant to all 
plant growth factors, whose synthesis could involve P450 enzymes, was 
tested. 
Plant growth factors including auxins (indole-3-acetic acid, 
a-naphthaleneacetic acid, 2,4-dichloro-phenoxyacetic acid), cytokinins 
(6-benzyl-aminopurine, 6-furfurylaminopurine, 
6-(.gamma.,.gamma.-dimethylallylamino)-purine riboside), abscisic acid, 
salicylic acid, methyl-jasmonate, as well as retinoic acid derivatives 
(vitamin A aldehyde, 9-cis-retinal, 13-cis-retinal, trans-retionoic acid, 
13-cis-retinoic acid and retinol) were used at final concentrations of 
0.01, 0.05, 0.1, 0.5 or 1 .mu.M, whereas gibberellins (gibberellic acid 
GA3, GA4, GA7 and GA13) were applied at 0.1, 1, 10, and 100 .mu.M 
concentrations in MSAR seed germination media. 
Brassinosteroids as listed in FIG. 1 and epi-isomers of teasterone, 
typhasterol, castasterone and brassinolide were obtained from A. Sakurai 
and S. Fujioka (Institute of Physical and Chemical Research (RIKEN), 
Japan) and G. Adam (Institute for Plant Biochemistry, Halle, Germany). BRs 
were tested at similar concentrations (0.005, 0.01, 0.05, 0.1, 0.5 and 1 
.mu.M) in MSAR media used for seed germination under aseptic conditions 
(Koncz et al. (1994), loc. cit. ). The bioassays were evaluated after 1, 
2, 5 and 10 days of germination by measurement of the length of hypocotyls 
and roots, as well as by visual inspection and photography of seedlings. 
Mutant plants grown in soil were sprayed with 0.1 or 1 .mu.M aqueous 
solutions of castasterone or brassinolide. 
Histological analyses were performed according to standard procedures 
(Feder and O'Brien, Am. J. Bot. 55 (1968), 123-142). Tissues were fixed in 
formalin:acetic acid:ethanol (90:5:5), embedded in 2-hydroxyethyl 
methacrylate, sectioned at 10 .mu.m using a rotary microtome, and stained 
by toluidine-blue. To prepare contact imprints, seedlings were placed in 
3% molten agarose and carefully removed from the solidified carrier before 
taking pictures. 
In these bioassays auxins, gibberellins, cytokinins, abscisic acid, 
ethylene, methyl-jasmonate, salicylic acid and different retinoid acid 
derivatives failed to promote the hypocotyl elongation of the cpd mutant 
grown in the dark or light (data not shown). However, brassinolide, an 
ecdysone-like plant steroid (used at concentrations of 0.005 to 
1.times.10.sup.-6 M), was found to restore cell elongation in the 
hypocotyl, leaves and petioles of cpd mutant seedlings in both dark and 
light. Brassinolide treatment also restored the male fertility of the 
mutant, allowing the production of homozygous seeds. 
When grown in the presence of C23-hydroxylated brassinosteroid (BR) 
precursors (0.1 to 1.times.10.sup.-6 M) of brassinolide, such as 
teasterone, 3-dehydroteasterone, typhasterol, and castasterone (Fujioka et 
al., Biosci. Biotech. Biochem. 59 (1995), 1543-1547), the cpd mutant was 
also indistunguisable from wild type in both dark and light (FIG. 7). 
However, cathasterone and its precursor campesterol (as well as 
campestanol, 6.alpha.-hydroxycampestanol and 6-oxocampestanol, 
.DELTA..sup.22 -6-oxocampestanol and 
22.alpha.,23.alpha.-epoxy6-oxocampestanol, data not shown), which do not 
carry hydroxyl moiety at the C23 position, did not alter the cpd 
phenotype, suggesting a deficiency of cathasterone C23-hydroxylation to 
teasterone in the cpd mutant. From the synthetic 22R,23R,24R!-derivatives 
of BRs (Adam and Marquardt, (1986), loc. cit.) epi-teasterone was found to 
be inactive, whereas epi-castasterone and epi-brassinolide rescued the cpd 
phenotype as well as their 22R,23R,24S!-stereoisomers. 
Remarkably, the hypocotyl elongation response of wildtype seedlings was 
unaffected by brassinosteroids in the dark (FIG. 7), indicating a possible 
saturation of this growth response. In contrast, treatments of wildtype 
seedlings with castasterone and brassinolide in the light promoted 
hypocotyl elongation (albeit with different efficiencies). When applied at 
higher concentrations (0.1 to 1.times.10.sup.-6 M), castasterone and 
brassinolide (as well as their epi-stereoisomers, but not other BRs 
precursors) caused aberrant leaf expansion, epinasty, senescence and 
retarded development in both wild type and mutant plants grown in the 
light (FIG. 7). 
EXAMPLE 8 
Identification Of Other Mutants Affected In Brassinosteroid Responses 
Physiological data indicate that the biosynthesis of gibberellins and 
steroids involve common precursors (Davies, Plant hormones and their role 
in plant growth and development (1987), Dordrecht, The netherlands: 
Martinus Nijhoff Publ.) and that BRs stimulate ethylene biosynthesis in 
the light (Mandava (1988), loc. cit.). Nonetheless, mutants affected in 
ethylene production (eto1), gibberellin biosynthesis (ga) and perception 
(gai) do not respond to BRs in the dark, and BRs promote only a weak 
hypocotyl elongation response in the ethylene resistant etr1 mutant. Thus, 
mutants affected in ethylene, gibberellin and BR responses can clearly be 
distinguished. The BR-bioassays performed with cpd mutant and wild type 
Arabidopsis seedlings in the dark show that BR-deficiency can result in a 
short hypocotyl phenotype, although BRs do not stimulate hypocotyl 
elongation in the wildtype. Mutants deficient in BR biosynthesis are 
expected therefore to develop short hypocotyls, which should be restored 
to wildtype by brassinolide and BR precursors. One can also predict that 
mutants defective in BR-perception and/or signaling will show short 
hypocotyl and a partial or complete insensitivity to BRs. The de-etiolated 
mutant det2 appears to be a BR biosynthetic mutant. The DET2 gene codes 
for a homolog of animal steroid-5a-reductases which is probably required 
for the conversion of campesterol to campestanol in the first step of 
brassinolide biosynthesis (Li, J., P. Nagpal, V. Vitart and J. Chory, 
personal com.). In other de-etiolated and constitutive photomorphogenic 
mutants, such as det1, cop1-16, fus4, fus5, fus6, fus7, fus8, fus9, fus11, 
and fus12, BRs stimulate hypocotyl elongation only in the dark. The 
cop1-13 mutant, which produces no COP1 protein (McNellis et al., Plant 
Cell 6 (1994), 487-500), is apparently insensitive to BRs. In contrast, 
the less severe cop1-16 mutant (Misera et al., Mol. Gen. Genet. 244 
(1994), 242-252; McNellis et al. (1994) loc. cit.), synthesizing an 
immunologically detectable amount of mutant COP1 protein, responds to BRs 
by hypocotyl growth. The fus6 mutant displays similar allelic differences, 
whereas the det3 mutant shows a complete insentivity to BRs. It is 
therefore possible that these mutations affect regulatory functions 
involved in BR perception and/or signaling. 
The effect of castasterone and brassinolide (and their epi-isomers) on 
different Arabidopsis mutants impaired in hypocotyl elongation was 
similarly tested. To avoid complexity resulting from negative regulation 
of the hypocotyl elongation by light, the mutants were germinated in the 
presense or absence of BRs in the dark and their hypocotyl growth was 
compared to that of untreated and ergosterol-treated seedlings as controls 
(FIG. 8). Mutants in gibberellin biosynthesis (ga5) or perception (gai), 
showing dwarfism and inhibition of hypocotyl and/or epicotyl growth in the 
light (Finkelstein and Zeevaart, in Arabidopsis (1994), Meyerowitz and 
Sommerville (Eds.) Cold spring Harbor Laboratory Press; Cold spring 
Harbor, N.Y., 523-553), developed similar or shorter hypocotyls as the 
wild type, but did not respond to BRs by significant hypocotyl elongation 
(more than 20%) in the dark. The inhibition of hypocotyl growth in the 
dark-grown ethylene overproducing mutant eto1 (Ecker, Science. 268 (1995), 
667-675) was also unaffected by BRs. In contrast, BR-treatments stimulated 
the rate of hypocotyl elongation by 50 to 80% in the ethylene resistant 
mutant etr1 (Ecker (1995), loc. cit.). The hypocotyl elongation of the 
auxin/ethylene resistant axr2 mutant (Estelle and Klee, in Arabidopsis 
(1994), Meyerowitz and Sommerville (Eds.) Cold spring Harbor Laboratory 
Press; Cold spring Harbor, N.Y., 555-578) was also increased 2 to 3-fold 
by BRs, which promoted the enlargement of cotyledons, but inhibited the 
root growth of axr2 seedlings. The wild type and the ga5, gai1, eto1, 
etr1, and axr2 mutants displayed comparable hypocotyl elongation (but 
different epicotyl/stem growth) responses to BRs in the light. 
As was observed for the cpd mutant, castasterone and brassinolide restored 
the phenotype of the dim mutant (Takahashi et al., Genes Dev. 9 (1995), 
97-107) to wild type in the dark, as well as in the light (data not 
shown). In contrast, the hypocotyl elongation of det1, cop1-16, fus4, 
fus5, fus6, fus7, fus8, fus9, fus11, and fus12 mutants (Chory and Susek, 
in : Arabidopsis (1994), Meyerowitz and Sommerville (Eds.) Cold spring 
Harbor Laboratory Press; Cold spring Harbor, N.Y., 579-614; Deng, Cell 76 
(1994), 423-426; Misera et al. (1994), loc. cit.) was stimulated 3 to 
10-fold by BRs only in the dark. BRs inhibited the elongation of roots in 
these mutants. BRs also stimulated the cell enlargement and decreased the 
accumulation of anthocyanins in the cotyledons of det1 and fus9 mutants. 
In comparison to their allelles, the cop1-13 and fus6-G mutants showed no, 
or respectively a minimal (10 to 20%), hypocotyl elongation response to 
castasterone and brassinolide, whereas the det3 mutant (Chory and Susek 
(1994), loc. cit.) was found to be completely insensitive to Brs. 
The data presented by the present application clearly provide evidence that 
brassinosteroids are of crucial importance for plant growth and 
development. Since their discovery (Grove et al., Nature 281 (1979), 
216-217), brassinosteroids (BRs) have been considered to be nonessential 
plant hormones, because their concentration is extremely low in most plant 
species and their action spectrum is redundant with those of ubiquitous 
growth factors auxin, gibberellin, ethylene and cytokinin. A major 
argument supporting this view is that BRs are inactive in hypocotyl 
elongation assays performed in the dark, which are used as standard tests 
to monitor the activity of photoreceptors and phytohormones controlling 
cell elongation (for review see Davies (1987), loc. cit.; Kendrick and 
Kronenberg, Photomorphogenesis in plants; Dordrecht, The Netherlands: 
Kluwer Academic Publ. (1994)). The data described in the present 
application clearly undermine this argument, since they demonstrate that 
the phenotype of a hypocotyl elongation mutant can be restored to wild 
type by brassinolide and its precursors, but not by other known plant 
growth factors. The BR-precursor feeding experiments suggest that the 
hypocotyl elongation defect in the cpd mutant results from a deficiency in 
brassinolide biosynthesis. Brassinolide has been observed in many plant 
species to stimulate the longitudinal arrangement of cortical microtubuli 
and cellulose microfilaments, leaf unrolling, xylem differentiation and 
hypocotyl elongation in the light. Brassinolide is also reported to 
inhibit root elongation, radial growth of the stem, anthocyanin synthesis, 
and de-etiolation (Mandava (1988), loc. cit.). Phenotypic traits of the 
cpd mutant--such as the inhibition of longitudinal cell elongation in most 
organs, the transverse arrangement of cellulose microfilaments on the 
surface of epidermal cells, the inhibition of leaf unrolling and xylem 
differentiation, and the induction of de-etiolation in the dark--are 
consistent with a phenotype expected for a mutant in brassinolide 
synthesis. In addition, the conservation of exon-intron boundaries between 
the CPD gene and CYP21 gene family of progesterone side-chain 
hydroxylases, the homology of the CYP90 protein with all conserved domains 
of functional P450 monooxygenases, and the similarity of CYP90 domains 
with the substrate binding regions of CYP2 testosterone hydroxylases also 
suggest that the CPD gene may code for a cytochrome P450 steroid 
hydroxylase. 
Cytochrome P450s are known to use a wide range of artificial substrates in 
vitro, but perform well-defined stereo-specific reactions in vivo. Because 
their substrate specificity can be altered by mutations affecting the 
substrate binding domains, the specificity of P450 enzymes can only be 
determined by in vivo feeding experiments with labeled substrates (Nebert 
and Gonzalez (1987), loc. cit.). Because it usually cannot be excluded 
that multiple cytochrome P450s contribute to a given metabolic conversion 
in vivo, such an analysis requires either the overexpression of cytochrome 
P450s in transgenic organisms, or mutants deficient in particular P450s. 
The cpd mutant and CPD overexpressing transgenic plants therefore provide 
a suitable material to confirm the requirement of CYP90 for 
C23-hydroxylation of cathasterone in brassinolide biosynthesis (Fujioka et 
al. (1995), loc. cit.). 
The cpd and det2 mutations result in similar phenotypic traits, including 
the induction of de-etiolation and expression of light-induced RBCS and 
CAB genes in the dark. Thus, cpd can be considered to be a new type of det 
mutation. Genetic analyses of detlhy double mutants suggest that det1 and 
det2 are epistatic to the hy mutations of photoreceptors. Therefore, det1 
and det2 have been proposed to act in parallel light signaling pathways as 
negative regulators of de-etiolation (Chory and Susek (1994), loc. cit.). 
In the det1 pathway, the products of DET1, COP1, and some FUS genes are 
thought to function as nuclear repressors of light-regulated genes in the 
dark (Deng (1994), loc. cit.); Quail et al., Science 268 (1995), 675-680). 
Now, the putative det2 light signaling pathway (Chory and Susek (1994), 
loc. cit.) appears to be a brassinosteroid pathway, because det2 as well 
as cpd and dim mutants are restored to wild type by BRs. This is 
consistent with data indicating that BRs inhibit de-etiolation in the dark 
(Mandava (1988), loc. cit.). Our data also show that the cpd mutation 
results in the activation of stress-regulated chalcone synthase (CHS), 
alcohol dehydrogenase, heat shock 18.2, lipoxygenase, 
S-adenosyl-methionine synthase genes in the light. This correlates with 
the observations showing that BRs suppress anthocyanin synthesis (i.e. 
controlled by CHS; Mandava (1988), loc. cit.) and that the CHS gene is 
also induced in the det2 mutant (Chory et al., Plant Cell 3 (1991), 
445-459). The CPD function (and thus the det2/BR-pathway) appears 
therefore to negatively regulate stress signaling, possibly via the 
modulation of lipoxygenase involved in the generation of lipid 
hydroperoxide signals (i.e jasmonate), which are known to control defense 
and stress responses in plants (Farmer, Plant Mol. Biol. 26 (1994), 
1423-1437). Cytokinin treatment of wild type Arabidopsis has been observed 
to result in a phenocopy of the det2 mutation (Chory et al., Plant 
Physiol. 104 (1994), 339-347). In agreement, our data show that the 
transcription of the CPD gene is downregulated by cytokinin, which may 
thus control BR-biosynthesis. The expression of the CPD gene is also 
modulated by light and the availability of carbon source (e.g. sucrose), 
suggesting complex regulatory interactions between light and BR signaling. 
It is therefore possible that the cpd and det2 mutations only indirectly 
affect the expression of light-regulated genes (e.g. through the 
regulation of stress responses). Studies of the dim mutant indicate that 
inhibition of the hypocotyl elongation may not influence the expression of 
light-induced RBCS, CAB and CHS genes in the dark (Takahashi et al. 
(1995), loc. cit.). This is intriguing, because the phenotypic traits of 
the dim mutant are nearly identical with those of the cpd and det2 
mutants, and our precursor feeding experiments suggest that dim causes a 
deficiency before typhasterol in BR-biosynthesis (unpublished). A 
comparative analysis of det2, cpd, and dim mutants, including their 
combinations with hy loci, is therefore necessary to clarify how the 
regulation of light-induced genes is affected by brassinolide and/or its 
brassinosteroid precursors. Unlike det2, the dim mutation has been 
proposed to control cell elongation by specific regulation of the tubulin 
TUB1 gene expression (Takahashi et al. (1995), loc. cit.). In fact, the 
available genetic data do not prove that the signaling pathways identified 
by the det1 and det2 mutations are exclusively involved in light signaling 
(Millar et al., Ann. Rev. Genet. 28 (1994), 325-349). Therefore, DET, COP, 
FUS, and CPD genes can also be considered to act as positive regulators of 
cell elongation, because their mutations result in the inhibition of 
hypocotyl elongation in the dark. The fact, that BRs can compensate the 
cell elongation defects caused by the det1, cop1 and fus mutations 
suggests a close interaction between the det1 and det2 pathways, as 
proposed by the genetic model (Chory and Susek (1994), loc. cit.). 
BR-insensitivity of the cop1-13 mutant may in fact point to a possible 
involvement of the COP1 WD-protein (Deng et al., Cell 71 (1992), 791-801) 
in BR-responses. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 4 
- (2) INFORMATION FOR SEQ ID NO: 1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 1608 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA to mRNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: A. thalia - #na 
- (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: lambda gt1 - #0 
(B) CLONE: C204 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION:48..1466 
#1: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
#GCC TTC 56TCTCTT CTTCTCTCAT CATCATCTTC TTCTTCA ATG 
#Ala Phe Met 
#1 
- ACC GCT TTT CTC CTC CTC CTC TCT TCC ATC GC - #C GCC GGC TTC CTC CTC 
104 
Thr Ala Phe Leu Leu Leu Leu Ser Ser Ile Al - #a Ala Gly Phe Leu Leu 
# 15 
- CTA CTC CGC CGT ACA CGT TAC CGT CGG ATG GG - #T CTG CCT CCG GGA AGC 
152 
Leu Leu Arg Arg Thr Arg Tyr Arg Arg Met Gl - #y Leu Pro Pro Gly Ser 
# 35 
- CTT GGT CTC CCT CTG ATA GGA GAG ACT TTT CA - #G CTG ATC GGA GCT TAC 
200 
Leu Gly Leu Pro Leu Ile Gly Glu Thr Phe Gl - #n Leu Ile Gly Ala Tyr 
# 50 
- AAA ACA GAG AAC CCT GAG CCT TTC ATC GAC GA - #G AGA GTA GCC CGG TAC 
248 
Lys Thr Glu Asn Pro Glu Pro Phe Ile Asp Gl - #u Arg Val Ala Arg Tyr 
# 65 
- GGT TCG GTT TTC ATG ACG CAT CTT TTT GGT GA - #A CCG ACG ATT TTC TCA 
296 
Gly Ser Val Phe Met Thr His Leu Phe Gly Gl - #u Pro Thr Ile Phe Ser 
# 80 
- GCT GAC CCG GAA ACG AAC CGG TTT GTT CTT CA - #G AAC GAA GGG AAG CTT 
344 
Ala Asp Pro Glu Thr Asn Arg Phe Val Leu Gl - #n Asn Glu Gly Lys Leu 
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392 
Phe Glu Cys Ser Tyr Pro Ala Ser Ile Cys As - #n Leu Leu Gly Lys His 
100 1 - #05 1 - #10 1 - 
#15 
- TCT CTG CTT CTT ATG AAA GGT TCT TTG CAT AA - #A CGT ATG CAC TCT CTC 
440 
Ser Leu Leu Leu Met Lys Gly Ser Leu His Ly - #s Arg Met His Ser Leu 
# 130 
- ACC ATG AGC TTT GCT AAT TCT TCA ATC ATT AA - #A GAC CAT CTC ATG CTT 
488 
Thr Met Ser Phe Ala Asn Ser Ser Ile Ile Ly - #s Asp His Leu Met Leu 
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- GAT ATT GAC CGG TTA GTC CGG TTT AAT CTT GA - #T TCT TGG TCT TCT CGT 
536 
Asp Ile Asp Arg Leu Val Arg Phe Asn Leu As - #p Ser Trp Ser Ser Arg 
# 160 
- GTT CTC CTC ATG GAA GAA GCC AAA AAG ATA AC - #G TTT GAG CTA ACG GTG 
584 
Val Leu Leu Met Glu Glu Ala Lys Lys Ile Th - #r Phe Glu Leu Thr Val 
# 175 
- AAG CAG TTG ATG AGC TTT GAT CCA GGG GAA TG - #G AGT GAG AGT TTA AGG 
632 
Lys Gln Leu Met Ser Phe Asp Pro Gly Glu Tr - #p Ser Glu Ser Leu Arg 
180 1 - #85 1 - #90 1 - 
#95 
- AAA GAG TAT CTT CTT GTC ATC GAA GGC TTC TT - #C TCT CTT CCT CTC CCT 
680 
Lys Glu Tyr Leu Leu Val Ile Glu Gly Phe Ph - #e Ser Leu Pro Leu Pro 
# 210 
- CTC TTC TCC ACC ACT TAC CGC AAA GCC ATC CA - #A GCG CGG AGG AAG GTG 
728 
Leu Phe Ser Thr Thr Tyr Arg Lys Ala Ile Gl - #n Ala Arg Arg Lys Val 
# 225 
- GCG GAG GCG TTG ACG GTG GTG GTG ATG AAA AG - #G AGG GAG GAG GAG GAA 
776 
Ala Glu Ala Leu Thr Val Val Val Met Lys Ar - #g Arg Glu Glu Glu Glu 
# 240 
- GAA GGA GCG GAG AGA AAG AAA GAT ATG CTT GC - #G GCG TTG CTT GCG GCG 
824 
Glu Gly Ala Glu Arg Lys Lys Asp Met Leu Al - #a Ala Leu Leu Ala Ala 
# 255 
- GAT GAT GGA TTT TCC GAT GAA GAG ATT GTT GA - #C TTC TTG GTG GCT TTA 
872 
Asp Asp Gly Phe Ser Asp Glu Glu Ile Val As - #p Phe Leu Val Ala Leu 
260 2 - #65 2 - #70 2 - 
#75 
- CTT GTC GCC GGT TAT GAA ACA ACC TCC ACG AT - #C ATG ACT CTC GCC GTC 
920 
Leu Val Ala Gly Tyr Glu Thr Thr Ser Thr Il - #e Met Thr Leu Ala Val 
# 290 
- AAA TTT CTC ACC GAG ACT CCT TTA GCT CTT GC - #T CAA CTC AAG GAA GAG 
968 
Lys Phe Leu Thr Glu Thr Pro Leu Ala Leu Al - #a Gln Leu Lys Glu Glu 
# 305 
- CAT GAA AAG ATT AGG GCA ATG AAG AGT GAT TC - #G TAT AGT CTT GAA TGG 
1016 
His Glu Lys Ile Arg Ala Met Lys Ser Asp Se - #r Tyr Ser Leu Glu Trp 
# 320 
- AGT GAT TAC AAG TCA ATG CCA TTC ACA CAA TG - #T GTG GTT AAT GAG ACG 
1064 
Ser Asp Tyr Lys Ser Met Pro Phe Thr Gln Cy - #s Val Val Asn Glu Thr 
# 335 
- CTA CGA GTG GCT AAC ATC ATC GGC GGT GTT TT - #C AGA CGT GCA ATG ACG 
1112 
Leu Arg Val Ala Asn Ile Ile Gly Gly Val Ph - #e Arg Arg Ala Met Thr 
340 3 - #45 3 - #50 3 - 
#55 
- GAT GTT GAG ATC AAA GGT TAT AAA ATT CCA AA - #A GGG TGG AAA GTA TTC 
1160 
Asp Val Glu Ile Lys Gly Tyr Lys Ile Pro Ly - #s Gly Trp Lys Val Phe 
# 370 
- TCA TCG TTT AGA GCG GTT CAT TTA GAC CCA AA - #C CAC TTC AAA GAT GCT 
1208 
Ser Ser Phe Arg Ala Val His Leu Asp Pro As - #n His Phe Lys Asp Ala 
# 385 
- CGC ACT TTC AAC CCT TGG AGA TGG CAG AGC AA - #C TCG GTA ACG ACA GGC 
1256 
Arg Thr Phe Asn Pro Trp Arg Trp Gln Ser As - #n Ser Val Thr Thr Gly 
# 400 
- CCT TCT AAT GTG TTC ACA CCG TTT GGT GGA GG - #G CCA AGG CTA TGT CCC 
1304 
Pro Ser Asn Val Phe Thr Pro Phe Gly Gly Gl - #y Pro Arg Leu Cys Pro 
# 415 
- GGT TAC GAG CTG GCT AGG GTT GCA CTC TCT GT - #T TTC CTT CAC CGC CTA 
1352 
Gly Tyr Glu Leu Ala Arg Val Ala Leu Ser Va - #l Phe Leu His Arg Leu 
420 4 - #25 4 - #30 4 - 
#35 
- GTG ACA GGC TTC AGT TGG GTT CCT GCA GAG CA - #A GAC AAG CTG GTT TTC 
1400 
Val Thr Gly Phe Ser Trp Val Pro Ala Glu Gl - #n Asp Lys Leu Val Phe 
# 450 
- TTT CCA ACT ACA AGA ACG CAG AAA CGG TAC CC - #G ATC TTC GTG AAG CGC 
1448 
Phe Pro Thr Thr Arg Thr Gln Lys Arg Tyr Pr - #o Ile Phe Val Lys Arg 
# 465 
- CGT GAT TTT GCT ACT TGA AGAAGAAGAG ACCCATCTGA TT - #TTATTTAT 
1496 
Arg Asp Phe Ala Thr * 
470 
- AGAACAACAG TATTTTTCAG GATTAATTTC TTCTTCTTTT TTTGCCTCCT TG - #TGGGTCTA 
1556 
- GTGTTTGACA ATAAAAGTTA TCATTACTCT ATAAAGCCTT AGCTTCTGTG TA - # 
1608 
- (2) INFORMATION FOR SEQ ID NO: 2: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 472 ami - #no acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
#2: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Met Ala Phe Thr Ala Phe Leu Leu Leu Leu Se - #r Ser Ile Ala Ala Gly 
# 15 
- Phe Leu Leu Leu Leu Arg Arg Thr Arg Tyr Ar - #g Arg Met Gly Leu Pro 
# 30 
- Pro Gly Ser Leu Gly Leu Pro Leu Ile Gly Gl - #u Thr Phe Gln Leu Ile 
# 45 
- Gly Ala Tyr Lys Thr Glu Asn Pro Glu Pro Ph - #e Ile Asp Glu Arg Val 
# 60 
- Ala Arg Tyr Gly Ser Val Phe Met Thr His Le - #u Phe Gly Glu Pro Thr 
# 80 
- Ile Phe Ser Ala Asp Pro Glu Thr Asn Arg Ph - #e Val Leu Gln Asn Glu 
# 95 
- Gly Lys Leu Phe Glu Cys Ser Tyr Pro Ala Se - #r Ile Cys Asn Leu Leu 
# 110 
- Gly Lys His Ser Leu Leu Leu Met Lys Gly Se - #r Leu His Lys Arg Met 
# 125 
- His Ser Leu Thr Met Ser Phe Ala Asn Ser Se - #r Ile Ile Lys Asp His 
# 140 
- Leu Met Leu Asp Ile Asp Arg Leu Val Arg Ph - #e Asn Leu Asp Ser Trp 
145 1 - #50 1 - #55 1 - 
#60 
- Ser Ser Arg Val Leu Leu Met Glu Glu Ala Ly - #s Lys Ile Thr Phe Glu 
# 175 
- Leu Thr Val Lys Gln Leu Met Ser Phe Asp Pr - #o Gly Glu Trp Ser Glu 
# 190 
- Ser Leu Arg Lys Glu Tyr Leu Leu Val Ile Gl - #u Gly Phe Phe Ser Leu 
# 205 
- Pro Leu Pro Leu Phe Ser Thr Thr Tyr Arg Ly - #s Ala Ile Gln Ala Arg 
# 220 
- Arg Lys Val Ala Glu Ala Leu Thr Val Val Va - #l Met Lys Arg Arg Glu 
225 2 - #30 2 - #35 2 - 
#40 
- Glu Glu Glu Glu Gly Ala Glu Arg Lys Lys As - #p Met Leu Ala Ala Leu 
# 255 
- Leu Ala Ala Asp Asp Gly Phe Ser Asp Glu Gl - #u Ile Val Asp Phe Leu 
# 270 
- Val Ala Leu Leu Val Ala Gly Tyr Glu Thr Th - #r Ser Thr Ile Met Thr 
# 285 
- Leu Ala Val Lys Phe Leu Thr Glu Thr Pro Le - #u Ala Leu Ala Gln Leu 
# 300 
- Lys Glu Glu His Glu Lys Ile Arg Ala Met Ly - #s Ser Asp Ser Tyr Ser 
305 3 - #10 3 - #15 3 - 
#20 
- Leu Glu Trp Ser Asp Tyr Lys Ser Met Pro Ph - #e Thr Gln Cys Val Val 
# 335 
- Asn Glu Thr Leu Arg Val Ala Asn Ile Ile Gl - #y Gly Val Phe Arg Arg 
# 350 
- Ala Met Thr Asp Val Glu Ile Lys Gly Tyr Ly - #s Ile Pro Lys Gly Trp 
# 365 
- Lys Val Phe Ser Ser Phe Arg Ala Val His Le - #u Asp Pro Asn His Phe 
# 380 
- Lys Asp Ala Arg Thr Phe Asn Pro Trp Arg Tr - #p Gln Ser Asn Ser Val 
385 3 - #90 3 - #95 4 - 
#00 
- Thr Thr Gly Pro Ser Asn Val Phe Thr Pro Ph - #e Gly Gly Gly Pro Arg 
# 415 
- Leu Cys Pro Gly Tyr Glu Leu Ala Arg Val Al - #a Leu Ser Val Phe Leu 
# 430 
- His Arg Leu Val Thr Gly Phe Ser Trp Val Pr - #o Ala Glu Gln Asp Lys 
# 445 
- Leu Val Phe Phe Pro Thr Thr Arg Thr Gln Ly - #s Arg Tyr Pro Ile Phe 
# 460 
- Val Lys Arg Arg Asp Phe Ala Thr 
465 4 - #70 
- (2) INFORMATION FOR SEQ ID NO: 3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 4937 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
#thaliana (A) ORGANISM: Arabidopsis 
(B) STRAIN: cv. Columbi - #a 
- (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: lambda gt1 - #0 
(B) CLONE: C204 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
#1680..1829, 1917..2165, 390368..1483, 
..3989, 4 - #084..4162, 4248..4354, 4446..4576, 4674 
..4773) 
#3: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- GGATCCAAAC AAAATGTAAT TATGGAACCA AAATTCTTGA CCTATGATTC AT - #CAGTTCCT 
60 
- CCATTTCTCT ACAATAATTA ATATTCAATA AGAATTTCAC ATTAACATCC TT - #TTAATATA 
120 
- TTTTAATTAT CTGTTGATGT CACTAGTTTG TTGATGCTAT CAACAAACCG AT - #CGATAATC 
180 
- AATGGATTAA AATTGGTTCG ATTTCTTTTC ACTTAAGTGT CTTTTGAAGT TA - #GCTAAGTC 
240 
- CAGTTACAAT CAAATTATCA TGACGAAATC AGAAGTTAAA AAAAAAAAAA AA - #TCAGAAGT 
300 
- TAAAAGTTGA ATAAATAATA TTTAGCATAT GCATGTGTGA GTTCTCTGCA AC - #CAAATACG 
360 
- AAAACACACT AAACCATAAA CATTCTGGTT CCAAAAATAA ACGGAATAAA GC - #TACCGGAA 
420 
- TTACTTTTTT ACCAGCAAAT GATATACAAT CCCAAATTAT ATAAATGATT CT - #ACATATAG 
480 
- TAAGAAAATC ATGATTCCAT TACCATGTGC ATAAAAGTTA ATAATATACA TA - #GACAACCC 
540 
- ACAAATTCAT CTATATTTAC TATAATTAAT TTCGTACATG CCAAATATGT TT - #TAGTTATA 
600 
- ATACAGAAAA AATATAACTC TTTAAGGCAC TAAATCTTTT AAATTATAGA AT - #TTGCTCTC 
660 
- TGATAAATTT GAAAATCTGT GTGTTAGAGA TGTTTGAAAC AAAATTTAGA AT - #AGTATCGA 
720 
- AAATATTTTA TCCTTATTTA AAAAATTCAT ATTTTATGAA GAAGTTATTA TT - #CACTGCTT 
780 
- ACTGTATTTT AGAAAATTAC TTATAATTTA GAAGAAAAAG AAAAGAAAAG AA - #GAAGAATG 
840 
- CAAAAGAGTA TAATGATGAA AGGTCCTACT TTATGCAGAA ACCCCCCGTG TG - #CCCACTCT 
900 
- CCCCTTCTCC ATTAATACTC TCTCTCCCTC ATCCTCTCTT CTTCTCTCAT CA - #TCATCTTC 
960 
#CTC TCT TCC ATC GCC 1009 TTT CTC CTC CTC 
#Leu Leu Leu Leu Ser Ser Ile Ala 
# 10 
- GCC GGC TTC CTC CTC CTA CTC CGC CGT ACA CG - #T TAC CGT CGG ATG GGT 
1057 
Ala Gly Phe Leu Leu Leu Leu Arg Arg Thr Ar - #g Tyr Arg Arg Met Gly 
# 30 
- CTG CCT CCG GGA AGC CTT GGT CTC CCT CTG AT - #A GGA GAG ACT TTT CAG 
1105 
Leu Pro Pro Gly Ser Leu Gly Leu Pro Leu Il - #e Gly Glu Thr Phe Gln 
# 45 
- CTG ATC GGA GCT TAC AAA ACA GAG AAC CCT GA - #G CCT TTC ATC GAC GAG 
1153 
Leu Ile Gly Ala Tyr Lys Thr Glu Asn Pro Gl - #u Pro Phe Ile Asp Glu 
# 60 
- AGA GTA GCC CGG TAC GGT TCG GTT TTC ATG AC - #G CAT CTT TTT GGT GAA 
1201 
Arg Val Ala Arg Tyr Gly Ser Val Phe Met Th - #r His Leu Phe Gly Glu 
# 75 
- CCG ACG ATT TTC TCA GCT GAC CCG GAA ACG AA - #C CGG TTT GTT CTT CAG 
1249 
Pro Thr Ile Phe Ser Ala Asp Pro Glu Thr As - #n Arg Phe Val Leu Gln 
# 90 
- AAC GAA GGG AAG CTT TTT GAG TGT TCT TAT CC - #T GCT TCC ATT TGT AAC 
1297 
Asn Glu Gly Lys Leu Phe Glu Cys Ser Tyr Pr - #o Ala Ser Ile Cys Asn 
#110 
- CTT TTG GGG AAA CAC TCT CTG CTT CTT ATG AA - #A GGT TCT TTG CAT AAA 
1345 
Leu Leu Gly Lys His Ser Leu Leu Leu Met Ly - #s Gly Ser Leu His Lys 
# 125 
- CGT ATG CAC TCT CTC ACC ATG AGC TTT GCT AA - #T TCT TCA ATC ATT AAA 
1393 
Arg Met His Ser Leu Thr Met Ser Phe Ala As - #n Ser Ser Ile Ile Lys 
# 140 
- GAC CAT CTC ATG CTT GAT ATT GAC CGG TTA GT - #C CGG TTT AAT CTT GAT 
1441 
Asp His Leu Met Leu Asp Ile Asp Arg Leu Va - #l Arg Phe Asn Leu Asp 
# 155 
- TCT TGG TCT TCT CGT GTT CTC CTC ATG GAA GA - #A GCC AAA AAG 
#1483 
Ser Trp Ser Ser Arg Val Leu Leu Met Glu Gl - #u Ala Lys Lys 
# 170 
- GTAACCAAAA AAATTCTTGC TTATCAAAAA CATTATATTA TTATTTTATT CG - #GCCTTCTC 
1543 
- ACTTATGTTT TTTTTATAAT AAAAATAAAA TAAAAATCCC GGACCGAGTT TG - #TGACTCAG 
1603 
- TGAGTCAGGC CGAGTCACCA CCGCATGCAT GCATGCATAG ATTGATGATT AT - #TAATGATG 
1663 
#AAG CAG TTG ATG 1712 TTT GAG CTA ACG GTG 
#Ile Thr Phe Glu Leu Thr Val Lys Gln Leu M - #et 
# 180 
- AGC TTT GAT CCA GGG GAA TGG AGT GAG AGT TT - #A AGG AAA GAG TAT CTT 
1760 
Ser Phe Asp Pro Gly Glu Trp Ser Glu Ser Le - #u Arg Lys Glu Tyr Leu 
# 195 
- CTT GTC ATC GAA GGC TTC TTC TCT CTT CCT CT - #C CCT CTC TTC TCC ACC 
1808 
Leu Val Ile Glu Gly Phe Phe Ser Leu Pro Le - #u Pro Leu Phe Ser Thr 
200 2 - #05 2 - #10 2 - 
#15 
- ACT TAC CGC AAA GCC ATC CAA GTATATATTT CGTTTCATT - #T ACTAATTCTT 
1859 
Thr Tyr Arg Lys Ala Ile Gln 
220 
- TCTTATTTCA ATCATATTTT GAGAATATAT ATCCTAATAT ATGTGTGTGT AT - #TTTAG 
1916 
- GCG CGG AGG AAG GTG GCG GAG GCG TTG ACG GT - #G GTG GTG ATG AAA AGG 
1964 
Ala Arg Arg Lys Val Ala Glu Ala Leu Thr Va - #l Val Val Met Lys Arg 
# 235 
- AGG GAG GAG GAG GAA GAA GGA GCG GAG AGA AA - #G AAA GAT ATG CTT GCG 
2012 
Arg Glu Glu Glu Glu Glu Gly Ala Glu Arg Ly - #s Lys Asp Met Leu Ala 
# 250 
- GCG TTG CTT GCG GCG GAT GAT GGA TTT TCC GA - #T GAA GAG ATT GTT GAC 
2060 
Ala Leu Leu Ala Ala Asp Asp Gly Phe Ser As - #p Glu Glu Ile Val Asp 
255 2 - #60 2 - #65 2 - 
#70 
- TTC TTG GTG GCT TTA CTT GTC GCC GGT TAT GA - #A ACA ACC TCC ACG ATC 
2108 
Phe Leu Val Ala Leu Leu Val Ala Gly Tyr Gl - #u Thr Thr Ser Thr Ile 
# 285 
- ATG ACT CTC GCC GTC AAA TTT CTC ACC GAG AC - #T CCT TTA GCT CTT GCT 
2156 
Met Thr Leu Ala Val Lys Phe Leu Thr Glu Th - #r Pro Leu Ala Leu Ala 
# 300 
- CAA CTC AAG GTAATTTTCC CATTTTTGGT AAATAATCTC TCTACTTAT - #T 
2205 
Gln Leu Lys 
305 
- TATATACATG GTTCGTATTT AATTAATAAA GAATAACTTT GAGAAAAATA TT - #CGATTTTA 
2265 
- GTATCGAATT TTGATTGAAT TATTTTTAAA AGAGTATACA CAGCGAATGA AA - #AACACGAC 
2325 
- ACGTATGAAT GAAATTTTAG GTGTTATGTA GTTGGTTTGA TTGCGAATCA AC - #AAGATTTA 
2385 
- GTGTTTTGGA AAAGATATTA AAAAATTAAG ATTCGATCTA TTCAGTGTTC AC - #TACATTGC 
2445 
- ATCTCTGCAT GCAAACCGTT TTTTTGAAGG ACCACCGGCG CATGTTTTAC CC - #TGCTCTTG 
2505 
- CTTTATTTGG GGTTTAGGGT ATCAAAACAA AAATGGTTTT GTTTCTTTTC TT - #TGAAAACT 
2565 
- AATTAAATTA CATTTCTGTA CTTTCAACAA AATAACGAAA AGAGTGAAAA CA - #TTGAATTA 
2625 
- GAACACGGTG ATGTGTTGTT ATCAACTAAT ATGAACTTTT TCTTGTGGGC AC - #AATCTTAC 
2685 
- TGATTTAAGC TTATTTCATT TTCTTAAGTA ATTAAGAGAT GGGAAGAAGT AG - #TTGGGGGA 
2745 
- AAAATAAAAT TTAAGGTGAA AGAAAGAAAT GGGACAGAGA CTACAACAAT GG - #GAGCATAA 
2805 
- TGATATGTGC ATGTTGGCCT CTAAATTTCT CCATCATTTA CGTTTCACAC GG - #GTGTCTAG 
2865 
- ATTTTTTGGC AATTAATAAA AACTATTATA AAAAGGACAC ACACACATCA AT - #GAAACGGC 
2925 
- TTAGGTCTCC AATGAACTAC TAGTTCACAT AGCAAGTAAG CAACAGTACA AT - #CTAGTCGG 
2985 
- TTGATACTAA TAATTGATAG TAGCCAAAAA AAAAAGACTT TTTGTTTTTG GT - #TTAGAATA 
3045 
- AGGTTTTTGT TTATAGCCTT CAATCTTGGT TAATTAATGG TTAGGTATCA AG - #AAAATTAA 
3105 
- AATACGCGAC ATTAGCCGGG TAAGACGATC TAGTACTGCT ATTCACTATT TC - #AAATTATG 
3165 
- TATATCATAT ACTAAACTGG TTTCAAAGTT TTTGTTTTCC GTCAACAAAT AA - #TGAATTAG 
3225 
- AAAACGTAAG CTTTCATTCT ATTTGTCTAT TCGATGAGTT TATAATCTAA GA - #TTAAGCAT 
3285 
- ATTATTAAGT GGGTGTGAGC TTTTTGAAAG GTGAAAACTG AAAAGTGTAA AA - #GGTACTAA 
3345 
- AATTACCGTA AAAGTCAAAG TAGTCATTTT CGAAAATAGA CAACATCATC AC - #CTCAGTTT 
3405 
- TAGAGTTTTA TTTTAATAAG GAAATTGTAA AATGTAAGGA GTTACAGTCT CA - #GAGATTTG 
3465 
- ACTAATTTGT CTCCTGAACT GCATGCATAA TCACACTTTT ACCAAACCTC AT - #CTTCTTCT 
3525 
- TTTGTTTTGT TTTGTTTGTT TGCCAACAAC TTTCATCTTC TTTTTTTATC TT - #ACTTGTCC 
3585 
- GATTATCCCC CAATAAATCT CTCTTTACAT TAAAGATAAA AGTTTTATCA TA - #AATATGTT 
3645 
- TGTGCTATGC GCGACCGACA AGCTTCTCAT CCATTGGTTC TTAATATTTT AA - #TTATTTGT 
3705 
- TGATGTCACT AGTTTTGTTC CAAGGATGGT ACTACTATAT TCACTAGTTT AG - #TCATTTAC 
3765 
- TCATTAGTGC TTCGAATATG ACCAACCGGT TCAAAAAACG GTTGGACCGG TG - #ACCTAATT 
3825 
- AATTAATTTT GCTTTTACAC CTTGTTTCTT TCTTTTATTG TTGGTTGATT TG - #GTATTTGC 
3885 
#GCA ATG AAG AGT 3935AG CAT GAA AAG ATT AGG 
#Seru Glu His Glu Lys Ile Arg Ala Met Lys 
# 310 - # 315 
- GAT TCG TAT AGT CTT GAA TGG AGT GAT TAC AA - #G TCA ATG CCA TTC ACA 
3983 
Asp Ser Tyr Ser Leu Glu Trp Ser Asp Tyr Ly - #s Ser Met Pro Phe Thr 
# 330 
- CAA TGT GTAAGTGTAC TTACCTAAAG CTCTTAAGAA TTCTTGTCTT AT - #CTTCTTTC 
4039 
Gln Cys 
- TAGTCATTTC TCATCAGTAT CCTTATAAAC CTATTTTGAT TCAG GTG G - #TT AAT GAG 
4095 
#Glu Val Val Asn 
# 335 
- ACG CTA CGA GTG GCT AAC ATC ATC GGC GGT GT - #T TTC AGA CGT GCA ATG 
4143 
Thr Leu Arg Val Ala Asn Ile Ile Gly Gly Va - #l Phe Arg Arg Ala Met 
# 350 
#AAATGAGTAA 4192 G GTAAAATAAT CTAACTTTTA 
Thr Asp Val Glu Ile Lys 
355 3 - #60 
- AAAGAGTCCA TTCTGTATCA AAAACTTAAC ATTTAGAAAA CTGGAACAAA AC - #CAG GT 
4249 
# Gly 
- TAT AAA ATT CCA AAA GGG TGG AAA GTA TTC TC - #A TCG TTT AGA GCG GTT 
4297 
Tyr Lys Ile Pro Lys Gly Trp Lys Val Phe Se - #r Ser Phe Arg Ala Val 
# 375 
- CAT TTA GAC CCA AAC CAC TTC AAA GAT GCT CG - #C ACT TTC AAC CCT TGG 
4345 
His Leu Asp Pro Asn His Phe Lys Asp Ala Ar - #g Thr Phe Asn Pro Trp 
# 390 
- AGA TGG CAG GTTTGTATTT TAAGCCCTGA ACTTGGTTTG GGTGTTCTT - #T 
4394 
Arg Trp Gln 
395 
#AGC AAC 4451ATTTTGA GTTATTGAAC GATTGCAATT CTGTGGAACA G 
# Ser Asn 
- TCG GTA ACG ACA GGC CCT TCT AAT GTG TTC AC - #A CCG TTT GGT GGA GGG 
4499 
Ser Val Thr Thr Gly Pro Ser Asn Val Phe Th - #r Pro Phe Gly Gly Gly 
# 410 
- CCA AGG CTA TGT CCC GGT TAC GAG CTG GCT AG - #G GTT GCA CTC TCT GTT 
4547 
Pro Arg Leu Cys Pro Gly Tyr Glu Leu Ala Ar - #g Val Ala Leu Ser Val 
415 4 - #20 4 - #25 4 - 
#30 
#GTATATATAC CTTCACATAG 4596 TTC AG 
Phe Leu His Arg Leu Val Thr Gly Phe Ser 
# 440 
- AAGATAGTAG CTCTGTTTTC CATTTCAAAA GGCTAAAGAG ACTGATTTGA TT - #TTGTTTTG 
4656 
#GAC AAG CTG GTT TTC 4707TT CCT GCA GAG CAA 
# Trp Val Pro Ala Glu Gln Asp Lys Leu - # Val Phe 
# 450 
- TTT CCA ACT ACA AGA ACG CAG AAA CGG TAC CC - #G ATC TTC GTG AAG CGC 
4755 
Phe Pro Thr Thr Arg Thr Gln Lys Arg Tyr Pr - #o Ile Phe Val Lys Arg 
# 465 
- CGT GAT TTT GCT ACT TGA AGAAGAAGAG ACCCATCTGA TT - #TTATTTAT 
4803 
Arg Asp Phe Ala Thr * 
470 
- AGAACAACAG TATTTTTCAG GATTAATTTC TTCTTCTTTT TTTGCCTCCT TG - #TGGGTCTA 
4863 
- GTGTTTGACA ATAAAAGTTA TCATTACTCT ATAAAGCCTT AGCTTCTGTG TA - #CATAAAAA 
4923 
# 4937 
- (2) INFORMATION FOR SEQ ID NO: 4: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 472 ami - #no acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
#4: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Met Ala Phe Thr Ala Phe Leu Leu Leu Leu Se - #r Ser Ile Ala Ala Gly 
# 15 
- Phe Leu Leu Leu Leu Arg Arg Thr Arg Tyr Ar - #g Arg Met Gly Leu Pro 
# 30 
- Pro Gly Ser Leu Gly Leu Pro Leu Ile Gly Gl - #u Thr Phe Gln Leu Ile 
# 45 
- Gly Ala Tyr Lys Thr Glu Asn Pro Glu Pro Ph - #e Ile Asp Glu Arg Val 
# 60 
- Ala Arg Tyr Gly Ser Val Phe Met Thr His Le - #u Phe Gly Glu Pro Thr 
# 80 
- Ile Phe Ser Ala Asp Pro Glu Thr Asn Arg Ph - #e Val Leu Gln Asn Glu 
# 95 
- Gly Lys Leu Phe Glu Cys Ser Tyr Pro Ala Se - #r Ile Cys Asn Leu Leu 
# 110 
- Gly Lys His Ser Leu Leu Leu Met Lys Gly Se - #r Leu His Lys Arg Met 
# 125 
- His Ser Leu Thr Met Ser Phe Ala Asn Ser Se - #r Ile Ile Lys Asp His 
# 140 
- Leu Met Leu Asp Ile Asp Arg Leu Val Arg Ph - #e Asn Leu Asp Ser Trp 
145 1 - #50 1 - #55 1 - 
#60 
- Ser Ser Arg Val Leu Leu Met Glu Glu Ala Ly - #s Lys Ile Thr Phe Glu 
# 175 
- Leu Thr Val Lys Gln Leu Met Ser Phe Asp Pr - #o Gly Glu Trp Ser Glu 
# 190 
- Ser Leu Arg Lys Glu Tyr Leu Leu Val Ile Gl - #u Gly Phe Phe Ser Leu 
# 205 
- Pro Leu Pro Leu Phe Ser Thr Thr Tyr Arg Ly - #s Ala Ile Gln Ala Arg 
# 220 
- Arg Lys Val Ala Glu Ala Leu Thr Val Val Va - #l Met Lys Arg Arg Glu 
225 2 - #30 2 - #35 2 - 
#40 
- Glu Glu Glu Glu Gly Ala Glu Arg Lys Lys As - #p Met Leu Ala Ala Leu 
# 255 
- Leu Ala Ala Asp Asp Gly Phe Ser Asp Glu Gl - #u Ile Val Asp Phe Leu 
# 270 
- Val Ala Leu Leu Val Ala Gly Tyr Glu Thr Th - #r Ser Thr Ile Met Thr 
# 285 
- Leu Ala Val Lys Phe Leu Thr Glu Thr Pro Le - #u Ala Leu Ala Gln Leu 
# 300 
- Lys Glu Glu His Glu Lys Ile Arg Ala Met Ly - #s Ser Asp Ser Tyr Ser 
305 3 - #10 3 - #15 3 - 
#20 
- Leu Glu Trp Ser Asp Tyr Lys Ser Met Pro Ph - #e Thr Gln Cys Val Val 
# 335 
- Asn Glu Thr Leu Arg Val Ala Asn Ile Ile Gl - #y Gly Val Phe Arg Arg 
# 350 
- Ala Met Thr Asp Val Glu Ile Lys Gly Tyr Ly - #s Ile Pro Lys Gly Trp 
# 365 
- Lys Val Phe Ser Ser Phe Arg Ala Val His Le - #u Asp Pro Asn His Phe 
# 380 
- Lys Asp Ala Arg Thr Phe Asn Pro Trp Arg Tr - #p Gln Ser Asn Ser Val 
385 3 - #90 3 - #95 4 - 
#00 
- Thr Thr Gly Pro Ser Asn Val Phe Thr Pro Ph - #e Gly Gly Gly Pro Arg 
# 415 
- Leu Cys Pro Gly Tyr Glu Leu Ala Arg Val Al - #a Leu Ser Val Phe Leu 
# 430 
- His Arg Leu Val Thr Gly Phe Ser Trp Val Pr - #o Ala Glu Gln Asp Lys 
# 445 
- Leu Val Phe Phe Pro Thr Thr Arg Thr Gln Ly - #s Arg Tyr Pro Ile Phe 
# 460 
- Val Lys Arg Arg Asp Phe Ala Thr 
465 4 - #70 
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