Isolated DNA Encoding plant histidinol dehydrogenase

The present invention comprises cDNA coding for histidinol dehydrogenase from plants, the final step in histidine biosynthesis. The invention also comprises a novel method of purifying histidinol dehydrogenase from plants to essential honogeneity, the purified histidinol dehydrogenase, an assay for identifying inhibitors of histidinol dehydrogenase, an assay to identify mutants of histidinol dehydrogenase that are not inhibited by inhibitors of wild-type histidinol dehydrogenase, the inhibitors so identified as well as herbicide compositions containing them, the non-inhibited mutants of histidinol dehydrogenase, transgenic crop plants containing the non-inhibited mutants of histidinol dehydrogenase, and methods of treating weeds utilizing the application of histidinol dehydrogenase inhibitors to the transgenic crops containing the non-inhibited mutants of histidinol dehydrogenase.

BACKGROUND OF THE INVENTION 
Histidinol dehydrogenase [L-Histidinol-NAD Oxido-Reductase (EC 1.1.1.23)] 
catalyzes the final two steps in the biosynthesis of the amino acid 
histidine. This reaction is an oxidation of histidinol to histidinal to 
histidine, which is coupled to reduction of two moles of the required 
cofactor NAD per mole of histidine formed. 
EQU Histidinol+NAD+.fwdarw.Histidine+2 NADH 
While the enzyme and gene encoding it have been well characterized in the 
bacteria Salmonella typhimurium [Yourno and Ino, J. Biol. Chem. 
243:3273-3276 (1968); Gorisch and Holke, Eur. J. Biochem. 150:305-308 
(1985); and Grubmeyer et al., Biochem. 28:8174-8180 (1989)] and 
Escherichia coli [Bitar et al., Biochem. Biophys. Acta 493:429-440 (1977)] 
and the yeast Saccharomyces cereviseae [Schaffer et al., Brookhaven Symp. 
Biol. 23:250-270 (1972); and Keesey et al., J. Biol. Chem. 254:7427-7433 
(1979)], this enzyme has not been previously purified to homogeneity from 
any plant sources. This is in part because enzymes for primary metabolic 
functions such as amino acid biosynthesis are especially difficult to 
purify due to their low concentration in the source tissue. In plants, 
this difficulty is compounded by the existence of phenolic compounds and 
other secondary metabolites that can react with proteins throughout their 
purification. 
Histidine biosynthesis in general in higher plants has not been studied 
well. Some evidence for the existence in plants of a biosynthetic pathway 
similar to that in microorganisms has been obtained from in vivo 
experiments using various different plants and a blue-green algae [Dougall 
and Fulton, Plant Physiol. 42:941-945 (1967); Negrutiu et al., Mol. Gen. 
Genet. 199:330-337 (1985); Helm et al., Plant Physiol. 91:1226-1231 
(1989); Yavada, Mol. Gen. Genet. 170:109-111 (1979)]. 
Histidinol dehydrogenase activity has been detected in ten different plant 
species: asparagus, cabbage, cucumber, egg plant, lettuce, radish [Wong 
and Mazalis, Phytochrom. 20:1831-1834 (1981)], rose, squash [Wong and 
Mazalis, Phytochem. 20:1831-1834 (1981)], turnip [Wong and Mazalis, 
Phytochem. 20:1831-1834 (1981)], and wheat [Wong and Mazalis, Phytochem. 
20:1831-1834 (1981) and this work] and in 5 preparations of distinct cell 
differentiation: germ [[Wong and Mazalis, Phytochrom. 20:1831-1834 (1981)] 
and this work), root [Wong and Mazalis, Phytochrom. 20:1831-1834 (1981)], 
fruit [Wong and Mazalis, Phytochrom. 20:1831-1834 (1981) and this work], 
shoot, and cultured cell. 
Only a few attempts have been made to study the enzymes involved in 
histidine (His) biosynthesis in higher plants. The hitherto impossible 
task of purifying to homogeneity any histidine biosynthesis enzyme from 
plants seems to be the limiting step in biochemical investigations. In 
crude extracts from shoots of barley, oats and peas, the activities of 
ATP-phosphoribosyl transferase, imidazoleglycerol phosphate dehydratase 
and histidinol phosphate phosphatase were detected [Wiater et al., Acta 
Biochim. Polonica 18:299-307 (1971)]. Histidinol dehydrogenase activity 
has been found in crude extracts of different plant species [Wong et al., 
Phytochem. 20:1831-1834 (1981)]. These data suggest that histidine 
biosynthesis in plants follows the same pathway as in microorganisms, 
although no protein has ever been isolated from plants and identified as 
an enzyme involved directly in histidine biosynthesis. 
The pathways for biosynthesis of the ten amino acids essential to the human 
diet are of special interest as targets for development of novel 
inhibitory compounds that could act as herbicides. This interest is due to 
the likelihood that a specific inhibitor of any enzyme from these pathways 
will be completely non-toxic to vertebrates, which lack the biosynthetic 
pathways for the essential amino acids. 
SUMMARY OF THE INVENTION 
The present invention comprises cDNA coding for histidinol dehydrogenase 
from plants, the final step in histidine biosynthesis. 
The invention also comprises a novel method of purifying histidinol 
dehydrogenase from plants to essential homogeneity comprising: 
(a) exposing a protein extract to an ion exchange chromatography column to 
produce a first eluate in the presence of a linear gradient; 
(b) exposing the first eluate to an affinity chromatography column to 
produce a second eluate in the presence of a displacing solvent, wherein 
said affinity chromatography column comprises a ligand specific for 
histidinol dehydrogenase; 
(c) storing the second eluate. 
Another aspect of the present invention is the purified histidinol 
dehydrogenase, preferably having the amino acid sequence identical to or 
substatiolly homologous to the amino acid sequence of FIG. 3. 
Another aspect of the present invention is an assay to identify inhibitors 
of histidinol dehydrogenase activity comprising: 
(a) incubating a first sample of histidinol dehydrogenase and its 
substrate; 
(b) measuring an uninhibited reactivity of the histidinol dehydrogenase 
from step (a); 
(c) incubating a first sample of histidinol dehydrogenase and its substrate 
in the presence of a second sample comprising an inhibitor compound; 
(d) measuring an inhibited reactivity of the histidinol dehydrogenase from 
step (c); and 
(e) comparing the inhibited reactivity to the uninhibited reactivity of the 
histidinol dehydrogenase. 
Another aspect of the present invention is an assay to identify less 
functional histidinol dehydrogenase mutants comprising: 
(a) incubating a first sample of histidinol dehydrogenase and its substrate 
in the presence of a second sample comprising a histidinol dehydrogenase 
inhibitor; 
(b) measuring an unmutated reactivity of the histidinol dehydrogenase from 
step (a); 
(c) incubating a first sample of a mutated histidinol dehydrogenase and its 
substrate in the presence of a second sample comprising a histidinol 
dehydrogenase inhibitor; 
(d) measuring a mutated reactivity of the mutated histidinol dehydrogenase 
from step (c); and 
(e) comparing the mutated reactivity to the unmutated reactivity of the 
histidinol dehydrogenase. 
Other aspects of the present invention are the inhibitors so identified as 
well as herbicide compositions containing them, the non-inhibited mutants 
of histidinol dehydrogenase, transgenic crop plants containing the 
non-inhibited mutants of histidinol dehydrogenase, and methods of treating 
weeds utilizing the application of histidinol dehydrogenase inhibitors to 
the transgenic crops containing the non-inhibited mutants of histidinol 
dehydrogenase.

DETAILED DESCRIPTION OF THE INVENTION 
To assist in interpreting the meaning and scope of the present invention, 
the following terms are intended to have the meanings as described below, 
unless otherwise indicated. All references cited in this application are 
hereby incorporated by reference for their relevant teachings. 
Coding DNA Sequence: A DNA sequence which, when transcribed and translated, 
results in the formation of a cellular polypeptide. 
Gene: A discrete chromosomal region comprising regulatory DNA sequences 
responsible for the control of expression, i.e., transcription and 
translation, and of a coding sequence which is transcribed and translated 
to give a distinct polypeptide. 
Histidinol Dehydrogenase Inhibitory Effective Amount or Significant 
Decrease in Histidinol Dehydrogenase Activity: A significant decrease in 
the ability of histidinol dehydrogenase to convert substrates such as 
histidinol into products such as histidine as measured by quantifying 
enzymatic activity as generally known to those of skill in the art, 
including equilibrium constants, reaction velocities of the appearance of 
reaction products or the consumption of reaction substrates, reaction 
kinetics, thermodynamics of reaction, spectrophotometric analysis of 
reaction products, detection of labelled reaction components, etc, with 
the preferred method measurement being the spectrophotometric measurement 
of NAD+as a reaction byproduct. A significant decrease in enzyme activity 
is any decrease that is larger than the margin of error inherent in the 
measurement technique, preferably a decrease by 50 percent, more 
preferably a decrease by 90%, more preferably a decrease by 99%, and most 
preferably a decrease to essentially undetectable levels of enzyme 
activity. 
Substantial Sequence Homology: Substantial functional and/or structural 
equivalence between sequences of nucleotides or amino acids. Functional 
and/or structural differences between sequences having substantial 
sequence homology will be de minimis. The sequences that differ from the 
natural sequences are usually variants of the natural sequence. A variant 
of a natural sequence is a modified form of the natural sequence that 
performs the same function. The variant may be a mutation, or may be a 
synthetic sequence. A de minimis functional difference results from a 
nucleotide or amino acid sequence that codes for a protein having 
essentially the same characteristics as the native protein. Such 
characteristics can include, for example, immunological reactivity, enzyme 
activity, structural protein integrity, etc. Structural differences are 
considered de minimis if there is a significant amount of sequence overlap 
or similarity between two or more different sequences or if the different 
sequences exhibit similar physical properties. In the case of a nucleotide 
sequence, the different sequences will preferably have at least 60 
percent, more preferably 75 percent, most preferably 90 percent or more 
sequence similarity between them. In the case of amino acid sequences, the 
different sequences have at least 70 percent, more preferably 80 percent, 
more preferably 90 percent, and most preferably at least 99 percent or 
more similarity between the polypeptides coded for by the amino acid 
sequences. Physical properties that may be similar include, for example, 
electrophoretic mobility, chromatography similarities, sediment gradient 
coefficients, spectrophotometric properties, etc. 
Transgenic Plant: A plant having stably incorporated exogenous DNA in its 
genetic material. Various different methods of introducing the exogenous 
DNA into protoplasts are known in the art and can be used to produce the 
transgenic protoplasts, transgenic cells or transgenic plants of the 
present invention. 
Weeds: Any undesired species of plant growing among a culture of cultivated 
plants such as crops, preferably any individual of a species that is 
heterogeneous to the species of a homogenous population of plants. 
The present invention relates to methods of purifying a previously 
unpurified protein from plants, the protein purified by such a method, DNA 
coding for such a protein, methods of using the protein and DNA to assay 
for specific inhibitors of the protein, and methods of using the specific 
inhibitors to assay for altered forms of the protein that are less 
functional forms of the protein so as to produce an insensitivity to the 
inhibitors. 
The method of purification utilizes an affinity chromatography column that 
is specific for histidinol dehydrogenase. Various known methods can be 
employed previous to and subsequent to the affinity chromatography to 
produce a crude extract to apply to the affinity column and to 
characterize and store the protein isolated by the affinity column. 
Such previous steps can include homogenization, filtration, centrifugation, 
ammonium salt precipitation, desalting, ion exchange chromatography, 
sonication, etc. Preferably, the plant tissue from which the histidinol 
dehydrogenase is obtained, is homogenized using a blender. The homogenate 
is then preferably passed through course filtration such as a filtration 
cloth. The filtrate is then preferably adequately centrifuged, preferably 
at about 30,000 x g for at least 20 minutes. The supernatant is then 
preferably precipitated using ammonium sulfate at from about 40 to 70% 
saturation, more preferably at about 45 to 60% saturation, and the 
precipitate is collected and desalted using an appropriate desalting 
technique such as dialysis or gel filtration, preferably using gel 
filtration with G-25 Sephadex with about 50 mM TRIS/HCL at pH from about 7 
to 7.5, preferably pH 7.4. 
The desalted protein extract can then be further prepared for affinity 
chromatography using an appropriate ion exchange column. A preferred ion 
exchange column is a Diethyl-amino-ethyl (DEAE) derivatized resin, such as 
DEAE-cellulose, more preferably DEAE-Toyopearl, equilibrated with a 
suitable buffer such as the buffer used in the desalting step, most 
preferably 50 mM TRIS/HCL at about pH 7.4. Elution from the ion exchange 
column can be accomplished using a linear gradient containing the 
equilibration buffer and a suitable salt gradient. A preferred salt 
gradient is NaCl from 0 to at least 500 mM. The eluate from the ion 
exchange column is then an appropriate crude extract for application to 
the affinity chromatography column. 
Appropriate ligands for the chromatography column are any substrate 
specific for the enzyme, histidinol dehydrogenase, that is to be isolated. 
Attachment of the ligand to the column resin will preferably occur at a 
position on the ligand that will not significantly interfere with the 
binding of the ligand to the enzyme to be isolated. Preferred ligands for 
histidinol dehydrogenase are therefore histidinol and histidinal, more 
preferably histidinol. Any suitable resin may be used to attach the 
ligand, with a preferred resin being Sepharose-4B. Methods of attaching 
ligands to chromatography resins are generally known in the art. 
A preferred method of preparing a suitable affinity column resin is as 
follows. Sepharose-4B is activated with epichlorohydrin as previously 
described [Matsumoto et al., J. Biochem. 85:1091-1098 (1979)]. 
Epoxyactivated Sepharose-4B gel is suspended in 0.1N NAOH containing 0.5 g 
glucose per g of gel and incubated at 40.degree. C. on a shaker for 24 h 
[Kanamori et al., J. Chromatog. 363:231-242 (1986). After washing 
extensively with water, the gel is suspended in prechilled sodium 
metaperiodate and the suspension shaken for 1 h at 40.degree. C. The gel 
is washed with water, suspended in 0.1M HCl and incubated at room 
temperature for 1 h. Again, the gel is washed extensively with water, 
followed by thorough washing with 10 mM sodium phosphate buffer, pH 7.0 at 
room temperature. 
The formyl carrier gel is suspended in 10 mM sodium phosphate buffer, pH 
7.0 containing 100 mM of histidinol dihydrochloride, which is previously 
neutralized with NAOH, and the suspension incubated at 40.degree. C. 
overnight. Five milligrams of sodium cyanoborohydride per g of gel is then 
added to the suspension and incubated with shaking at room temperature for 
6 h. The gel is extensively washed with distilled water and treated with 5 
mg sodium borohydride per g of gel for 3 h at 4.degree. C. to convert the 
remaining formyl groups into hydroxymethyl groups. 
After preparation of a suitable affinity chromatography column resin, the 
resin is placed in a suitable column, preferably about 2.5.times.8 cm. The 
crude extract of protein is placed on the affinity column and preferably 
extensively washed with a suitable buffer, preferably containing an 
adequate ionic strength of salt, e.g., 10 mM TRIS/HCl buffer, about pH 
7.3, containing about 100 to 200 mM NaCl, preferably about 140 mM NaCl. 
The bound enzyme can be eluted using a suitable displacing solvent. A 
suitable displacing solvent is one containing a substance that will have 
an affinity to the enzyme to be isolated that is similar to the ligand 
used in the affinity column. When the enzyme is histidinol dehydrogenase 
and the ligand is histidinol, a preferred displacing solvent is one 
containing imidazole, more preferably one containing about 50 mM TRIS/HC1, 
pH about 7.3, and imidazole. 
Subsequent to the affinity chromatography methods can be employed to 
characterize and store the protein isolated by the affinity column. These 
include desalting the eluate from the affinity column into a suitable 
buffer to remove the displacing solvent. The desalted protein can then be 
further characterized or concentrated and stored cryogenically, preferably 
below -50.degree. C., more preferably at about -80.degree. C. 
The protein can be characterized using standard techniques such as gel 
filtration (molecular mass), paolyacrylamide gel electrophoresis [Laemmli, 
Nature 227:680-685 (1970)] (purity and molecular weight), isoelectric 
focusing (isoelectric point), amino acid sequencing, appropriate enzyme 
assays (to measure enzymatic activity), etc. 
The enzyme activity is assayed spectrophotometrically by measuring the 
increase in absorbence at 340 nm due to the reduction of NAD+in a Hitachi 
U-3120 spectrophotomer. The reaction mixture contained 150 mM Gly/NaOH 
buffer, pH 9.2, 0.5 mM, MnCl , 2 mM NAD+, 5 mM histidinol, and 2-5 mU of 
enzyme sample in total volume of 0.5 ml. The reaction is started with the 
addition of histidinol, and incubated at 30.degree. C. A reference sample 
containing water instead of histidinol is always run. One unit of enzyme 
catalyzes the formation of 2 .mu.mol of NADH per min under the assay 
conditions. 
In some experiments the activity is also followed by measuring the 
formation of His with a Hitachi L-850 amino acid analyzer. The enzyme 
reaction is terminated with formic acid (final concentration, 30%). The 
sample is then evaporated, and the precipitate dissolved in 0.2N HC1. This 
preparation is used for His analysis, amino acid sequencing, enzymatic 
activity, etc. 
Determination of amino acid sequencing can be accomplished by known 
methods, e.g., manual or automated Edman degradation reaction using 
commercially available instrumentation (Applied Biosystems, Foster City, 
Calif.). Amino acid sequencing can be performed on the entire full length 
protein, or sequencing can be performed on fragments or portions of the 
protein. Such fragments include N-terminal sequencing, sequencing after 
digestion with Lys-C (Wako Pure Chemical Co., Osaka, Japan), sequencing 
after digestion with cyanogen-bromide etc. 
Using the amino acid sequence(s), portions of the amino acid sequence that 
display minimal degeneration of the nucleotide sequences coding for the 
protein can be used to synthesize oligonucleotide probes. The 
oligonucleotide probes can be synthesized by phosphoramidite chemistry, 
e.g., as performed by an Applied Biosystems 380B DNA synthesizer (Foster 
City, Calif.) using reagents available from the manufacturer. 
Examples of the useful portions of the amino acid sequence obtained from 
cabbage that can be used to synthesize oligonucleotide probes include: 
______________________________________ 
Lys-C #17 
TELSFAK 
Lys-C #18 
TVVLATPPTK 
Lys-C #32 
EAFDVAYDNIYAFHLAQK 
Lys-C #45 
KFMTVQSLTEEGLRNLGPYVA 
TMAEIEGLDA 
Lys-C #48 
ALSHSFTVFARDMIEAITFSN 
LYAPEK 
CNBr #6 MAEIEGLDAHKRAVTLR?KDIE 
CNBr #18 LAIPANIAGRKTVVLATP 
N-terminal 
MKIYRLSELSF?NVENLKAR? 
?ID 
______________________________________ 
wherein the amino acid terminology is the standard abbreviations. 
After labelling with P-32, these oligonucleotide probes can be used to 
probe a cDNA library of the MRNA from the organism from which the 
histidinol dehydrogenase is obtained. Such a library (a cDNA library) of 
poly A(+) RNA from the source organism can be constructed in an 
appropriate cloning vector, preferably a cloning vector such as 
lambdaZAPII (available from Stratagene, La Jolla, Calif. using the 
Uni-ZAPXR cDNA kit, also available from Stratagene) utilizing methods that 
are known in the art. Positive hybridization of the oligonucleotide probes 
to a cDNA clone from the library identifies that clone as potentially 
containing the nucleotide sequence corresponding to the amino acid 
sequence for histidinol dehydrogenase. 
Alternatively, total DNA from the cDNA library can be prepared and used as 
a template for a PCR reaction with primers representing low degeneracy 
portions of the amino acid sequence. Preferably, the primers used will 
generate PCR products that represent a significant portion of the 
nucleotide sequence. Preferred primers that can be used to probe total DNA 
from the cDNA library include: 
______________________________________ 
EW25 5' TTAAGATTCTAYGAYAAYATHTAYGC 3' 
JR03 5' CCYTCDATYTCNGCCAT 3' 
______________________________________ 
where D indicates any the bases A, T, or G; H indicates the bases A, T, or 
C; R indicates the purines A or G; Y indicates the pyrimidines T or C. The 
PCR products can be further probed to determine if they correspond to a 
portion of the histidinol dehydrogenase gene using a synthetic 
oligonucleotide probe corresponding to an amino acid fragment sequence 
located in the interior or middle region of the histidinol dehydrogenase 
protein. An example of such a probe would include the .sup.32 P labelled 
probe represented by the formula: 
EQU JR02 5' ATNGCYTCDATCATRTC 3' 
where D indicates any the bases A, T, or G; H indicates the bases A, T, or 
C; R indicates the purines A or G; Y indicates the pyrimidines T or C. 
The PCR product can thus be used, for example, to select and isolate 
further DNA clones using standard techniques (Maniatis et al., Molecular 
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring 
Harbor, N.Y. (1982). Nucleotide sequencing of the full length DNA coding 
for histidinol dehydrogenase can then be obtained using standard 
techniques (Maxam and Gilbert, Proc. Nat. Acad. Sci. USA 74:560-564 
(1977); and Sanger, Proc. Nat. Acad. Sci. USA 74:5463-5467 (1977)] or 
using commercially available nucleotide sequencing instrumentation 
(available from Applied Biosystems, Foster City, Calif. and Dupont, 
Wilmington, Del.). 
The purified histidinol dehydrogenase can then be used in an assay to 
identify inhibitors of the histidinol dehydrogenase activity. 
Alternatively, purified histidinol dehydrogenase can be obtained from 
transgenic expression of the DNA coding for the enzyme, i.e., placing the 
gene for histidinol dehydrogenase into an appropriate bacterial, yeast or 
other cell expression system. 
An assay to identify inhibitors of histidinol dehydrogenase activity 
comprises: 
(a) incubating a first sample of histidinol dehydrogenase and its 
substrate; 
(b) measuring an uninhibited reactivity of the histidinol dehydrogenase 
from step (a); 
(c) incubating a first sample of histidinol dehydrogenase and its substrate 
in the presence of a second sample comprising an inhibitor compound; 
(d) measuring an inhibited reactivity of the histidinol dehydrogenase from 
step (c); and 
(e) comparing the inhibited reactivity to the uninhibited reactivity of the 
histidinol dehydrogenase. 
Suitable histidinol dehydrogenase for the above assay can be obtained using 
the purification methods of the present invention. Suitable substrates are 
histidinol or structural analogs that are capable of being converted into 
an analog of histidine by the histidinol dehydrogenase enzyme, with the 
preferred substrate being histidinol. Preferably, the substrate is at a 
concentration of about 1 to about 100 mM, more preferably at about 1 to 
about 10 mM, most preferably at about 5 mM. 
In addition to the histidinol dehydrogenase enzyme and a suitable 
substrate, the reaction mixture can contain a suitable buffer, suitable 
cofactors and suitable divalent cations as a cofactor. A suitable buffer 
includes any suitable biological buffer that can provide buffering 
capability at a pH conducive to the reaction requirements of the enzyme. 
Preferably, the buffer provides buffering capability in the pH range 7.5 
to 10.0, more preferably in the pH range of about 9.1 to about 10.0, most 
preferably at a pH of about 9.2, e.g., 150 mM Gly/NaOH (glycine/sodium 
hydroxide) at pH 9.2. A preferred cofactor for the histidinol 
dehydrogenase enzyme is NAD, preferably at a concentration of about 1 to 
about 100 mM, more preferably at about 2 mM. Preferably, the divalent 
cation is a divalent metal cation, more preferably manganese. The reaction 
is carried out at a suitable temperature to allow the reaction to proceed. 
Such a suitable temperature is about 4.degree. C. to about 40.degree. C., 
more preferably from about room temperature to about 35.degree. C., most 
preferably at about 30.degree. C. The most preferred reaction mixture 
contains 150 mM Gly/NaOH buffer, pH 9.2, 0.5 mM, MnCl.sub.2, 2 mM NAD+, 5 
mM histidinol, and 2-5 mU of enzyme sample in total volume of 0.5 ml at 
30.degree. C. Preferably, the reaction is started with the addition of 
histidinol. 
Uninhibited reactivity of the histidinol dehydrogenase is any measure of 
enzymatic activity of the histidinol dehydrogenase enzyme while in the 
presence of a suitable substrate. Such measures of enzymatic activity are 
generally known to those of skill in the art, including equilibrium 
constants, reaction velocities of the appearance of reaction products or 
the consumption of reaction substrates, reaction kinetics, thermodynamics 
of reaction, spectrophotometric analysis of reaction products, detection 
of labelled reaction components, etc. See, generally, Segel, Biochemical 
Calculations, 2nd Edition, John Wiley and Sons, New York (1976); Suelter, 
A Practical Guide to Enzymology, John Wiley and Sons, New York (1985). The 
preferred method of measuring enzymatic activity is spectophotometrically 
by measuring the increase in absorbence at 340 nm due to the resuction of 
NAD+, e.g., using a Hitachi U-3120 spectrophotometer. 
Suitable inhibitor compounds are identified using the above methods. To 
date at least seven compounds have been so identified. 
Inhibited reactivity is determined in the same manner as uninhibited 
reactivity with the addition of an inhibitor of histidinol dehydrogenase. 
The concentration of the inhibitor may vary depending on the inhibitory 
activity, but generally it will be in an amount ranging from about 10 nm 
to about 200 mM, more preferably about 0.1 mM to about 100 mM, more 
preferably about 1 to about 10 mM. Generally, the histidinol or other 
substrate will be added to a mixture containing the enzyme and inhibitor 
and then an enzyme activity determined as described previously. 
Comparing the inhibited reactivity to the uninhibited reactivity of the 
histidinol dehydrogenase includes determining whether a significant 
decrease in enzyme activity is observed in the inhibited reactivity 
compared to the uninhibited reactivity. A significant decrease is a 
decrease in enzymatic activity that is larger than the margin of error 
inherent in the measurement technique, preferably a decrease by about 50% 
of the activity in the absence of the inhibitor, more preferably a 
decrease by about 90%, more preferably a decrease by about 99%, most 
preferably a decrease to a level of enzymatic activity that is essentially 
undetectable. 
Once an herbicidal compound is identified that inhibits function of the 
wild-type enzyme, thus killing the organism containing the enzyme, 
resistant mutants can be isolated by mutagenizing a population of the 
organism in question, growing the mutagenized population in the presence 
of a concentration of the inhibitor sufficient to inhibit growth of the 
wild-type organism, and selecting individuals from the population that are 
able to grow more rapidly than wild-type organisms. 
Mutagenesis in vivo can be by any of several means, including chemical, 
e.g., ethyl methanesulfonate [Miller, Experiments in Molecular Genetics, 
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1972); Davis et 
al. Advanced Bacterial Genetics, Cold Spring Harbor Laboratory, Cold 
Spring Harbor, N.Y. (1980); Sherman et al., Methods in Yeast Genetics, 
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1983); and 
Somerville and Ogren, In Edelman et al. (eds) Methods in Chloroplast 
Molecular Biology, Elsevier Biomedical Press, pp. 129-138 (1982)], 
ultraviolet mutagenesis [Miller, Experiments in Molecular Genetics, Cold 
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1972)], X-ray 
mutagenesis (e.g., Hake and Freeling, Nature 320, 621-623 (1986); and 
Poethig, Nature 336, 82-83 (1988)] and gamma irradiation. Alternatively, 
the sequence encoding histidinol dehydrogenase can be mutagenized in vitro 
by any of several chemical or enzymatic treatments [e.g., Zoller and 
Smith, Meth. Enzymol. 100, 468-500 983); Leung et al., Technique 1:11-15 
(1989); and Shortle and Bolstein, Method. Enzymol. 100:457-468 (1983)]. 
Such mutated sequences are re-introduced into the host organism, 
preferably a microbe, and the population containing a mixture of 
reintroduced mutated sequences is grown on a toxic concentration of the 
inhibitor. Individuals are directly selected for their ability to grow in 
the presence of this toxic amount of inhibitor, which ability is conferred 
by the introduced mutated sequence. 
The mutant individuals that result from in vivo mutagenesis, having an 
ability to tolerate normally toxic concentrations of the inhibitor, can be 
identified and genetically purified (either by streaking to single 
colonies several times for the case of a microbe, or by repeatedly 
selecting a single seed, growing to maturity, and self-pollinating for the 
case of a plant), the gene encoding histidinol dehydrogenase can be 
rapidly isolated from the mutant by the polymerase chain reaction and its 
DNA sequence determined and translated into a predicted amino acid 
sequence. Amino acids found in the mutant to differ from wild type can be 
assumed to be responsible for the inhibitor-resistant phenotype. 
Preferably, the causal nature of the amino acid changes can be directly 
tested by expressing the mutated sequence in a microbial or plant system, 
and demonstrating that introduction of the mutated sequence is sufficient 
to confer an inhibitor-resistant phenotype on an otherwise wild-type 
individual. 
An assay to identify inhibitor-resistant histidinol dehydrogenase mutants 
comprises: 
(a) incubating a first sample of histidinol dehydrogenase and its substrate 
in the presence of a second sample comprising a histidinol dehydrogenase 
inhibitor; 
(b) measuring an unmutated reactivity of the histidinol dehydrogenase from 
step (a); 
(c) incubating a first sample of a mutated histidinol dehydrogenase and its 
substrate in the presence of a second sample comprising a histidinol 
dehydrogenase inhibitor; 
(d) measuring a mutated reactivity of the mutated histidinol dehydrogenase 
from step (c); and 
(e) comparing the mutated reactivity to the unmutated reactivity of the 
histidinol dehydrogenase. 
The reaction mixture and the reaction conditions are the same as for the 
assay to identify inhibitors of histidinol dehydrogenase (inhibitor assay) 
with the following modifications. First, a histidinol dehydrogenase 
mutant, obtained as described above, is substituted in one of the reaction 
mixtures for the wild-type histidinol dehydrogenase of the inhibitor 
assay. Second, an inhibitor of wild-type histidinol dehydrogenase is 
present in both reaction mixtures. Third, mutated reactivity (enzyme 
activity in the presence of inhibitor and mutated histidinol 
dehydrogenase) and unmutated reactivity (enzyme activity in the presence 
of inhibitor and wild-type histidinol dehydrogenase) are compared to 
determine whether a significant increase in enzyme activity is observed in 
the mutated reactivity when compared to the unmutated reactivity. Mutated 
reactivity is any measure of enzymatic activity of the mutated histidinol 
dehydrogenase enzyme while in the presence of a suitable substrate and the 
inhibitor. Unmutated reactivity is any measure of enzymatic activity of 
the wild-type histidinol dehydrogenase enzyme while in the presence of a 
suitable substrate and the inhibitor. A significant increase is an 
increase in enzymatic activity that is larger than the margin of error 
inherent in the measurement technique, preferably an increase by about 
2-fold of the activity of the wild-type enzyme in the presence of the 
inhibitor, more preferably an increase by about 10-fold, most preferably 
an increase by about 100-fold. 
A mutated histidinol dehydrogenase-encoding DNA sequence that confers 
herbicide resistance can then be introduced into a crop species of 
interest, allowing the crop to survive in the presence of a concentration 
of the herbicide that kills all plants lacking the resistant form of the 
enzyme. 
The coding DNA sequence to be introduced into the plant cell can be 
introduced into the plant cell in a number of ways that are well known to 
those of skill in the art. For example, methods of transforming plant 
cells include microinjection (Crossway et al., BioTechniques 4:320-334 
(1986)], electroporation (Riggs et al., Proc. Nat. Acad. Sci. USA 
83:5602-5606 (1986)], Agrobacterium mediated transformation (Hinchee et 
al. Biotechnology 6:915-921 (1988)], direct gene transfer (Paszkowski et 
al., EMBO J. 3:2717-2722 (1984)], and ballistic particle acceleration 
using devices available from Agracetus, Inc., Madison, Wis. and Dupont, 
Inc., Wilmington, Del. (see, for example, Sanford et al., U.S. Pat. No. 
4,945,050; and McCabe et al., Biotechnology 6:923-926 (1988)]. Also see, 
Weissinger et al., Annual Rev. Genet. 22:421-477 (1988); Sanford et al., 
Particulate Science and Technology 5:27-37 (1987)(onion); Christou et al., 
Plant Physiol. 87:671-674 (1988)(soybean); McCabe et al., Bio/Technology 
6:923-926 (1988)(Soybean); Datta et al., Bio/Technology 8:736-740 
(1990)(rice); Klein et al., Proc. Nat. Acad. Sci. USA 85:4305-4309 
(1988)(maize); Klein et al, Bio/Technology 6:559-563 (1988)(maize); Klein 
et al., Plant Physiol. 91:440-444 (1989)(maize); Fromm et al., 
Bio/Technology 8:833-839 (1990); Gordon-Kamm et al., Plant Cell 2:603-618 
(1990)(maize); and Pace et al., copending U.S. patent application Ser. No. 
573,105, filed Aug. 24, 1990, entitled Production of Transgenic Elite 
Maize Plants and Their Progeny (elite maize). 
Transformation of the plant cells includes separating transformed cells 
from those that have not been transformed. One convenient method for such 
separation or selection is to incorporate into the material to be inserted 
into the transformed cell a gene for a selection marker. As a result, only 
those cells that have been successfully transformed will contain the 
marker gene. The translation product of the marker gene will then confer a 
phenotypic trait that will make selection possible. Usually the phenotypic 
trait is the ability to survive in the presence of some chemical agent, 
such as an antibiotic, e.g., kanamycin, G418, paromomycin, etc., which is 
placed in a selection media. 
Once the transformed plant cells have been cultured on the selection media, 
surviving cells are selected for further study and manipulation. Selection 
methods and materials are well known to those of skill in the art, 
allowing one to choose surviving cells with a high degree of 
predictability that the chosen cells will have been successfully 
transformed with exogenous DNA. 
After transformation of the plant cell or plant, those plant cells or 
plants transformed by the Ti plasmid so that the enzyme is expressed, can 
be selected by an appropriate phenotypic marker. These phenotypical 
markers include, but are not limited to, antibiotic resistance. Other 
phenotypic markers are known in the art and may be used in this invention. 
All plants from which protoplasts can be isolated and cultured to give 
whole regenerated plants can be transformed by the present invention so 
that whole plants are recovered which contain the DNA coding sequence. 
Some suitable plants include, for example, species from the genera 
Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, 
Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, 
Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, 
Solanum, Petunia, Dactylis, Majorana, Cichorium, Helianthus, Lactuca, 
Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, 
Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, 
Browallia, Glycine, Lolium, Zea, Triticum, Sorghum and Datura. 
There is an increasing body of evidence that practically all plants can be 
regenerated from cultured cells or tissues, including but not limited to, 
all major cereal crop species, sugarcane, sugar beet, cotton, fruit and 
other trees, legumes and vegetables. Limited knowledge presently exists on 
whether all of these plants can be transformed by Agrobacterium. Species 
which are a natural plant host for Agrobacterium may be transformable in 
vitro. Monocotyledonous plants, and in particular, cereals and grasses, 
are not natural hosts to Agrobacterium. Attempts to transform them using 
Agrobacterium have been successful recently (Hooykas-Van Slogteren et al., 
Nature 311:763-764 (1984)]. There is growing evidence now that certain 
monocots can be transformed by Agrobacterium. Using current experimental 
approaches that have now become available, cereal and grass species may be 
transformable. 
Additional plant genera that may be transformed by Agrobacterium include 
Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, 
Santalum, Allium, Lilium, Narcissus, Ananas, Arachis, Phaseolus and Pisum. 
Plant regeneration from cultural protoplasts is described in Evans et al., 
"Protoplast Isolation and Culture," in Handbook of Plant Cell Culture 
1:124-176 (MacMillan Publishing Co., New York 1983); M. R. Davey, "Recent 
Developments in the Culture and Regeneration of Plant Protoplasts," 
Protoplasts 1983); P. J. Dale, "Protoplast Culture and Plant Regeneration 
of Cereals and Other Recalcitrant Crops," in Protoplasts 1983--Lecture 
Proceedings, pp. 31-41 (Birkhauser, Basel, 1983); and H. Binding, 
"Regeneration of Plants," in Plant Protoplasts, pp. 21-37 (CRC Press, Boca 
Raton, 1985). 
Regeneration varies from species to species of plants, but generally a 
suspension of transformed protoplasts containing multiple copies of the 
toxin gene is first provided. Embryo formation can then be induced from 
the protoplast suspensions, to the stage of ripening and germination as 
natural embryos. The culture media will generally contain various amino 
acids and hormones, such as auxin and cytokinins. It is also advantageous 
to add glutamic acid and proline to the medium, especially for such 
species as corn and alfalfa. Shoots and roots normally develop 
simultaneously. Efficient regeneration will depend on the medium, on the 
genotype, and on the history of the culture. If these three variables are 
controlled, then regeneration is fully reproducible and repeatable. 
The mature plants, grown from the transformed plant cells, are selfed to 
produce an inbred plant. The inbred plant produces seed containing the 
hybrid gene. These seeds can be grown to produce plants that have the 
hybrid gene. Such transformed plants and seeds are known as transgenic 
plants and seeds. Preferred transgenic plants are transgenic crops, which 
are plants suitable for human cultivation, more preferably plants all or 
part of which are fit for consumption by animals raised as a source of 
human food, e.g., livestock, poultry, fish, etc. 
Such transformed seeds can then be used to improve crop yields by growing 
transformed plants from the transformed seeds in the presence of a 
histidinol dehydrogenase inhibitor that would be herbicidal to 
nontransformed plants growing among the transformed crops (i.e., weeds). 
Genetically engineering crops in this fashion to become resistant to a 
herbicide with no vertebrate toxicity would encourage the use of safe 
herbicides on this type as an alternative to more broadly toxic herbicides 
of questionable environmental safety and vertebrate toxicity. 
The active ingredients of the present invention are normally applied in the 
form of compositions together with one or more agriculturally acceptable 
carriers, and can be applied to the crop area or plant to be treated, 
simultaneously or in succession, with further compounds. These compounds 
can be both fertilizers or micronutrient donors or other preparations that 
influence plant growth. They can also be selective herbicides, 
insecticides, fungicides, bactericides, nematicides, mollusicides or 
mixtures of several of these preparations, if desired together with 
further carriers, surfactants or application-promoting adjuvants 
customarily employed in the art of formulation. Suitable carriers and 
adjuvants can be solid or liquid and correspond to the substances 
ordinarily employed in formulation technology, e.g. natural or regenerated 
mineral substances, solvents, dispersants, wetting agents, tackifiers, 
binders or fertilizers. 
A preferred method of applying active ingredients of the present invention 
or an agrochemical composition which contains at least one of the active 
ingredients is leaf application. The number of applications and the rate 
of application depend on the intensity of infestation by the corresponding 
weed. However, the active ingredients can also penetrate the plant through 
the roots via the soil (systemic action) by impregnating the locus of the 
plant with a liquid composition, or by applying the compounds in solid 
form to the soil, e.g. in granular form (soil application). The active 
ingredients may also be applied to seeds (coating) by impregnating the 
seeds either with a liquid formulation containing active ingredients, or 
coating them with a solid formulation. In special cases, further types of 
application are also possible, for example, selective treatment of the 
plant stems or buds. 
The active ingredients are used in unmodified form or, preferably, together 
with the adjuvants conventionally employed in the art of formulation, and 
are therefore formulated in known manner to emulsifiable concentrates, 
coatable pastes, directly sprayable or dilutable solutions, dilute 
emulsions, wettable powders, soluble powders, dusts, granulates, and also 
encapsulations, for example, in polymer substances. Like the nature of the 
compositions, the methods of application, such as spraying, atomizing, 
dusting, scattering or pouring, are chosen in accordance with the intended 
objectives and the prevailing circumstances. Advantageous rates of 
application are normally from 50 g to 5 kg of active ingredient (a.i.) per 
hectare ("ha", approximately 2.471 acres), preferably from 100 g to 2 kg 
a.i./ha, most preferably from 200 g to 500 g a.i./ha. 
The formulations, compositions or preparations containing the active 
ingredients and, where appropriate, a solid or liquid adjuvant, are 
prepared in known manner, for example by homogeneously mixing and/or 
grinding the active ingredients with extenders, for example solvents, 
solid carriers and, where appropriate, surface-active compounds 
(surfactants). 
Suitable solvents include aromatic hydrocarbons, preferably the fractions 
having 8 to 12 carbon atoms, for example, xylene mixtures or substituted 
naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, 
aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and 
glycols and their ethers and esters, such as ethanol, ethylene glycol 
monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly 
polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or 
dimethyl formamide, as well as epoxidized vegetable oils such as 
epoxidized coconut oil or soybean oil; or water. 
The solid carriers used e.g. for dusts and dispersible powders, are 
normally natural mineral fillers such as calcite, talcum, kaolin, 
montmorillonite or attapulgite. In order to improve the physical 
properties it is also possible to add highly dispersed silicic acid or 
highly dispersed absorbent polymers. Suitable granulated adsorptive 
carriers are porous types, for example pumice, broken brick, sepiolite or 
bentonite; and suitable nonsorbent carriers are materials such as calcite 
or sand. In addition, a great number of pregranulated materials of 
inorganic or organic nature can be used, e.g. especially dolomite or 
pulverized plant residues. 
Depending on the nature of the active ingredient to be used in the 
formulation, suitable surface-active compounds are nonionic, cationic 
and/or anionic surfactants having good emulsifying, dispersing and wetting 
properties. The term "surfactants" will also be understood as comprising 
mixtures of surfactants. 
Suitable anionic surfactants can be both water-soluble soaps and 
water-soluble synthetic surface-active compounds. 
Suitable soaps are the alkali metal salts, alkaline earth metal salts or 
unsubstituted or substituted ammonium salts of higher fatty acids (chains 
of 10 to 22 carbon atoms), for example the sodium or potassium salts of 
oleic or stearic acid, or of natural fatty acid mixtures which can be 
obtained for example from coconut oil or tallow oil. The fatty acid 
methyltaurin salts may also be used. 
More frequently, however, so-called synthetic surfactants are used, 
especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole 
derivatives or alkylarylsulfonates. 
The fatty sulfonates or sulfates are usually in the form of alkali metal 
salts, alkaline earth metal salts or unsubstituted or substituted 
ammoniums salts and have a 8 to 22 carbon alkyl radical which also 
includes the alkyl moiety of alkyl radicals, for example, the sodium or 
calcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixture of 
fatty alcohol sulfates obtained from natural fatty acids. These compounds 
also comprise the salts of sulfuric acid esters and sulfonic acids of 
fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole 
derivatives preferably contain 2 sulfonic acid groups and one fatty acid 
radical containing 8 to 22 carbon atoms. Examples of alkylarylsulfonates 
are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic 
acid, dibutylnapthalenesulfonic acid, or of a naphthalenesulfonic 
acid/formaldehyde condensation product. Also suitable are corresponding 
phosphates, e.g. salts of the phosphoric acid ester of an adduct of 
p-nonylphenol with 4 to 14 moles of ethylene oxide. 
Non-ionic surfactants are preferably polyglycol ether derivatives of 
aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty 
acids and alkylphenols, said derivatives containing 3 to 30 glycol ether 
groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 
6 to 18 carbon atoms in the alkyl moiety of the alkylphenols. 
Further suitable non-ionic surfactants are the water-soluble adducts of 
polyethylene oxide with polypropylene glycol, ethylenediamine propylene 
glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in 
the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether 
groups and 10 to 100 propylene glycol ether groups. These compounds 
usually contain 1 to 5 ethylene glycol units per propylene glycol unit. 
Representative examples of non-ionic surfactants re 
nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, 
polypropylene/polyethylene oxide adducts, 
tributylphenoxypolyethoxyethanol, polyethylene glycol and 
octylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan 
and polyoxyethylene sorbitan trioleate are also suitable non-ionic 
surfactants. 
Cationic surfactants are preferably quaternary ammonium salts which have, 
as N-substituent, at least one C.sub.8 -C.sub.22 alkyl radical and, as 
further substituents, lower unsubstituted or halogenated alkyl, benzyl or 
lower hydroxyalkyl radicals. The salts are preferably in the form of 
halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium 
chloride or benzyldi(2-chloroethyl)ethylammonium bromide. 
The surfactants customarily employed in the art of formulation are 
described, for example, in "McCutcheon's Detergents and Emulsifiers 
Annual," MC Publishing Corp. Ringwood, N.J., 1979, and Sisely and Wood, 
"Encyclopedia of Surface Active Agents," Chemical Publishing Co., Inc. New 
York, 1980. 
The agrochemical compositions usually contain from about 0.1 to about 99%, 
preferably about 0.1 to about 95%, and most preferably from about 3 to 
about 90% of the active ingredient, from about 1 to about 99.9%, 
preferably from about 1 to about 99%, and most preferably from about 5 to 
about 95% of a solid or liquid adjuvant, and from about 0 to about 25%, 
preferably about 0.1 to about 25%, and most preferably from about 0.1 to 
about 20% of a surfactant. 
Whereas commercial products are preferably formulated as concentrates, the 
end user will normally employ dilute formulations. 
EXAMPLES 
The following examples are intended to exemplify specific embodiments of 
the present invention without limiting the scope in any way. 
EXAMPLE 1 
Materials and Strains 
Sepharose-4B is purchased from Pharmacia LKB. Histidinol dihydrochloride is 
from Aldrich (Milwaukee, Wis.), and NAD+, sodium metaperiodate, 
epichlorohydrin and nitroblue tetrazolium are from Sigma (St. Louis, Mo.). 
Sodium cyanoborohydride is purchased from Nakarai, and phenazine 
methosulfate from Wako Pure Chemical Industries (Osaka, Japan). All other 
chemicals used are of analytical grade. 
Cabbage (Brassica oleracea), cucumber (Cucumis sativus), asparagus 
(Asparagus officinalis), eggplant (Solanum melongena), lettuce (Lactuca 
saliva), and pimento (Capsicum annum) are cultivated in a growth chamber 
with 16 h/8 h light/dark cycle at 25.degree. C. during illumination period 
and 15.degree. C. during dark period. The relative humidity is constant at 
80%. Cell cultures of Rosa "Paul's Scarlet" are a gift of Andre Strauss, 
CIBA-GEIGY Ltd., Switzerland, and cultured in suspension as previously 
described [Strauss et al, Planta 163:554-562 (1985)]. Cell cultures of 
tobacco (Nicotiana tabacum L. cv Samsun NN) and suspension cultures of 
wheat (Triticum aestivum var Chinese Spring) are kindly provided by 
Yaguyuki Yamanda, Kyoto University, Japan, and grown as described 
elsewhere [Kumpaisal et al., Plant Physiol. 85:145-151 (1987); and Yamada 
and Sato, Plant & Cell Physiol. 19:691-699 (1978)]. Mature spring cabbage 
(Brassica oleracea L. var capitata L.) is purchased from a local grocer. 
Wheat germ is obtained from Sigma (St. Louis, Mo.). 
EXAMPLE 2 
Preparation of Enzyme Extract 
Two week old plants, whole cabbage heads or well grown cell tissues are 
used as the enzyme sources. The plant material is homogenized with a 
Polytron blender in cold 100 mM sodium phosphate buffer, pH7.2 (buffer A), 
and the homogenate passed through a filtration cloth. After centrifugation 
at 30,000.times.g for 20 min, the supernatant is fractionated with 45-60% 
saturation of ammonium sulfate on ice. The precipitate is collected by 
centrifugation, dissolved in buffer A and the solution desalted on 
Sephadex G-25. This protein enriched extract is used for the determination 
of histidinol dehydrogenase activity in various plants. 
EXAMPLE 3 
Purification of Histidinol Dehydrogenase 
Spring cabbage heads are homogenized using a kitchen mixer and suspension 
processed as described above, except that desalting is performed by 
extensively dialyzing against 50 mM Tris/HCl buffer, pH7.4 (buffer B). All 
subsequent procedures are carried out at 4.degree. C. 
Dialyzed protein extract is applied to a DEAE-Toyopearl column equilibrated 
with buffer B. After washing the column with buffer B, protein is eluted 
with a linear gradient of buffer B containing sodium chloride (0-500 mM). 
The protein fraction with activity is eluted from the DEAE-Toyopearl 
column before the main protein peak, at a concentration of 150 mM sodium 
chloride in the buffer. 
Fractions containing enzyme activity are pooled and directly applied to a 
histidinol-Sepharose-4B column (2.5.times.8 cm) (EXAMPLE 7) at a flow rate 
of 36 ml/h. Unabsorbed protein is eluted by extensively washing with 10 mM 
Tris/HCl buffer, pH 7.3 containing 140 mM NaCl (buffer C). Bound 
histidinol dehydrogenase is eluted with 50 mM Tris/HC1, pH 7.3 containing 
imidazole. 
Several unsuccessful attempts were made to purify histidinol dehydrogenase 
from mature spring cabbage heads by conventional purification methods. The 
development of an affinity gel, highly specific for this enzyme finally 
made it possible to purify the protein in three steps with a high yield to 
apparent homogeneity (Table I). All activity is bound to the gel, probably 
very tightly, since only 40% of the activity could be eluted specifically 
with a one step addition of high concentration of imidazole (700 mM) in 
the elution buffer (FIG. 1). By this step alone, the specific enzyme 
activity is increased by a factor of 350 to 10.16 U/mg of protein, showing 
this chromatography to be very specific for histidinol dehydrogenase. The 
over-all 2116-fold purification does not include the first step of 
fractionated ammonium sulfate precipitation, since no activity could be 
detected in crude extract. A heavily loaded SDS polyacrylamid gel showed 
one major band. 
TABLE I 
______________________________________ 
Purification of histidinol dehydrogenase. In this typical purifi- 
cation, 10 kg of cabbage heads are processed. All activities are 
measured after desalting with either Sephadex G-25 or dialysis. 
The activity in the crude extract could not be detected (n.d.). 
Total Total 
pro- acti- Specific 
tein vity Recovery 
activity 
Purity- 
Purification step 
mg U % U/mg fold 
______________________________________ 
Crude extract 
n.d. n.d. n.d. n.d. n.d. 
Ammonium 4750 23 100 0.0048 1 
sulfate 
fractionation 
DEAE-Toyo- 
560 20.1 87 0.029 6 
pearl ion- 
exchange 
chromato- 
graphy 
Histidinol- 
0.75 7.6 33 10.16 2116 
Sepharose-4B 
affinity chrom- 
atography 
______________________________________ 
After desalting all fractions on Sephadex G-25, the enzyme activity in each 
fraction is determined. Active fractions are pooled and concentrated on an 
Amicon ultrafiltration membrane (YM-10). This enzyme preparation is stored 
at -80.degree. C. and used for all experiments, except for the activity 
screening in plants. 
Storage of the enzyme for more than 4 months at -80.degree. C. did not 
change significantly the activity. At 4.degree. C., the enzyme lost 10% of 
its activity during one week. By the addition of glycerol to a final 
concentration of 20%, full activity could be maintained at 40.degree. C. 
for more than one week. 
EXAMPLE 4 
Gel Filtration 
A Sepharose 12 column (Pharmacia LKB, Piscataway, N.J.) is equilibrated 
with purification buffer C at room temperature. Samples of purified enzyme 
are applied to the column and eluted with buffer C at a flow rate of 0.5 
ml/min. The molecular mass is calculated using a molecular weight marker 
kit (Sigma, St. Louis, Mo.). The native molecular weight of the enzyme is 
determined by Superose 12 gel filtration to be 103,000. 
EXAMPLE 5 
Polyacrylamide Gel Electrophoresis 
SDS-polyacrylamide gel electrophoresis is performed as described elsewhere 
[Laemmli, Nature 227:680-685 (1970)], using 10-20% acrylamide. Proteins in 
sample buffer are put in a heating block at 100.degree. C. for 10 min. For 
the calculation of the molecular mass of denatured and reduced protein, an 
electrophoresis calibration kit for molecular weight determination 
(Pharmacia, Piscataway, N.J.) is used. Under denaturing and reducing 
conditions, the enzyme migrated as a single band of 52 kdaltons if exposed 
to SDS-polyacrylamide gel electrophoresis, suggesting a dimeric quaternary 
structure of the enzyme. 
The molecular weight of 103,000 of native cabbage enzyme is considerably 
higher than that of the enzyme from S. typhimurium (82,000 [Burger et al., 
Biochem. J. 181:771-774 (1979)] and 90,000 [Eccleston et al., J. Biol. 
Chem. 254:11399-11404 (1979)]). The enzymes from cabbage and S. 
typhimurium are each composed of two subunits with a similar molecular 
weight of 52,000 and 43,000 [Burger et al., Biochem. J. 181:771-774 
(1979)], respectively. However, the subunit molecular weight of the 
trifunctional enzyme from S. cerevisiae is calculated from the nucleotide 
sequence to be 87,935 [Donahue et al., Gene 18:47-59 (1982)] and estimated 
on SDS gels as about 95,000 [Keesey et al., J. Biol. Chem. 254:7427-7433 
(1979)]. According to these data, the enzymes from yeast, S. typhimurium 
and cabbage differ significantly in their molecular weights. 
EXAMPLE 6 
Isoelectric Focusing 
PhastGel (Pharmacia LKB, Piscataway, N.J.) with a ph-range of 4-6.5 is used 
for isoelectric focusing, performed on a Phast System (Pharmacia LKB, 
Piscataway, N.J.) according to the instruction manual of the supplier. The 
isoelectric point of the protein is determined using an isoelectric point 
calibration kit (Pharmacia LKB, Piscataway, N.J.). 
The exposure of purified enzyme to analytical isoelectric focusing lead to 
its separation into six protein bands within the ph-range 5.1-5.4. Five 
bands showed histidinol dehydrogenase activity by employing activity 
staining. The sixth band had only very slight or no activity. In control 
experiments lacking either histidinol or NAD+in the reaction mixture, 
.rho.-nitro blue tetrazolium is not reduced to formazan. The activity of 
each of the five bands correlated with their colour intensities obtained 
by protein staining. 
To rule out artifacts of the purification procedure, several independent 
purifications are carried out, starting from cabbage heads in different 
growth stages. In all cases, six protein bands are obtained. 
EXAMPLE 7 
Preparation of Histidinol-Sepharose-4B Gel 
Sepharose-4B is activated with epichlorohydrin as previously described 
(Matsumoto et al., J. Biochem. 85:1091-1098 (1979)]. Epoxyactivated 
Sepharose-4B gel is suspended in 0.1N NAOH containing 0.5 g glucose per g 
of gel and incubated at 40.degree. C. on a shaker for 24 h [Kanamori et 
al., J. Chromatog. 363:231-242 (1986). After washing extensively with 
water, the gel is suspended in prechilled sodium metaperiodate and the 
suspension shaken for 1 h at 4.degree. C. The gel is washed with water, 
suspended in 0.1M HCl and incubated at room temperature for 1 h. Again, 
the gel is washed extensively with water, followed by thorough washing 
with 10 mM sodium phosphate buffer, pH 7.0 at room temperature. 
The formyl carrier gel is suspended in 10 mM sodium phosphate buffer, pH 
7.0 containing 100 mM of histidinol dihydrochloride, which is previously 
neutralized with NAOH, and the suspension incubated at 4.degree. C. 
overnight. Five milligrams of sodium cyanoborohydride per g of gel is then 
added to the suspension and incubated with shaking at room temperature for 
6 h. The gel is extensively washed with distilled water and treated with 5 
mg sodium borohydride per g or gel for 3 h at 4.degree. C. to convert the 
remaining formyl groups into hydroxymethyl groups. 
EXAMPLE 8 
Enzyme Assay 
The enzyme activity is assayed spectrophotometrically by measuring the 
increase in absorbance at 340 nm due to the reduction of NAD+in a Hitachi 
U-3120 spectrophotometer. The reaction mixture contained 150 mM Gly/NaOH 
buffer, pH9.2, 0.5 mM, MnCl.sub.2, 2 mM NAD +, 5 mM histidinol, and 2-5 mU 
of enzyme sample in total volume of 0.5 ml. The reaction is started with 
the addition of histidinol, and incubated at 30.degree. C. A reference 
sample containing water instead of histidinol is always run. One unit of 
enzyme catalyzing the formation of 2 .mu.mol of NADH per min under the 
assay conditions. 
In some experiments the activity is also followed by measuring the 
formation of His with a Hitachi L-850 amino acid analyzer. The enzyme 
reaction is terminated with formic acid (final concentration, 30%). The 
sample is then evaporated, and the precipitate dissolved in 0.2N HCl. This 
preparation is used for His analysis. 
Distribution of Histidinol Dehydrogenase in eleven plant species or 
preparations are examined for histidinol dehydrogenase activity. Enzyme 
activity could not be detected in any crude extract, but is found in 
enriched extracts from various monocotyledon and dicotyledon species 
(Table I). Cultured rose cells and spring cabbage showed the highest 
specific enzyme activities. Wheat germ had an extremely high extractable 
activity probably due to its low content of water, however, its specific 
activity is modest. Also shoots of cabbage and cucumber, and spring 
cabbage heads contained a high extractable activity. No activity could be 
detected in 2 week old pimento shoots and cultured tobacco cells. 
Histidinol dehydrogenase in microorganisms catalyzes the two last reactions 
to form the endproduct His. To verify this catalytic role for the isolated 
protein from cabbage, its catalyzed reaction is analyzed stoichimetrically 
with regard to His formation and NAD+reduction. The amount of 
enzymatically produced NADH is determined from the absorbence at 340 nm, 
using the absorbence coefficient of 6220. The other formed product is 
identified by amino acid analysis as His. Authentic His added to reacted 
sample is co-eluted with enzymatically formed His. Neither His nor NADH 
are formed in the absence of NAD+, histidinol, or by using cooked enzyme. 
Two moles of NADH are formed for 0.99 mole of His after a reaction time of 
2 min. (Table III). The formation of both products histidine and NADH is 
time dependent and correlated. 
Optimum pH for the activity is at 9.2 in 50 mM Gly/NaOH buffer (FIG. 2). At 
pH values below 9, activity decreased in either Gly/NaOH buffer or in 50 
mM HEPES buffer. At pH9.2-10, almost full activity is maintained, but 
above 10, a rapid loss of activity occurred. 
The apparent K.sub.m values of the enzyme for L-histidinol and NAD+are 
determined under standard assay conditions such as 15.5 and 42 .mu.M, 
respectively. 
The influence of divalent metal ions on the enzyme reaction is shown in 
Table IV. The reaction is stimulated 26% by the addition of Mn.sup.2+ 
compared to control conditions without the addition of any divalent metal 
ion to the reaction buffer (150 mM Gly/NaOH, pH9.2). The addition of 
Ba.sup.2+, Mg.sup.2+, Ni.sup.2+, C.sup.2+, Zn.sup.2+ and Cu.sup.2+ 
caused inhibition of the reaction. 
Histidinol dehydrogenase from cabbage is the first His biosynthesis enzyme 
that has been purified to homogeneity from higher plants. By using a new 
affinity chromatography, the purification of this enzyme and its 
investigation became possible. Direct evidence that the purified protein 
is histidinol dehydrogenase is obtained from activity staining of the 
protein on the gel after isoelectic focusing. The enzymatically formed 
product starting from histidinol is identified as His. His and NADH are 
produced stoichiometrically, showing the same reduction of 2 mole of 
NAD+for 1 mole of formed His as previously described for this reaction in 
microorganisms [Adams, J. Biol. Chem. 217:325-344 (1955)]. The present 
results demonstrate that the last two reaction steps in the His 
biosynthetic pathway in cabbage are identical to those in microorganisms 
and are catalyzed by the same single enzyme. The K.sub.m of this enzyme 
for L-histidinol (15.5 .mu.m at pH 9.2) is very similar to those (16 and 
8.8 .mu.m) of the pure enzyme from S. typhimurium [Burger et al., Eur. J. 
Biochem. 116:137-142 (1981)] and the partially purified one from wheat 
germ (Wong and Mazelis, Phytochem. 20:1831-1834 (1981)]. The K.sub.m (42 
.mu.M) for NAD+is considerably lower than that of the enzymes from S. 
typhimurium (1 mM) (24) and from wheat germ (140 .mu.M, using partially 
purified enzyme (Wong and Mazelis, Phytochem. 20:1831-1834 (1981)]). The 
ph-optimum of the reaction between pH 9.2-9.4 is identical to that of 
histidinol dehydrogenase from other organisms. No other divalent metal ion 
than Mn.sup.2 +showed a stimulation of the enzyme activity. This is in 
line with the results obtained with the enzyme from S. typhimurium, whose 
catalyzed reaction is also stimulated in the presence of Mn.sup.2 
+[Grubmeyer et al., Arch. Biochem. Biophys. 272:311-317 (1989)]. 
TABLE II 
______________________________________ 
Distribution of histidinol dehydrogenase in different plant 
species and preparations. For the calculation of the 
extractable activity, the wet weight of the plant material is 
used. The weight of cells from suspension tissue cultures is 
determined after collection by filtration with a cloth. *) 
mU/mg: units of activity per mg. of protein; **) mU/g: units 
of activity per g of plant material. 
Specific Extractable 
Activity Activity 
Plant source (mU/mg*) (mU/mg**) 
______________________________________ 
Cabbage shoots (Brassica 
1.6 3.3 
oleracea L. var capitata L.) 
Aparagus shoots (Asparagus 
0.9 1.6 
officinalis) 
Lettuce shoots (Lactuca sativa) 
0.4 0.3 
Egg plants shoots (Solanum 
0.4 0.9 
melonena) 
Cucumber shoots (Cucumis sativus) 
0.3 3.3 
Pimento shoots (Capsicum annum) 
0 0 
Spring cabbage (Brassica oleracea 
6.1 2.8 
L. car capitata L.) 
Wheat germ 2.1 91 
Wheat cell culture (Triticum 
0.5 0.4 
aestivum var Chinese Spring) 
Rose cell culture (Rosa "Paul's 
6.2 0.9 
Scarlet") 
Tobacco cell culture (Nicotiana 
0 0 
tabacum L. cv Samsun (NN) 
______________________________________ 
TABLE III 
______________________________________ 
Stoichiometry of histidinol dehydrogenase reaction with regard 
to the formation of His and NAD.sup.+. The enzyme reaction is 
stopped after 2, 5 and 30 min reaction time and the sample 
analyzed by automated amino acid analysis. Histidinol does not 
react with ninhydrin and cannot be analyzed by amino acid 
analysis (at 50 nmole). 
Reaction time 
His concentration 
NADA concentration 
min .mu.mole/ml (a) 
.mu.mole/ml (b) 
b/a 
______________________________________ 
0 0 0 -- 
2 17.6 35.6 2.02 
5 36.6 80.0 2.18 
30 143.7 316.0 2.2 
______________________________________ 
TABLE IV 
______________________________________ 
Effect of divalent metal ions on histidinol dehydrogenase 
activity. The concentration of metal ion is 0.5 mM. The 
activity is determined after 3 min reaction time, while the 
velocity is still constant. 
Addition Relative Activity % 
______________________________________ 
None 100 
Mn.sup.2+ 126 
Ba.sup.2+ 65 
Mg.sup.2+ 54 
Ni.sup.2+ 46 
Ca.sup.2+ 36 
Zn.sup.2+ 34 
Cu.sup.2+ 24 
______________________________________ 
EXAMPLE 9 
Activity Staining 
Isoelectric point calibration proteins and purified enzyme (in 
quadruplicate) are applied on a PhastGel (pH-range 4-6.5) to give five 
lanes. After running the isoelectric focusing, the gel is cut into four 
pieces. The one containing marker proteins in addition to enzyme is 
stained with Coomassie Brilliant Blue R250. Another piece of gel 
containing only enzyme is subjected to activity staining as described 
elsewhere [Skyring et al., Anal. Biochem., 36:511-520 (1970)], with a 
slight modification. The gel is immersed in 10 ml of reaction mixture 
containing 5 mM histidinol and 2 mM NAD+. By the addition of 150 .mu.l of 
nitroblue tetrazolium and 150 .mu.l of phenazine methosulfate to give a 
final concentration of 0.5 mM and 0.13 mM, respectively, the staining 
process is started. Incubation is carried out at 30.degree. C. for 30 min. 
Two other pieces of gel containing only enzyme are identically incubated, 
but either without histidinol or without NAD.sup.+. 
EXAMPLE 10 
Protein Determination 
The concentration of the protein is determined by Bradford protein assay 
method using bovine serum albumin as a standard [Bradford, Anal. Biochem. 
72:248-254 (1976)]. 
EXAMPLE 11 
Amino Acid Sequencing 
Approximately 50 .mu.g purified histidinol dehydrogenase is dried and 
resuspended in 0.1% trifluoroacetic acid and is subjected directly to 
automated Edman degradation with an Applied Biosystems 477A 
gas-liquid-phase protein sequencer (Strickler et al., Anal. Biochem. 
140:553-566 (1984)]. The phenylthiohydantion (PTH) amino acid derivatives 
are separated and identified with an on-line PTH analyzer (Applied 
Biosystems, Foster City, Calif.) with PTH-Cl.sub.8 column. 
The first sequence run showed a recovery of 15% of the protein 
concentration determined by amino acid analysis, indicating that 85% of 
the N-terminus is blocked for unknown reason. The first N-terminal amino 
acid of the 15% of unblocked enzyme could not be determined unambiguously 
in both experiments. Ala and Lys are found most abundantly, but also Ser, 
Gly, Glu, Val and Leu are detected, all in similar concentrations. 
The remainder of the N-terminal amino acid sequence is determined to be: 
EQU M K I Y R L S E L S F ? Q V E N L K A R ?? I D 
where question marks indicate residues that could not be determined from 
the amino acid sequencing data. 
A further 160 ug of purified histidinol dehydrogenase protein is dried and 
resuspended in 415 ul of 20 mM Tris-HCl, pH 8.0. Lysyl endopeptidase [EC 
3.4.21.50; Wako Chemicals, Osaka, Japan) is added to achieve a final 
concentration of approximately 33 ug/ml. This is equivalent to a weight 
ratio of approximately 1:500, peptidase:protein substrate. The digestion 
is incubated at room temperature for 24 hours, and the resulting peptides 
are separated by reverse phase HPLC on an Aquapore RP-300 (C-8) column 
(2.1.times.220 mM). The column is eluted with a 45 minute linear gradient 
of 0-70% acetonitrile in 0.1% TFA at a flow rate of 220 ul/minute. 
Peptides eluting at 17, 18, 32, 45, and 48 minutes are collected and 
analyzed by automated Edman degradation as described above. The following 
amino acid sequences are obtained: 
______________________________________ 
Lys-C #17 
TELSFAK 
Lys-C #18 
TVVLATPPTK 
Lys-C #32 
EAFDVAYDNIYAFHLAQK 
Lys-C #45 
KFMTVQSLTEEGLRNLGPYVA 
TMAEIEGLDA 
Lys-C #48 
ALSHSFTVFARDMIEAITFSN 
LYAPEK 
______________________________________ 
A further 90 ug of purified histidinol dehydrogenase protein is dried and 
resuspended in 200 ul of 2% CNBR in 70% formic acid. The protein is 
digested at room temperature for 24 hours. The resulting peptides are 
purified exactly as described above, except that a 0-100% gradient of 
acetonitrle is used. Peptides eluting at 6 and 18 minutes are collected 
and subjected to amino acid sequencing by automated Edman degradation as 
described above. The following sequences are obtained: 
______________________________________ 
CNBr #6 MAEIEGLDAHKRAVTLR?KDIE 
CNBr #18 
LAIPAQIAGRKTVVLATP 
______________________________________ 
EXAMPLE 12 
Obtaining the cDNA Sequence for Histidinol Dehydrogenase 
Three oligonucleotides that correspond to portions of the above amino acid 
sequences are synthesized by automated phosphoramidite synthesis on an 
Applied Biosystems (Foster City, Calif.) Model 380B DNA Synthesizer. 
Their sequences and the amino acid residues they correspond to are 
indicated below: 
______________________________________ 
EW25 5' TTAAGAATTCTAYGAYAAYATHTAYGC 3' 
JR03 5' CCYTCDATYTCNGCCAT 3' 
JR02 5' ATNGCYTCDATCATRTC 3' 
______________________________________ 
where D indicates any the bases A, T, or G; H indicates the bases A, T, or 
C; R indicates the purines A or G; Y indicates the pyrimidines T or C. 
EW25 is a mixture of 48 oligonucleotides comprising all possible DNA 
sequences coding for the amino acid sequence Y D N I Y A, found within 
peptide Lys-C #32. JR03 is a mixture of 48 oligonucleotides comprising the 
complements of all possible DNA sequences coding for the amino acid 
sequence M A E I E G, found within both peptides Lys-C #45 and CNBR #6. 
JR02 is a mixture of 48 oligonucleotides comprising the complementary 
strands of all possible DNA sequences coding for the amino acid sequence D 
M I E A I, found within peptide Lys-C #48. 
The sequences for the cabbage peptides are aligned with the translated 
sequence for the yeast HIS4C gene [Donahue et al., Gene 18:47-59 (1982)], 
which encodes histidinol dehydrogenase. The cabbage peptides are found to 
have approximately 45% identity to the yeast sequence in their shared 
regions. Using such an alignment, and assuming that the cabbage sequence 
is approximately co-linear with the yeast sequence, Lys-C #32 is found to 
lie upstream (i.e. toward the N-terminus of the protein) relative to Lys-C 
#45 and CNBR #6, which lie near the C-terminus of the protein 
approximately 320 amino acids downstream. Lys-C #48 is found to lie 
between these peptides. Thus, EW25 should be relatively near the 5' end of 
the cDNA, JR03 should be near the 3' end of the cDNA, and JR02 should lie 
between these oligonuceotides. 
Total RNA is prepared from frozen cabbage leaf tissue by phenol extraction 
followed by lithium chloride precipitation as described by Lagrimini et 
al. [Proc. Natl. Acad. Sci. USA 84, 7542-7546 (1987)]. Poly(A)+MRNA is 
isolated from the total RNA using a poly(A) quick MRNA isolation kit 
(Stratagene, La Jolla, Calif.) exactly as described in the manufacturer's 
instructions. The MRNA is enzymatically converted into cDNA and cloned in 
the lambda ZAP II vector using a ZAP-cDNA gigapack II gold synthesis kit 
(Stratagene, La Jolla, Calif.) using instructions supplied with the kit by 
the manufacturer. The resulting cDNA library is propagated and amplified 
as described by the manufacturer of the kit. 
Total DNA is prepared from the library using the Lambdasorb reagent 
(Promega Biotech, Madison, Wis.) by the method described by the 
manufacturer. A polymerase chain reaction [PCR; Saiki et al., Science 
239:487-491 (1988)] is performed using the library DNA as template, and 
EW25 and JR03 as primers. The reaction is performed in a total volume of 
50 ul, with 10 ug of template DNA, 200 pmole of each primer mixture, 100 
uM each of DATP, DCTP, DGTP, and DTTP, 1x PCR buffer supplied by the 
manufacturer (Perkin Elmer Cetus, Norwalk, Conn.) and 2.5u AmpliTaq DNA 
polymerase (Perkin Elmer Cetus). The reaction is cycled through the 
following temperature profile 40 times, in a DNA Thermocycler (Perkin 
Elmer Cetus): 94.degree. C. for 45 seconds, 42.degree. C. for 45 seconds, 
and 72.degree. C. for 45 seconds. At each cycle, the 72.degree. C. step is 
increased by 2 seconds. 
The PCR products are separated on a 2% low-gelling-temperature agarose gel 
(Nu-Sieve, FMC BioProducts, Rockland, Me.). Several fragments are detected 
by ethidium bromide stain, one of which is approximately 975 bp in length, 
the approximate size expected. The gel is blotted to nylon membrane 
(GeneScreen Plus, NEN Research Products, Boston, Mass.) in 0.4 M NAOH as 
described by Reed and Mann (Nucleic Acids Res. 13:7207-7221 (1985)]. The 
gel blot is hybridized to the JR02 oligonucleotide mixture which has been 
.sup.32 P-labeled using T4 polynucleotide kinase (New England Biolabs, 
Beverly, Mass.) and gamma- 32 P ATP (New England Nuclear, Boston, Mass.). 
The approximately 975 bp fragment is specifically detected by the JR02 
probe, strongly indicating that it comprises a portion of the cDNA for 
histidinol dehydrogenase. 
The PCR fragment is excised from a low-gelling-temperature agarose gel, 
digested with EcoRI, and subcloned into the pbluescript plasmid 
(Stratagene, La Jolla, Calif.). The subcloned PCR insert is labeled with 
.sup.32 P by random priming using the PrimeTime kit (International 
Biotechnologies, New Haven, Conn.), and used to probe plaque lifts of the 
lambda ZAP II cabbage cDNA library described above. Positively-hybridizing 
plaques are purified and their inserts excised into the pbluescript 
plasmid in vivo using the method described by the manufacturer of the 
lambda ZAP II cloning vector (Stratagene, La Jolla, Calif.). The DNA 
sequences of the plasmid cDNA inserts are determined by the dideoxy method 
using the Sequenase kit (United States Biochemical, Cleveland, OH). One 
plasmid subclone, designated pBSACabHdH7, is found to contain a 1613 bp 
insert that encodes a predicted protein that matches the sequences of the 
peptides derived from histidinol dehydrogenase, including the N-terminal 
peptide, indicating a full-length, or nearly full-length cDNA (see, FIG. 3 
for the DNA sequence). 
EXAMPLE 13 
Transformation of Maize by Microprojectile Bombardment of Friable, 
Embryogenic Culture of the Elite Genotype 0274 with the Genes for 
Histidinol Dehydrogenase, a Scorable Marker and a Selectable Marker 
The target is a friable, embryogenic culture of the Funk genotype 0274 
(CIBA-GEIGY Seeds, Greensboro, NC). Each target contained approximately 
250 milligrams of this culture plated onto Durapore filters, which are 
placed on top of the medium designated '2N63S'[N6 media of Chu et al., 
Scientia Sinica, Vol. XVIII(NO. 5):659-668 (1975) supplemented with 3 
percent sucrose and 2 milligrams per liter of 2,4-dichlorophenoxyacetic 
acid). A 2X gene dose of GUS gene `equivalents` of the plasmide pCHN1761 
(as described in copending U.S. patent Ser. No. 573,105, filed Aug. 24, 
1990, wherein the histidinol dehydrogenase gene is placed into the vector; 
or the histidinol dehydrogenase clones contained therein) and plasmids 
containing selectable markers such as phosphinothricin resistance, 
hygromycin resistance, paromomycin resistance or scorable markers such as 
glucouronidase (GUS) or luciferase are co-precipitated onto tungsten 
particles according to the CaCl.sub.2 - Spermidine precipitation method of 
Klein et al. (Proc. Nat. Acad. Sci. USA, 85:4305, 1988). Five microliters 
of the DNA-tungsten preparation is pipetted onto the macroprojectile and 
propelled toward the target using a number 1 gray cartridge essentially as 
described in the operating instructions for the bombardment device. 
After 5 days, the cells are transferred to fresh filters and placed onto 
media containing selection agents at a concentration of 1 to 500 
milligrams of per liter, depending on the agent used. The filters with the 
cells are transferred approximately weekly to fresh medium containing the 
selection agent. The concentration of the agent may be increased during 
the course of selection. After 6 weeks of selection, colonies appeared 
randomly on the filters. Colonies are isolated over the course of three 
weeks, each one being transferred to fresh medium containing the selection 
agent but without a filter for a secondary selection period.