Use of anthocyanin genes to maintain male sterile plants

This invention is directed to a method to maintain male sterility in corn by the genetic linkage via genetic engineering of a homozygous male-fertility gene with a color linked restorer and plant comprising said genes. Color genes disclosed as useful in this invention include those involved in anthocyanin biosynthesis, which in corn are under the control of as many as 20 or more genes. In particular, the use of the anthocyanin color genes R, B and C1, and specific regulatory elements sequences are taught.

BACKGROUND OF THE INVENTION 
In many, if not most plant species, the development of hybrid cultivars is 
highly desired because of their generally increased productivity due to 
heterosis: the superiority of performance of hybrid individuals compared 
with their parents (see e.g., Fehr, 1987, Principles of cultivar 
development, Vol. 1: Theory and Technique, MacMillan Publishing Co., New 
York; Allard, 1960, Principles of Plant Breeding, John Wiley and Sons, 
Inc.). 
The development of hybrid cultivars of various plant species depends upon 
the capability of achieving essentially almost complete cross-pollination 
between parents. This is most simply achieved by rendering one of the 
parent lines male sterile (i.e. bringing them in a condition so that 
pollen is absent or nonfunctional) either manually, by removing the 
anthers, or genetically by using, in the one parent, cytoplasmic or 
nuclear genes that prevent anther and/or pollen development (for a review 
of the genetics of male sterility in plants see Kaul, 1988, `Male 
Sterility in Higher Plants`, Springer Verlag). 
For hybrid plants where the seed is the harvested product (e.g. corn, 
oilseed rape) it is in most cases also necessary to ensure that fertility 
of the hybrid plants is fully restored. In systems in which the male 
sterility is under genetic control this requires the existence and use of 
genes that can restore male fertility. The development of hybrid cultivars 
is mainly dependent on the availability of suitable and effective 
sterility and restorer genes. 
Endogenous nuclear loci are known for most plant species that may contain 
genotypes which effect male sterility, and generally, such loci need to be 
homozygous for particular recessive alleles in order to result in a 
male-sterile phenotype. The presence of a dominant `male fertile` allele 
at such loci results in male fertility. 
Recently it has been shown that male sterility can be induced in a plant by 
providing the genome of the plant with a chimeric male-sterility gene 
comprising a DNA sequence (or male-sterility DNA) coding, for example, for 
a cytotoxic product (such as an RNase) and under the control of a promoter 
which is predominantly active in selected tissue of the male reproductive 
organs. In this regard stamen-specific promoters, such as the promoter of 
the TA29 gene of Nicotiana tabacum, have been shown to be particularly 
useful for this purpose (Mariani et al., 1990, Nature 347:737, European 
patent publication ("EP") 0,344,029). By providing the nuclear genome of 
the plant with such a male-sterility gene, an artificial male-sterility 
locus is created containing the artificial male-sterility genotype that 
results in a male-sterile plant. 
In addition it has been shown that male fertility can be restored to the 
plant with a chimeric fertility-restorer gene comprising another DNA 
sequence (or fertility-restorer DNA) that codes, for example, for a 
protein that inhibits the activity of the cytotoxic product or otherwise 
prevents the cytotoxic product from being active in the plant cells 
(European patent publication "EP" 0,412,911). For example the barnase gene 
of Bacillus amyloliquefaciens codes for an RNase, called barnase, which 
can be inhibited by a protein, barstar, that is encoded by the barstar 
gene of B. amylolicruefaciens. The barnase gene can be used for the 
construction of a sterility gene while the barstar gene can be used for 
the construction of a fertility-restorer gene. Experiments in different 
plant species, e.g. oilseed rape, have shown that a chimeric barstar gene 
can fully restore the male fertility of male sterile lines in which the 
male sterility was due to the presence of a chimeric barnase gene (EP 
0,412,911, Mariani et al., 1991, Proceedings of the CCIRC Rapeseed 
Congress, Jul. 9-11, 1991, Saskatoon, Saskatchewan, Canada; Mariani et 
al., 1992, Nature 357:384). By coupling a marker gene, such as a dominant 
herbicide resistance gene (for example the bar gene coding for 
phosphinothricin acetyl transferase (PAT) that converts the herbicidal 
phosphinothricin to a non-toxic compound De Block et al., 1987, EMBO J. 
6:2513!), to the chimeric male-sterility and/or fertility-restorer gene, 
breeding systems can be implemented to select for uniform populations of 
male sterile plants (EP 0,344,029; EP 0,412,911). 
The production of hybrid seed of any particular cultivar of a plant species 
requires the: 1) maintenance of small quantities of pure seed of each 
inbred parent, and 2) the preparation of larger quantities of seed of each 
inbred parent. Such larger quantities of seed would normally be obtained 
by several (usually two) seed multiplication rounds, starting from a small 
quantity of pure seed ("basic seed") and leading, in each multiplication 
round, to a larger quantity of pure seed of the inbred parent and then 
finally to a stock of seed of the inbred parent (the "parent seed" or 
"foundation seed") which is of sufficient quantity to be planted to 
produce the desired quantities of hybrid seed. Of course, in each seed 
multiplication round larger planting areas (fields) are required. 
In order to maintain and enlarge a small stock of seeds that can give rise 
to male-sterile plants it is necessary to cross the male sterile plants 
with normal pollen-producing parent plants. In the case in which the 
male-sterility is encoded in the nuclear genome, the offspring of such 
cross will in all cases be a mixture of male-sterile and male-fertile 
plants and the latter have to be removed from the former. With 
male-sterile plants containing an artificial male-sterility locus as 
described above, such removal can be facilitated by genetically linking 
the chimeric male sterility gene to a suitable marker gene, such as the 
bar gene, which allows the easy identification and removal of male-fertile 
plants (e.g. by spraying of an appropriate herbicide). 
However, even when suitable marker genes are linked to male-sterility 
genotypes, the maintenance of parent male-sterile plants still requires at 
each generation the removal from the field of a substantial number of 
plants. For instance in systems using a herbicide resistance gene (e.g. 
the bar gene) linked to a chimeric male-sterility gene, as outlined above, 
only half of the parent stock will result in male-sterile plants, thus 
requiring the removal of the male-fertile plants by herbicide spraying 
prior to flowering. In any given field, the removal of male-fertile plants 
effectively reduces the potential yield of hybrid seed or the potential 
yield of male-sterile plants during each round of seed multiplication for 
producing parent seed. In addition removal of the male-fertile plants may 
lead to irregular stands of the male-sterile plants. For these reasons 
removal of the male-fertile plants is economically unattractive for many 
important crop species such as corn and oilseed rape. 
Anthocyanins are pigments that are responsible for many of the red and blue 
colors in plants. The genetic basis of anthocyanin biosynthesis has been 
well characterized, particularly in corn, Petunia, and Antirrhinium 
(Dooner et al, 1991, Ann.Rev.Genet. 25:179-199; Jayaram and Peterson, 
1990, Plant Breeding Reviews 2:91-137; Coe, 1994, In `The Maize Handbook`, 
Freeling and Walbot, eds. Springer Verlag New York Inc., p. 279-281). In 
corn anthocyanin biosynthesis is apparently under control of 20 or more 
genes. The structural loci C2, Whp, A1, A2, Bz1, and Bz2 code for various 
enzymes involved in anthocyanin biosynthesis and at least 6 regulatory 
loci, acting upon the structural genes, have been identified in corn i.e. 
the R, B, C1, Pl, P and Vp1 loci. 
The R locus has turned out to be a gene family (in corn located on 
chromosome 10) comprising at least three different genes i.e. R (which 
itself may comprise duplicate genes organized in a tandem array), and the 
displaced duplicate genes R(Sn) and R(Lc). R typically conditions 
pigmentation of the aleurone but various alleles are known to confer 
distinct patterns of pigmentation. R(Lc) is associated with unique 
pigmentation of leaves and R(Sn) with unique pigmentation of the scutellar 
node. One state of R is associated with pigmentation of the whole plant 
(R(P)), while another is associated with pigmentation of the seeds (R(S)). 
Alleles of the unlinked B locus (in corn located on chromosome 2) rarely 
condition pigmentation of the aleurone, but are frequently associated with 
pigmentation of mature plant parts. The B-peru allele however, pigments 
the aleurone (like R(S)). Analysis at the molecular level has confirmed 
that the R and B loci are duplicate genes. 
In order that the R and B loci can color a particular tissue, the 
appropriate allele of C1 or Pl loci also needs to be present. The C1 and 
C1-S alleles, for instance, pigment the aleurone when combined with the 
suitable R or B allele. 
Alleles of the C1 locus have been cloned and sequenced. of particular 
interest are C1 (Paz-Ares et al, 1987, EMBO J. 6:3553-3558) and C1-S 
(Schleffer et al, 1994, Mol.Gen.Genet. 242:40-48). Analysis of the 
sequences revealed the presence of two introns in the coding region of the 
gene. The protein encoded by the C1 and C1-S alleles shares homology with 
mvb proto-oncogenes and is known to be a nuclear protein with DNA-binding 
capacity acting as transcriptional activators. 
The cDNA of the B-peru allele has also been analyzed and sequenced 
(Radicella et al, 1991, Plant Mol. Biol. 17:127-130). Genomic sequences of 
B-peru were also isolated and characterized based on the homology between 
R and B (Chandler et al., 1989, the Plant Cell 1:1175-1183; Radicella et 
al., 1992, Genes & Development 6:2152-2164). The tissue specificity of 
anthocyanin production of two different B alleles was shown to be due to 
differences in the promoter and untranslated leader sequences (Radicella 
et al, 1992, supra). 
Various alleles of the R gene family have also been characterized at the 
molecular level, e.g. Lc (Ludwig et al, 1989, PNAS 86:7092-7096), R-nj, 
responsible for pigmentation of the crown of the kernel (Dellaporta et al, 
1988, In "Chromosome Structure and Function,: Impact of New Concepts, 18th 
Stadeler Genetics Symposium, Gustafson and Appels, eds. (New York, Plenum 
press, pp. 263-282)), Sn (Consonni ei al, 1992, Nucl. Acids. Res. 20:373), 
and R(S) (Perrot and Cone, 1989, Nucl. Acids. Res. 17:8003). 
The proteins encoded by the B and R genes share homology with mVc 
proto-oncogenes and have characteristics of transcriptional activators. 
It has been shown that various structural and regulatory genes introduced 
in maize tissues by microprojectiles operate in a manner similar to the 
endogenous loci and can complement genotypes which are deficient in the 
introduced genes (Klein et al., 1989, PNAS 86:6681-6685; Goff et al., 
1990, EMBO J. 9:2517-2522). The Lc gene was also used as a visible marker 
for plant transformation (Ludwig et al., 1990, Science 247:449-450). Apart 
from the above other genes involved in anthocyanin biosynthesis have been 
cloned (Cone, 1994, In `The Maize Handbook`, Freeling and Walbot eds., 
Springer Verlag New York Inc., p. 282-285). 
In Barley, Falk et al (1981, In Barley Genetics IV, proceedings of the 4th 
International Barley Genetics symposium, Edinburgh University press, 
Edinburgh, pp. 778-785) have reported the coupling of a male-sterile gene 
to a xenia-expressing shrunken endosperm gene which makes it possible to 
select seeds, before planting, that will produce male-sterile plants. 
Problems associated with such proposal include complete linkage of the two 
genes (Stoskopf, 1993, Plant Breeding : Theory and Practice, Westview 
Press, Boulder, San Francisco, Oxford). In sweetcorn, a genetic system to 
produce hybrid corn seeds without detassling, which utilizes the closely 
linked genes y (white endosperm) and ms (male sterility) was suggested but 
was never used because of contamination from 5% recombination. Galinat 
(1975, J. Hered. 66:387-388) described a two-step seed production scheme 
that resolved this problem by using electronic color sorters to separate 
yellow from white kernels. This approach has not been utilized 
commercially (Kankis and Davis, 1986, in &lt;&lt;Breeding Vegetable Crops&gt;&gt;, the 
Avi Publishing Company Inc. Westport, Conn., U.S.A., p. 498). 
EP 0,198,288 and U.S. Pat. No. 4,717,219 describe methods for linking 
marker genes (which can be visible markers or dominant conditional 
markers) to endogenous nuclear loci containing nuclear male-sterility 
genotypes. 
EP 412,911 describes foreign restorer genes (e.g. barstar coding region 
under control of a stamen-specific promoter) that are linked to marker 
genes, including herbicide resistance genes and genes coding for pigments 
(e.g. the A1 gene) under control of a promoter which directs expression in 
specific cells, such as petal cells, leaf cells or seed cells, preferably 
in the outer layer of the seed. 
SUMMARY OF THE INVENTION 
The invention concerns a maintainer plant consisting essentially of cells 
which comprise in their genome: 
a homozygous male-sterility genotype at a first genetic locus; and 
a color-linked restorer genotype at a second genetic locus, which is 
heterozygous (Rf/-) for a foreign DNA Rf comprising: 
a) a fertility-restorer gene capable of preventing the phenotypic 
expression of said male-sterility genotype, and 
b) at least one anthocyanin regulatory gene involved in the regulation of 
anthocyanin biosynthesis in cells of seeds of said plant and which is 
capable of producing anthocyanin at least in the seeds of said plant, so 
that anthocyanin production in the seeds is visible externally. 
The invention also concerns an anthocyanin regulatory gene which is a 
shortened R, B or C1 gene or a combination of shortened R, B or C1 genes 
which is functional for conditioning and regulating anthocyanin production 
in the aleurone. 
The invention also includes a DNA such as a plasmid comprising a 
fertility-restorer gene capable of preventing the phenotypic expression of 
a male-sterility genotype in a plant and at least one anthocyanin 
regulatory gene involved in the regulation of anthocyanin biosynthesis in 
cells of seeds of a plant and which is capable of producing anthocyanin at 
least in the seeds of a plant, so that anthocyanin production in the seeds 
is visible externally. 
Also within the scope of the invention is a process to maintain a line of 
male-sterile plants, which comprises the following steps: 
i) crossing: 
a) a male-sterile parent plant of said line having, in a first genetic 
locus, a homozygous male-sterility genotype, and 
b) a maintainer parent plant of said line consisting essentially of cells 
which comprise, stably intergrated in their nuclear genome: 
a homozygous male-sterility genotype at a first genetic locus; and 
a colored-linked restorer genotype at a second genetic locus, which is 
heterozygous for a foreign DNA comprising: 
i) a fertility-restorer gene capable of preventing the phenotypic 
expression of said male-sterility genotype, and 
ii) at least one anthocyanin regulatory gene involved in the regulation of 
anthocyanin biosynthesis in cells of seeds of said plant which is capable 
of producing anthocyanin at least in the seeds of said plant, so that 
anthocyanin production in the seeds is visible externally, 
ii) obtaining the seeds from said parent plants, and 
iii) separating on the basis of color, the seeds in which no anthocyanin is 
produced and which grow into male-sterile parent plants. 
Preferably, the genome of the male-sterile parent plant does not contain at 
least one anthocyanin regulatory gene necessary for the regulation of 
anthocyanin biosynthesis in seeds of this plant to produce externally 
visible anthocyanin in the seeds. In one embodiment of the invention, the 
genome of the male-sterile parent plant contains a first anthocyanin 
regulatory gene and the genome of the maintainer plant a second 
anthocyanin regulatory gene which, when present with the first anthocyanin 
regulatory gene in the genome of a plant, is capable of conditioning the 
production of externally visible anthocyanin in seeds. 
The invention also concerns a process to maintain a line of maintainer 
plants, which comprises the following steps: 
i) crossing: 
a) a male-sterile parent plant as described previously, and 
b) a maintainer parent plant as described previously, 
ii) obtaining the seeds from said male-sterile parent plant, and 
iii) separating on the basis of color, the seeds in which anthocyanin is 
produced and which grow into maintainer parent plants. 
The invention also relates to a kit for maintaining a line of male-sterile 
or maintainer plants, said kit comprising: 
a) a male-sterile parent plant of said line as described previously, 
having, in a first genetic locus, a homozygous male-sterility genotype and 
which is incapable of producing externally visible anthocyanin in seeds, 
and 
b) a maintainer parent plant of said line as described previously. 
Also within the scope of the invention is a process to maintain the kit 
described previously which comprises: 
crossing said male-sterile parent plant with said maintainer parent plant; 
obtaining the seeds from said male-sterile parent plants and optionally the 
seeds from said maintainer parent plant in which no anthocyanin is 
produced; and 
optionally growing said seeds into male-sterile parent plants and 
maintainer parent plants. 
As mentioned above, the present invention provides means to maintain a line 
of male-sterile plants, particularly corn or wheat plants. These means can 
be in the form of a process which comprises the following steps: 
i) crossing A) a first parent plant of said line, which is male-sterile, 
and which is genetically characterized by the absence of at least one 
anthocyanin regulatory gene thereby being incapable of producing 
anthocyanin in seeds, particularly in the aleurone layer, and also by 
having at a first genetic locus a homozygous male-sterility genotype, and 
B) a second parent plant of said line, which is male-fertile, and which is 
genetically characterized by having at said first genetic locus, said 
homozygous male-sterility genotype, and at a separate second genetic locus 
the genotype Rf/-, 
whereby, 
Rf is a foreign chimeric DNA (the "color-linked restorer gene") stably 
integrated in the nuclear genome of said plant which comprises: 
a) a fertility-restorer gene that is capable of preventing the phenotypic 
expression, i.e. the male-sterility, of said male-sterility genotype. 
b) said at least one anthocyanin regulatory gene (the "color gene") 
involved in the regulation of the anthocyanin biosynthesis in cells of 
seeds of said cereal plant which is capable of producing anthocyanin at 
least in the seeds, particularly in the aleurone, of said cereal plant, 
ii) obtaining the seeds from said first parent plants 
iii) separating, on the basis of color, the seeds in which no anthocyanin 
is produced and in which the genotype at said first genetic locus is said 
homozygous male-sterility genotype and the genotype at said second genetic 
locus is -/-, and the seeds in which anthocyanin is produced and in which 
the genotype at said first genetic locus is said homozygous male-sterility 
genotype and the genotype at said second genetic locus is Rf/-. 
Of particular interest in the invention is a second parent plant in which 
said at least one anthocyanin regulatory gene comprises a gene derived 
from a genomic clone of an R or B gene, particularly an R or B gene that 
conditions anthocyanin production in the aleurone, preferably the B-peru 
allele (e.g. the shortened B-peru gene in pCOL13), and/or comprises a gene 
derived from a genomic clone of the C1 gene (e.g. the gene with the 
sequence of SEQ ID NO 1 or SEQ ID NO 5) or the C1-S gene. 
The first genetic locus can be endogenous to plants of said line (in which 
case the homozygous male-sterility genotype will be m/m), but is 
preferably a foreign locus with genotype S/S in which S is a foreign DNA 
which, when expressed in a plant is capable of rendering the plant 
male-sterile. A preferred foreign DNA comprises at least: 
s1) a male-sterility DNA encoding a RNA, protein or polypeptide which, when 
produced or overproduced in a cell of the plant, significantly disturbs 
the metabolism, functioning and/or development of the cell, and, 
s2) a sterility promoter capable of directing expression of the 
male-sterility DNA selectively in stamen cells, preferably tapetum cells, 
of the plant; the male-sterility DNA being in the same transcriptional 
unit as, and under the control of, the sterility promoter. 
In case such a foreign male-sterility genotype is used, the 
fertility-restorer gene in the foreign DNA Rf preferably comprises at 
least: 
a1) a fertility-restorer DNA encoding a restorer RNA, protein or 
polypeptide which, when produced or overproduced in the same stamen cells 
as said male-sterility gene S, prevents the phenotypic expression of said 
foreign male-sterility genotype comprising S, and, 
a2) a restorer promoter capable of directing expression of the 
fertility-restorer DNA at least in the same stamen cells in which said 
male-sterility gene S is expressed, so that the phenotypic expression of 
said male-sterility gene is prevented; the fertility-restorer DNA being in 
the same transcriptional unit as, and under the control of, the restorer 
promoter. 
In case of an endogenous male-sterility genotype which is homozygous for 
the recessive male-sterility allele m, the fertility restorer gene is 
preferably a DNA comprising the dominant allele M of said locus. 
The present invention also provides the novel foreign chimeric DNA Rf as 
used in the second parent plants, plasmids comprising these chimeric 
genes, and host cells comprising these plasmids. 
The present invention also provides the shortened B-peru gene in pCOL13 
(SEQ ID NO 6) and the shortened C1 gene, particularly the EcoRI-SfiI 
fragment of pCOL9 of SEQ ID NO 5. 
The present invention further provides plants the nuclear genome of which 
is transformed with the foreign chimeric DNA Rf, particularly the second 
parent plant.

DETAILED DESCRIPTION OF THE INVENTION 
A male-sterile plant is a plant of a given plant species which is 
male-sterile due to expression of a male-sterility genotype such as a 
foreign male-sterility genotype containing a male-sterility gene. A 
restorer plant is a plant of the same plant species that contains within 
its genome at least one fertility-restorer gene that is able to restore 
the male fertility in those offspring obtained from a cross between a 
male-sterile plant and a restorer plant and containing both a 
male-sterility genotype and a fertility-restorer gene. A restored plant is 
a plant of the same species that is male-fertile and that contains within 
its genome a male-sterility genotype and a fertility-restorer gene. 
A line is the progeny of a given individual plant. 
A gene as used herein is generally understood to comprise at least one 
coding region coding for an RNA, protein or polypeptide which is operably 
linked to suitable promoter and 3' regulatory sequences. A structural gene 
is a gene whose product is a e.g. an enzyme, a structural protein, tRNA or 
rRNA. For example anthocyanin structural genes encode-enzymes (e.g. 
chalcone synthase) directly involved in the biosynthesis of anthocyanins 
in plant cells. A regulatory gene is a gene which encodes a regulator 
protein which regulates the transcription of one or more structural genes. 
For example the R, B, and C1 genes are regulatory genes that regulate 
transcription of anthocyanin structural genes. 
For the purpose of this invention the expression of a gene, such as a 
chimeric gene, will mean that the promoter of the gene directs 
transcription of a DNA into a mRNA which is biologically active i.e. which 
is either capable of interacting with another RNA, or which is capable of 
being translated into a biologically active polypeptide or protein. 
The phenotype is the external appearance of the expression (or lack of 
expression) of a genotype i.e. of a gene or set of genes (e.g. 
male-sterility, seed color, presence of protein or RNA in specific plant 
tissues etc.) 
As used herein, a genetic locus is the position of a given gene in the 
nuclear genome, i.e. in a particular chromosome, of a plant. Two loci can 
be on different chromosomes and will segregate independently. Two loci can 
be located on the same chromosome and are then generally considered as 
being linked (unless sufficient recombination can occur between them). 
An endogenous locus is a locus which is naturally present in a plant. A 
foreign locus is a locus which is formed in the plant because of the 
introduction, by means of genetic transformation, of a foreign DNA. 
In diploid plants, as in any other diploid organisms, two copies of a gene 
are present at any autosomal locus. Any gene can be present in the nuclear 
genome in several variant states designated as alleles. If two identical 
alleles are present at a locus that locus is designated as being 
homozygous, if different alleles are present, the locus is designated as 
being heterozygous. The allelic composition of a locus, or a set of loci, 
is the genotype. Any allele at a locus is generally represented by a 
separate symbol (e.g. M and m, S and -, - representing the absence of the 
gene). A foreign locus is generally characterized by the presence and/or 
absence of a foreign DNA. A heterozygous genotype in which one allele 
corresponds to the absence of the foreign DNA is also designated as 
hemizygous (e.g. Rf/-). A dominant allele is generally represented by a 
capital letter and is usually associated with the presence of a 
biologically active gene product (e.g. a protein) and an observable 
phenotypic effect (e.g. R indicates the production of an active regulator 
protein and under appropriate conditions anthocyanin production in a given 
tissue while r indicates that no active regulator protein is produced 
possibly leading to absence of anthocyanin production). 
A plant can be genetically characterized by identification of the allelic 
state of at least one genetic locus. 
The genotype of any given locus can be designated by the symbols for the 
two alleles that are present at the locus (e.g. M/m or m/m or S/-). The 
genotype of two unlinked loci can be represented as a sequence of the 
genotype of each locus (e.g. S/S,Rf/-) 
The nuclear male-sterility genotype as used in this invention refers to the 
genotype of at least one locus, preferably only one locus, in the nuclear 
genome of a plant (the "male-sterility locus") the allelic composition of 
which may result in male sterility in the plant. A male-sterility locus 
may be endogenous to the plant, but it is generally preferred that it is 
foreign to the plant. 
Foreign male-sterility loci are those in which the allele responsible for 
male sterility is a foreign DNA sequence S (the "male-sterility gene") 
which when expressed in cells of the plant make the plant male-sterile 
without otherwise substantially affecting the growth and development of 
the plant. Such male-sterility gene preferably comprises at least: 
s1) a male-sterility DNA encoding a sterility RNA, protein or polypeptide 
which, when produced or overproduced in a stamen cell of the plant, 
significantly disturbs the metabolism, functioning and/or development of 
the stamen cell, and, 
s2) a sterility promoter capable of directing expression of the 
male-sterility DNA selectively in stamen cells (e.g. anther cells or 
tapetum cells) of the plant; the male-sterility DNA being in the same 
transcriptional unit as, and under the control of, the sterility promoter. 
The male-sterility locus preferably also comprises in the same genetic 
locus at least one first marker gene T which comprises at least: 
t1) a first marker DNA encoding a first marker RNA, protein or polypeptide 
which, when present at least in a specific tissue or specific cells of the 
plant, renders the plant easily separable from other plants which do not 
contain the first marker RNA, protein or polypeptide encoded by the first 
marker DNA at least in the specific tissue or specific cells, and, 
t2) a first marker promoter capable of directing expression of the first 
marker DNA at least in the specific tissue or specific cells: the first 
marker DNA being in the same transcriptional unit as, and under the 
control of, the first marker promoter. 
Such male-sterility gene is always a dominant allele at such a foreign 
male-sterility locus. The recessive allele corresponds to the absence of 
the male-sterility gene in the nuclear genome of the plant. 
Male-sterility DNAs and sterility promoters that can be used in the 
male-sterility genes in the first parent line of this invention have been 
described before (EP 0,344,029 and EP 0,412,911). For the purpose of this 
invention the expression of the male-sterility gene in a plant cell should 
be able to be inhibited or repressed for instance by means of expression 
of a suitable fertility-restorer gene in the same plant cell. In this 
regard a particular useful male-sterility DNA codes for barnase (Hartley, 
J.Mol. Biol. 1988 202:913). The sterility promoter can be any promoter but 
it should at least be active in stamen cells, particularly tapetum cells. 
Particularly useful sterility promoters are promoters that are selectively 
active in stamen cells, such as the tapetum-specific promoters of the TA29 
gene of Nicotiana tabacum (EP 0,344,029) which can be used in tobacco, 
oilseed rape, lettuce, cichory, corn, rice, wheat and other plant species; 
the PT72, the PT42 and PE1 promoters from rice which can be used in rice, 
corn, wheat, and other plant species (WO 92/13956) ; the PCA55 promoter 
from corn which can be used in corn, rice, wheat and other plant species 
(WO 92/13957); and the A9 promoter of a tapetum-specific gene of 
Arabidopsis thaliana (Wyatt et al., 1992, Plant Mol. Biol. 19:611-922). 
However, the sterility promoter may also direct expression of the 
sterility DNA in cells outside the stamen; particularly if the effect of 
expression of the male-sterility DNA is such that it will specifically 
disturb the metabolism, functioning and/or development of stamen cells so 
that no viable pollen is produced. One example of such a male-sterility 
DNA is the DNA coding for an antisense RNA which is complementary to the 
mRNA of the chalcone synthase gene (van der Meer et al (1992) The Plant 
Cell 4:253-262). In this respect a useful promoter is the 35S promoter 
(see EP 0,344,029), particularly a 35S promoter that is modified to have 
enhanced activity in tapetum cells as described by van der Meer et al 
(1992) The Plant Cell 4:253-262 (the "35S-tap promoter"). 
A preferred endogenous male-sterility locus is one in which a recessive 
allele (hereinafter designated as m) in homozygous condition (m/m) results 
in male sterility. At such loci male fertility is encoded by a 
corresponding dominant allele (M). In many plant species such endogenous 
male-sterility loci are known (see Kaul, 1988, supra (in corn see also 
recent issues of Maize Genetics Cooperation Newsletter, published by 
Department of Agronomy and U.S. Department of Agriculture, University Of 
Missouri, Columbia, Mo., U.S.A.). The DNA sequences in the nuclear genome 
of the plant corresponding to m and M alleles can be identified by gene 
tagging i.e. by insertional mutagenesis using transposons, or by means of 
T-DNA integration (see e.g. Wienand and Saedler, 1987, In `Plant DNA 
Infectious Agents`, Ed. by T. H. Hohn and J. Schell, Springer Verlag Wien 
New York, p. 205; Shepherd, 1988, In `Plant Molecular Biology: a Practical 
Approach`, IRL Press, p. 187; Teeri et al., 1986, EMBO J. 5:1755). It will 
be evident that in the first and second parent plant of this invention S/S 
can be replaced by m/m without affecting the outcome of the process. 
Indeed, one feature of the process of this invention is that the 
male-sterility locus is homozygous thus allowing the use of `recessive` 
male-sterility alleles. 
Fertility-restorer DNAs that can be used in the fertility restorer gene in 
the second parent line of this invention have been described before (EP 
0,412,911). 
In this regard, fertility-restorer genes in which the fertility-restorer 
DNA encodes barstar (Hartley, J.Mol. Biol. 1988 202:913) are particularly 
useful to inhibit the expression of a male-sterility DNA that encodes 
barnase. In this regard it is believed that a fertility-restorer DNA that 
codes for a mutant of the barstar protein, i.e. one in which the Cysteine 
residue at position 40 in the protein is replaced by serine (Hartley, 
1989, TIBS 14:450), functions better in restoring the fertility in the 
restored plants of some species. 
In principle any promoter can be used as a restorer promoter in the 
fertility restorer gene in the second parent line of this invention. The 
only prerequisite is that such second parent plant, which contains both 
the color gene and the fertility-restorer gene, should be phenotypically 
normal and male-fertile. This requires that the restorer promoter in the 
fertility-restorer gene should be at least active in those cells of a 
plant of the same species in which the sterility promoter of the 
corresponding male-sterility gene can direct expression of the 
male-sterility DNA. In this regard it will be preferred that the sterility 
promoter and the restorer promoter are the same; they can for example be 
both stamen-specific promoters (e.g. the TA29 promoter or the CA55 
promoter) or they can be both constitutive promoters (such as the 35S or 
35S-tap promoter). However, the sterility promoter may be active only in 
stamen cells while the restorer promoter is also active in other cells. 
For instance, the sterility promoter can be a stamen-specific (such as the 
TA29 or CA55 promoter) while the restorer promoter is the 35S-tap 
promoter. 
When the male sterility to be restored is due to the male-sterility 
genotype at an endogenous male-sterility locus being homozygous for a 
recessive allele m, it is preferred that the fertility-restorer gene is 
the dominant allele of that male-sterility locus, preferably under control 
of its own promoter. The DNA corresponding to such a dominant allele, 
including its natural promoter can be isolated from the nuclear genome of 
the plant by means of gene tagging as described above. 
The nature of the color gene that is used in the color-linked restorer gene 
in the second parent plant of this invention depends upon the genotype of 
the untransformed plants of the same line. Preferably, only cereal plants 
with a genotype that does not condition externally visible anthocyanin 
production in seeds, particularly in the aleurone can be used to produce 
the second parent plants. These plants usually have a genotype in which no 
functional copy of a suitable regulatory gene such as the R or B gene, 
and/or the C1 gene, is present. 
In corn, for instance, all of the currently used inbred lines in the U.S.A. 
are r-r (pink anthers, leaf tips, plant base) or r-g (green) and most of 
these are c1 and pl; at the B- locus the B-peru allele is very rare (Coe 
et al, 1988, In `Corn and Corn Improvement`, 3rd edition, G. F. Sprague 
and J. W. Dudley, eds. America Science of Agronomy, Inc. Publishers, 
Madison, Wis., U.S.A.). The result is that no anthocyanins are produced in 
the aleurone of these lines and that the kernels are yellow. This requires 
that when these lines are transformed with a color-linked restorer gene, 
the color gene should consist of a functional R or B gene which conditions 
anthocyanin production in aleurone, and usually also a functional C1 gene 
capable of conditioning anthocyanin production in aleurone. 
A useful R or B gene is the B-peru gene, but of course also other R genes 
could be used such as the R(S) gene (Perrot and Cone, 1989, Nucl. Acids 
Res. 17:8003). In this regard a gene derived from genomic clones of the 
B-peru gene (Chandler et al, 1989, The Plant Cell 1:1175-1183) is believed 
to be particularly useful. However the length of this genomic DNA (11 kbp) 
renders its practical manipulation and use for transformation by direct 
gene transfer, difficult, certainly in combination with other genes such 
as the restorer gene and the C1 gene. 
In one inventive aspect of this invention it was found that the B-peru gene 
could be considerably shortened while still retaining, under appropriate 
conditions, its capability of conditioning anthocyanin production in the 
aleurone of seeds of cereal plants such as corn. A preferred shortened 
B-peru gene is that of Example 2.2 and which is contained in plasmid 
pCOL13 (deposited under accession number LMBP 3041). 
A useful C1 gene is the genomic clone as described by Paz-Ares et al, 1987, 
EMBO J. 6:3553-3558. However the length of this genomic DNA (4 kbp) 
precludes its practical manipulation and use for transformation by direct 
gene transfer, certainly in combination with other genes such as the 
restorer gene and the B-peru gene. Nevertheless other variants of the C1 
gene can also be used. In this regard Scheffler et al, 1994, 
Mol.Gen.Genet. 242:40-48 have described the C1-S allele which differs from 
the C1 allele of Paz-Ares et al, supra by a few nucleotides in the 
promoter region near the CAAT box and which is dominant to the wild-type 
allele (C1) and shows enhanced pigmentation. The C1-S gene can be easily 
used in this invention by appropriate changes in the C1 gene. For example 
the TGCAG at positions 935 to 939 in SEQ ID NO 1 (respectively at 
positions 884-888 in SEQ ID NO 5) can be easily changed to TTAGG yielding 
a C1-S allele (respectively pCOL9S). 
In one inventive aspect of this invention it was found that the C1 gene 
(and the C1-S gene) could be considerably shortened while still retaining, 
under appropriate conditions, its capability of conditioning anthocyanin 
production in the aleurone of seeds of cereal plants such as corn. 
Preferred shortened C1 genes for instance are those of Example 2.1 such as 
comprised in pCOL9 which has the sequence of SEQ ID NO 5, particularly as 
comprised between the EcoRI and SfiI sites of pCOL9, and the corresponding 
shortened C1-S gene in pCOL9S. 
The transcribed region of the shortened B-peru and C1 genes still contain 
some small introns which can also be deleted without affecting the 
function of the genes. It is also believed that the shortened B-peru and 
C1 genes can be somewhat further truncated at their 5' and 3' ends, 
without affecting their expression in aleurone. In particular it is 
believed that the sequence between positions 1 and 3272 of SEQ ID NO 6 can 
also be used as a suitable B-peru gene. It is also believed that this gene 
can still be truncated at its 3' end down to a position between 
nucleotides 2940 and 3000 of SEQ ID No. 6. 
Athough the use of genomic sequences of the B-peru gene and the C1 gene, 
particularly the shortened B-peru and/or the shortened C1 of C1-S genes, 
is preferred, chimeric R, B, or C1 genes can also be used. For instance a 
chimeric gene can be used which comprises the coding region (e.g. obtained 
from the cDNA) of any functional R or B gene (i.e. which conditions 
anthocyanin production anywhere in the plant) which is operably linked to 
the promoter region of a R or B gene which conditions anthocyanin 
production in the aleurone (such as R(S) or B-peru). Since the presence of 
anthocyanin does not negatively affect growth, development and functioning 
of plant cells, a constitutive promoter (e.g. the 35S promoter), or a 
promoter which directs expression at least in the aleurone can also be 
used in such a chimeric gene. In this regard the promoter of the C1 gene 
can also be used to direct expression of a DNA comprising the coding 
region of suitable R or B gene, particularly the B-peru gene. 
Similarly the coding region (e.g. obtained from cDNA) of the C1 gene can be 
operably linked to the promoter of a gene that directs expression at least 
in the aleurone. In this regard, the promoter of the B-peru gene can also 
be used to direct expression of a DNA comprising the coding region of a 
suitable C1 gene such as that of the C1 gene of SEQ ID No. 1 or of the 
C1-S gene. 
In another inventive aspect of the invention it was found that the the 
promoters comprised in DNAs characterized by the sequences between 
positions 1 to 1077, particularly between positions 447 and 1077, quite 
particularly between positions 447 and 1061 of SEQ ID NO 1, between 
positions 396 and 1026 of SEQ ID NO 5, and between positions 1 to 575, 
particularly between position 1 to 188 of SEQ ID NO 6 are promoters that 
predominantly, if not selectively, direct expression of any DNA, 
preferably a heterologous DNA in the aleurone layer of the seeds of 
plants. 
Of course in those lines in which a functional C1 gene is already present 
in the genome the color gene can consist only of a suitable functional R 
or B gene (or a chimeric alternative). Alternatively if a line contains 
already a functional R or B gene which can condition anthocyanin 
production in the aleurone, but no functional C1 gene, only a functional 
C1 gene is required as a color gene. 
It is believed that the color genes of this invention are especially useful 
in cereal plants, and that they are of particular use in corn and wheat, 
and certainly in corn. 
For the purposes of this invention it is preferred that, in the second 
parent plants the "Rf" locus and the male-sterility (e.g. "S") locus are 
not linked and segregate separately. 
In the second parent plant, the fertility restorer gene, the B-peru gene 
and the C1 gene are preferably closely linked. This can of course be 
achieved by introducing these genes in the nuclear genome of the plants as 
a single transforming foreign DNA (the Rf DNA) thus forming a foreign Rf 
locus. Alternatively, the fertility restorer gene and the color gene can 
be separately introduced by cotransformation which usually results in 
single locus insertions in the plant genome. 
The color-linked restorer gene Rf as used in the second parent plant 
preferably also comprises at least c) a second marker gene which comprises 
at least: 
c1) a second marker DNA encoding a second marker RNA, protein or 
polypeptide which, when present at least in a specific tissue or specific 
cells of the plant, renders the plant easily separable from other plants 
which do not contain the second marker RNA, protein or polypeptide encoded 
by the second marker DNA at least in the specific tissue or specific 
cells, and, 
c2) a second marker promoter capable of directing expression of the second 
marker DNA at least in the specific tissue or specific cells: the second 
marker DNA being in the same transcriptional unit as, and under the 
control of, the second marker promoter. 
First and second marker DNAs and first and second marker promoters that can 
be used in the first and second marker genes of this invention are also 
well known (EP 0,344,029; EP 0,412,911). In this regard it is preferred 
that the first and second marker DNA are different, although the first and 
second marker promoter may be the same. 
Foreign DNA such as the fertility-restorer gene, the foreign male-sterility 
gene, the B-peru and the C1 genes, or the first or second marker gene 
preferably also are provided with suitable 3' transcription regulation 
sequences and polyadenylation signals, downstream (i.e. 3') from their 
coding sequence i.e. respectively the fertility-restorer DNA, the 
male-sterility DNA, the coding region of a color gene (such as a B-peru 
gene and/or a C1 gene) or the first or second marker DNA. In this regard 
either foreign or endogenous transcription 3' end formation and 
polyadenylation signals suitable for obtaining expression of the chimeric 
gene can be used. For example, the foreign 3' untranslated ends of genes, 
such as gene 7 (Velten and Schell (1985) Nucl. Acids Res. 13:6998), the 
octopine synthase gene (De Greve et al., 1982, J.Mol. Appl. Genet. 1:499; 
Gielen et al (1983) EMBO J. 3:835; Ingelbrecht et al., 1989, The Plant 
Cell 1:671) and the nopaline synthase gene of the T-DNA region of 
Agrobacterium tumefaciens Ti-plasmid (De Picker et al., 1982, J.Mol. Appl. 
Genet. 1:561), or the chalcon synthase gene (Sommer and Saedler, 1986, 
Mol.Gen.Genet. 202:429-434), or the CaMV 19S/35S transcription unit (Mogen 
et al., 1990, The Plant Cell 2:1261-1272) can be used. However, it is 
preferred that the color genes in this invention carry their endogenous 
transcription 3' end formation and polyadenylation signals. 
The fertility-restorer gene, the male-sterility gene, the color gene or the 
first or second marker gene in accordance with the present invention are 
generally foreign DNAs, preferably foreign chimeric DNA. In this regard 
"foreign" and "chimeric" with regard to such DNAs have the same meanings 
as described in EP 0,344,029 and EP 0,412,911. 
The cell of a plant, particularly a plant capable of being infected with 
Agrobacterium such as most dicotyledonous plants (e.g. Brassica napus) and 
some monocotyledonous plants, can be transformed using a vector that is a 
disarmed Ti-plasmid containing the male-sterility gene, the color linked 
restorer gene or both and carried by Agrobacterium. This transformation 
can be carried out using the procedures described, for example, in EP 
0,116,718 and EP 0,270,822. Preferred Ti-plasmid vectors contain the 
foreign DNA between the border sequences, or at least located to the left 
of the right border sequence, of the T-DNA of the Ti-plasmid. Of course, 
other types of vectors can be used to transform the plant cell, using 
procedures such as direct gene transfer (as described, for example, in EP 
0,233,247), pollen mediated transformation (as described, for example, in 
EP 0,270,356, PCT patent publication "WO" 85/01856, and U.S. Pat. No. 
4,684,611), plant RNA virus-mediated transformation (as described, for 
example, in EP 0,067,553 and U.S. Pat. No. 4,407,956) and 
liposome-mediated transformation (as described, for example, in U.S. Pat. 
No. 4,536,475). Cells of monocotyledonous plants such as the major cereals 
including corn, rice, wheat, barley, and rye, can be transformed (e.g. by 
electroporation) using wounded or enzyme-degraded intact tissues capable 
of forming compact embryogenic callus (such as immature embryos in corn), 
or the embryogenic callus (such as type I callus in corn) obtained 
thereof, as described in WO 92/09696. In case the plant to be transformed 
is corn, other recently developed methods can also be used such as, for 
example, the method described for certain lines of corn by From et al., 
1990, Bio/Technology 8:833; Gordon-Kamm et al., 1990, Bio/Technology 2:603 
and Gould et al., 1991, Plant Physiol. 95:426. In case the plant to be 
transformed is rice, recently developed methods can also be used such as, 
for example, the method described for certain lines of rice by Shimamoto 
et al., 1989, Nature 338:274; Datta et al., 1990, Bio/Technology 8:736; 
and Hayashimoto et al., 1990, Plant Physiol. 93:857. 
The transformed cell can be regenerated into a mature plant and the 
resulting transformed plant can be used in a conventional breeding scheme 
to produce more transformed plants with the same characteristics or to 
introduce the male-sterility gene, the color-linked restorer gene (or 
both), in other varieties of the same related plant species. Seeds 
obtained from the transformed plants contain the chimeric gene(s) of this 
invention as a stable genomic insert. Thus the male-sterility gene, or the 
color-linked restorer gene of this invention when introduced into a 
particular line of a plant species can always be introduced into any other 
line by backcrossing. 
The first parent plant of this invention contains the male-sterility gene 
as a stable insert in its nuclear genome (i.e. it is a male-sterile 
plant). For the purposes of this invention it is preferred that the first 
parent plant contains the male-sterility gene in homozygous condition so 
that it transmits the gene to all of its progeny. 
The second parent plant of this invention contains the male-sterility gene 
and the color-linked restorer gene as stable inserts in its nuclear genome 
(i.e. it is a restored plant). It is preferred that the male-sterility 
gene be in homozygous condition so that the second parent plant transmits 
the gene to all of its progeny and that the color-linked restorer gene be 
in heterozygous condition so that the second parent plant transmits the 
gene to only half of its progeny. 
It is preferred that the first and second parent plants are produced from 
the same untransformed line of a plant species, particularly from the same 
inbred line of that species. 
The first and second parent plants of this invention have the particular 
advantage that seeds of such plants can be maintained indefinitely, and 
can be amplified to any desired amount (e.g. by continuous crossing of the 
two plant lines). 
The color genes of this invention can be used as marker gene in any 
situation in which it is worthwhile to detect the presence of a foreign 
DNA (i.e. a transgene) in seeds of a transformed plant in order to isolate 
seeds which possess the foreign DNA. In this regard virtually any foreign 
DNA, particularly a chimeric gene can be linked to the color gene. 
Examples of such foreign DNAs are genes coding for insecticidal (e.g. from 
Bacillus thuringiensis), fungicidal or nematocidal proteins. Similarly the 
color-gene can be linked to a foreign DNA which is the male-sterility gene 
as used in this invention. 
However, the color genes are believed to be of particular use in the 
process of this invention in which they are present in a foreign DNA which 
comprises a fertility restorer gene (such as the barstar gene of Bacillus 
amylolicruefaciens) under control of a stamen-specific promoter (such as 
PTA29). In appropriate conditions the use of the color genes allows the 
easy separation of harvested seeds that will grow into male-sterile 
plants, and harvested seeds that will grow into male-fertile plants. In 
this regard the seeds are preferably harvested from male-sterile plants 
(the first parent plants) that. are homozygous at a male-sterility locus 
(such as a locus comprising the barnase gene under control of PTA29) and 
which have been pollinated by restorer plants (the second parent plants of 
this invention) which contain in their genome two unlinked gene loci one 
of which comprises the same male-sterility locus which is homozygous for 
the same male-sterility gene while the other is a foreign locus which 
comprises an appropriate fertility restorer gene (i.e. whose expression 
will counteract the expression of the male-sterility gene) and also the 
color gene of this invention, particularly an R or B gene that is 
expressed in the aleurone and/or a C1 gene, preferably the B-peru and C1 
gene (e.g. as described in the examples). First and second parent plants 
can be essentially produced as described in the examples and as summarized 
in FIG. 1. In step 8 of FIG. 1 it is demonstrated that the crossing of the 
first and second parent plants of this invention will give rise in the 
progeny to about 50% new first parent (i.e. male-sterile) plants and about 
50% new second parent (i.e. male-fertile) plants and that these two types 
of plants can already be separated at the seed stage on the basis of 
color. Red kernels will grow into male-fertile plants while yellow kernels 
will grow into male-sterile plants. 
Thus a line of male-sterile first parent plants of this invention can be 
easily maintained by continued crossing with the second parent plants of 
this invention with, in each generation, harvesting the seeds from the 
male-sterile plants and separation of the yellow and red kernels. Of 
course in this way any desired amount of seed for foundation seed 
production of a particular line, such as an inbred line, can also be 
easily obtained. 
The red and yellow seeds harvested from a cereal plant (e.g. the first 
parent plant of this invention) can be separated manually. However, such 
separation can also be effected mechanically. A color sorting machine for 
corn kernel and other granular products is for instance available from 
Xeltron U.S. (Redmond, Wash., U.S.A.) 
Unless otherwise indicated all experimental procedures for manipulating 
recombinant DNA were carried out by the standardized procedures described 
in Sambrook et al., 1989, "Molecular Cloning: a Laboratory Manual", Cold 
Spring Harbor Laboratory, and Ausubel et al, 1994, "Current Protocols in 
Molecular Biology", John Wiley & Sons. 
The polymerase chain reactions ("PCR") were used to clone and/or amplify 
DNA fragments. PCR with overlap extension was used in order to construct 
chimeric genes (Horton et al, 1989, Gene 77:61-68; Ho et al, 1989, Gene 
77:51-59). 
All PCR reactions were performed under conventional conditions using the 
Vent.sup.T M polymerase (Cat. No. 254L--Biolabs New England, Beverley, 
Mass. 01915, U.S.A.) isolated from Thermococcus litoralis (Neuner et al., 
1990, Arch.Microbiol. 153:205-207). Oligonucleotides were designed 
according to known rules as outlined for example by Kramer and Fritz 
(1968, Methods in Enzymology 154:350), and synthesized by the 
phosphoramidite method (Beaucage and Caruthers, 1981, Tetrahedron Letters 
22:1859) on an applied Biosystems 380A DNA synthesizer (Applied Biosystems 
B.V., Maarssen, Netherlands). 
In the following examples, reference will be made to the following sequence 
listing and figures: 
Sequence Listing 
SEQ ID NO 1: sequence of C1 gene 
SEQ ID NO 2: plasmid pTS256 
SEQ ID NO 3: EcoRI-HindIII region of pTS200 comprising the chimeric gene 
PCA55-barstar-3'nos (the omitted region of pTS200 is derived from pUC19. 
SEQ ID NO 4: oligonucleotide 1 
SEQ ID NO 5: pCOL9 containing the shortened C1 gene as a EcoRI-SfiI 
fragment 
SEQ ID NO 6: presumed sequence of the EcoRI-HindIII region of pCOL13 
containing the shortened B-peru gene (the rest of the plasmid is pUC19). 
The stretch of N nucleotides corresponds to a region of approximate length 
which is derived from the genomic clone of the B-peru gene but for which 
the sequence needs to be confirmed. 
SEQ ID NO 7: actual sequence of the EcoR1-HindIII region of pCOL13 
containing the shortened B-peru gene (the rest of the plasmid is pUC19). 
Figures 
FIG. 1: Breeding scheme to obtain the first and second parent plants of 
this invention 
FIG. 2: Schematic structure of pCOL25, pCOL26, pCOL27, pCOL28, pCOL100 and 
pDE110. 
EXAMPLES 
Example 1 
Construction of Plasmids Containing the Male-sterility Gene Comprising the 
TA29 Promoter and the Barnase Coding Region 
Plasmids useful for transformation of corn plants and carrying a 
male-sterility gene and a selectable marker gene have been described in WO 
92/09696 and WO 92/00275. 
Plasmid pVE107 contains the following chimeric genes: 1) 
PTA29-barnase-3'nos, i.e. a DNA coding for barnase of Bacillus 
amyloliquefaciens (barnase) operably linked to the stamen-specific 
promoter of the TA29 gene of (neo) operably linked to the 35S promoter of 
Cauliflower Mosaic Virus (P35S) and the 3' regulatory sequence containing 
the polyadenylation signal of the octopine synthase gene of Agrobacterium 
tumefaciens (3'ocs). 
Plasmid pVE108 contains the following chimeric genes: 1) 
PTA29-barnase-3'nos, and 2) P35S-bar-3'nos, i.e. the gene of Streptomyces 
hygroscopicus (EP 242236) coding for phosphinothricin acetyl transferase 
(bar) operably linked to the P35S and 3'nos. 
PTA29-barnase-3'nos is an example of a foreign chimeric male-sterility gene 
(S) used in this invention. 
Example 2 
Construction of a Plasmid Containing the Color-linked Restorer Gene 
2.1. Obtaining a shortened functional C1 gene 
The C1 gene of maize was cloned from transposable-induced mutants and its 
sequence was reported (Paz-Ares, 1987, EMBO J. 6:3553-3558). This sequence 
is reproduced in SEQ ID NO. 1. Plasmid p36 (alternatively designated as 
pC1LC5kb and further designated as plasmid pXX036) comprising a C1 genomic 
clone was obtained from Dr. H. Saedler and Dr. U. Wienand of the 
Max-Planck Institut fur Zuchtungsforschung, Koln, Germany. pXX036 was 
digested with SnabI and HindIII, filled-in with Klenow, and selfligated, 
yielding plasmid pCOL9. pCOL9 corresponds to pUC19 (Yanisch-Perron et al, 
1985, Gene 33:103-119) which contains, between its EcoRI and modified 
HindIII sites, the 2189 bp EcoRI-SnabI fragment (corresponding to the 
sequence between positions 448 and 2637 of SEQ ID NO 1) of pXX036. 
pXX036 was also digested with SfiI and HindIII and treated with Klenow to 
make blunt ends. After ligation the plasmid in which the DNA downstream 
from the SfiI site was deleted was designated as pCOL12. 
The sequence TGCAG in pCOL9, corresponding to the sequence at positions 884 
to 888 in SEQ ID NO 5, is changed to TTAGG, yielding pCOL9S which instead 
of a shortened C1 gene contains a shortened overexpressing C1-S gene 
(Schleffer et al, 1994, Mol.Gen.Genet. 242:40-48). A similar change is 
introduced in pCOL12, yielding pCOL12S. 
2.2. Obtaining a shortened functional B-peru gene 
Plasmid pBP2 (further designated as pXX004) is plasmid pTZ18U (Mead et al., 
1986, Protein Engineering 1:67; U.S. Biochemical Corp.) containing the 
genomic clone of the B-peru gene. Plasmid p35SBPcDNA (further designated 
as pXX002) is plasmid pMF6 (Goff et al, 1990, EMBO J. 9:2517-2522) 
containing the cDNA corresponding to the B-peru gene. Both plasmids were 
obtained from Dr. V. Chandler of the University of Oregon, Oregon, U.S.A. 
A 2660 bp sequence of the genomic clone around the translation initiation 
codon was reported (EMBL/Genbank/DDBJ databases; locus name ZMBPERUA, 
Accession number X70791; see also Radicella et al, 1992, Genes & 
Development 6:2152-2164). The sequence of the B-peru cDNA was also 
reported (Radicella et al, 1991, Plant Mol. Biol. 17:127-130). 
Substantial amounts of 5' and 3' flanking sequences were deleted from 
pXX004, and the MluI-MunI fragment in the coding region of the genomic 
clone was replaced by the 1615 bp MluI-MunI fragment of the cDNA clone. 
The resulting plasmid was designated as pCOL13 which was deposited at the 
Belgian Coordinated Collection of Microorganisms--LMBP Collection, 
Laboratory Molecular Biology, University of Ghent, K. L. Ledeganckstraat 
35, B-9000 Ghent, Belgium and was given the Accession Number LMBP 3041. A 
shortened but functional B-peru gene is contained in pCOL13 as an 
EcoRI-SalI fragment with an approximate length of 4 kbp (see SEQ ID NO 6). 
2.3. Combining the C1 and B-peru genes 
The C1 gene in pCOL9 and the B-peru gene in pCOL13 were then combined as 
follows. The 4 kbp EcoRI-SalI fragment of pCOL13 was introduced between 
the EcoRI and SalI sites of the vector pBluescript II SK(-) (Stratagene), 
yielding #7 B SK(-). pCOL9 was digested with SfiI, treated with Klenow to 
fill in protruding ends, and further digested with EcoRI. The 1978 bp 
SfiI(Klenow)/EcoRI was then introduced between the EcoRI and SmaI sites of 
#7 B SK(-), yielding #7 B+C SK(-). Finally the XhoI site in the C1 
sequence was removed as follows. The 950 bp EcoRI-SacII fragment of #7 B 
SK(-) (EcoRI site corresponding to the EcoRI site at position 1506 in SEQ 
ID NO 1; the SacII site from the pBluescript linker) was introduced 
between the EcoRI and SacII sites of the Phagescript Vector (Stratagene) 
to yield pCOL21. Single strands of pCOL21 were prepared and hybridized to 
the following synthetic oligonucleotide 1 (SEQ ID No. 4): 
5'-CGT TTC TCG AAT CCG ACG AGG-3' 
resulting in a silent change (CTCGAG.fwdarw.CTCGAA) and removal of the XhoI 
site. 
The 710 AatII-SacII fragment of #7 B SK(-) was then exchanged for the 
corresponding AatII-SacII fragment of the mutated pCOL21, yielding pCOL23. 
pCOL23 was then linearized with SacII, treated with Klenow, and ligated to 
XhoI linker sequence (Stratagene), yielding pCOL24. 
Using the same procedure as described above, the shortened C1-S gene of 
pCOL9S is combined with the shortened B-peru gene of pCOL23, yielding 
plasmid pCOL24S. 
2.4. Construction of vectors comprising the C1 and B-peru genes as well as 
male-sterility gene and a selectable marker gene 
pTS256 is derived from pUC19 and contains the following two chimeric genes: 
1) P35S-bar-3'nos, and 2) PTA29-barstar-3'nos, i.e. a DNA coding for 
barstar of Bacillus amyloliquefaciens (barstar or bar*) operably linked to 
PTA29 and 3'nos. The complete sequence of pTS256 is given in SEQ ID NO 2. 
pTS200 is derived from pUC19 and contains the following two chimeric genes 
: 1) P35S-bar-3'nos, and 2) PCA55-barstar-3'nos, i.e. barstar operably 
linked to the stamen-specific promoter PCA55 of Zea mays and 3'nos. The 
complete sequence of pTS200 is given in SEQ ID NO 3. 
pTS256 was modified by the inclusion of NotI linkers (Stratagene) in both 
the unique SspI and SmaI sites, yielding pTS256NN. The shorter BspEI-SacII 
fragment of pTS256NN was then replaced by the shorter BspEI-SacII fragment 
of pTS200, yielding pTS256+200. 
pTS256NN contains P35S-bar3'-nos and pTA29-barstar3'nos on a NotI cassette. 
pTS256NN+200 contains P35S-bar3'-nos and pCA55-barstar3'nos on a NotI 
cassette. 
he NotI cassette of pTS256NN was introduced in the NotI site of pCOL24, 
yielding pCOL25 and pCOL26 which differ with respect to the orientation of 
the P35S-bar3'-nos gene with respect to the shortened C1 gene (FIG. 2). 
The NotI cassette of pTS256NN+200 was introduced in the NotI site of 
pCOL24, yielding pCOL27 and pCOL28 which differ with respect to the 
orientation of the P35S-bar3'-nos gene with respect to the shortened C1 
gene (FIG. 2). 
Plasmids pCOL25, pCOL26, pCOL27 or pCOL28 contain a color-linked restorer 
gene Rf and a selectable marker gene (P35S-bar-3'nos). Rf comprises the 
shortened C1 and B-peru genes and a chimeric barstar gene (either 
PTA29-barstar-3'nos or PCA55- barstar-3'nos). 
Plasmids pCOL25S, pCOL26S, pCOL27S or pCOL28S, containing the shortened 
C1-S gene instead of the shortened C1 gene, are obtained in a similar way 
using pCOL24S instead of PCOL24. 
2.5. Construction of vectors comprising the C1 and B-peru genes as well as 
male-sterility gene 
Plasmid pTS59 can be obtained from plasmid pTS256 (of SEQ ID NO 2) by 
replacing the fragment extending from positions 1 to 1470 (comprising the 
chimeric gene P35S-bar-3'nos) with the sequence TATGATA. Then NotI linkers 
(Stratagene) were introduced in the EcoRV and SmaI sites of pTS59; 
yielding pTS59NN. Finally the NotI fragment comprising the chimeric gene 
PTA29-barstar-3'nos was introduced in the NotI site of #7 B+C SK(-), 
yielding pCOL100 (the general structure of pCOL100 and pDE110 is also 
presented in FIG. 2). 
2.6. Expression of shortened C1 and B-peru in aleurone in corn seeds 
Dry seeds were incubated overnight in water at room temperature and were 
then peeled and sliced in half. Four to six half kernels were placed with 
the cut side on wet filter paper and were bombarded with tungsten 
particles (diameter 0.7 .mu.m) which were coated with DNA. 
Particle bombardment was essentially carried out using the particle gun and 
procedures as described by Zumbrunn et al, 1989, Technique, 1:204-216. The 
tissue was placed at 10 cm from the stopping plate while a 100 .mu.m mesh 
was placed at 5 cm from the stopping plate. 
DNA of the following plasmids was used: 
pXX002: B-peru cDNA under control of the 35S promoter 
pXX201: C1 cDNA under control of the 35S promoter 
pCOL13: shortened B-peru gene as described in Example 2.2 
pCOL12: shortened C1 gene as described in Example 2.1 
pCOL100: shortened B-peru and shortened C1 and PTA29-barstar-3'nos as 
described in Example 2.5. 
After bombardment the tissue was incubated for 2 days on wet filter paper 
at 27.degree. C. and was then checked for the presence of red spots 
indicating anthocyanin production. 
TABLE 1 
__________________________________________________________________________ 
pXX002 
pXX00 
PXX201 
pXX201 
pCOL13 
pCOL12 
pCOL100 
__________________________________________________________________________ 
H99 r c1 
- - + nt nt nt 
Pa91 
r c1 
- - + nt nt nt 
B73 r c1 
- - + nt nt + 
inbred1 
r c1 
- - + nt nt nt 
inbred2 
r c1 
- - + nt nt nt 
inbred3 
r c1 
- - + nt nt nt 
inbred4 
r c1 
+ - + + - nt 
inbred5 
r c1 
+ - + + - nt 
inbred6 
r c1 
- - + nt nt nt 
inbred7 
r c1 
- - + nt nt nt 
inbred8 
r c1 
- - + nt nt nt 
inbred9 
r c1 
+ - + + - nt 
c-ruq 
R c1 
- + + - + nt 
__________________________________________________________________________ 
Note: + indicates that anthocyanin production was observed in at least on 
experiment; 
- indicates that no anthocyanin production was observed, 
nt = not tested. 
The results for three public lines (H99, Pa91, B73) and 9 different, 
commercially important, proprietary inbred lines from various sources are 
shown in Table 1. The line c-ruq is a tester line which is homozygous for 
a C1 allele that is inactivated by insertion of a receptor for the 
regulator Uq (Cormack et al., 1988, Crop Sci. 28:941-944). 
All lines which were r and c1 produced anthocyanin in the aleurone after 
introduction with both a functional B-peru and C1 gene. Lines which were R 
and c1 produced anthocyanin upon introduction of a functional C1 gene. 
Lines which were r and C1 produced anthocyanin upon introduction of a 
functional B-peru gene. This proves that the B-peru and C1 gene are 
sufficient for anthocyanin production in most corn lines. From the data in 
Table 1 it is also evident that even the shortened B-peru and C1 genes are 
still functional and are capable of producing anthocyanin in aleurone of 
corn lines with suitable genotypes. 
Example 3 
Production of First Parent Corn Plants by Transformation of Corn With the 
Plasmids of Example 1. 
Corn plants of line H99, transformed with a male-sterility gene comprising 
a DNA encoding barnase of Bacillus amyloliguefaciens under control of the 
promoter of the TA29 gene of Nicotiana tabacum have been described in WO 
92/09696. The transformed plants were shown to be male-sterile. 
Example 4 
Production of second Parent Corn Plants by Transformation of Corn With the 
Plasmids of Examples 2. 
Corn inbred lines H99 and Pa91 are transformed using the procedures as 
described in WO 92/09696 but using plasmids pCOL25, pCOL26, pCOL27 or 
pCOL28 of Example 2. Regenerated plants are selected that are male fertile 
and in which the shortened C1, the shortened B-peru gene, the 
P35S-bar-3'nos gene, and the PTA29-barstar-3'nos (or PCA55-barstar-3'nos) 
are expressed. 
Alternatively the male-sterile plants of Example 3 (already containing the 
S gene) can be transformed with plasmids pCOL25, pCOL26, pCOL27 or pCOL28 
of Example 2 on the condition that the S and Rf genes are linked to 
different selectable marker genes. 
Similarly, transformed corn plants are obtained using plasmids pCOL25S, 
pCOL26S, pCOL27S or pCOL28S of Example 2. 
In an alternative set of experiments the second parent plants of this 
invention were obtained by transforming corn plants of line H99, Pa91, and 
(Pa91xH99)x H99 with two separate plasmids one of which contained the 
color linked restorer gene (pCOL100), while the other contains an 
appropriate selectable marker gene such as a chimeric bar gene (pDE110) 
(alternatively a chimeric neo gene may also be used). pDE110 was described 
in WO 92/09696 and the construction of pCOL100 was descibed in Example 
2.5. 
In yet another set of experiments the second parent plants of this 
invention are obtained by transforming corn plants with a purified 
fragment of the plasmids of example 2.4. Such purified fragment is 
obtained by digestion of the plasmids of example 2.4 with XhoI and 
subsequent purification using conventional procedures such as gel 
filtration. 
Untransformed corn plants of lines H99 or Pa91 are detasseled and 
pollinated with pollen of the plants transformed with the Rf DNA. It is 
observed that the f gene segregates in a Mendelian way and that the seed 
that is harvested from these plants is colored and non-colored (yellow) in 
a 1:1 ratio. The red color of the seeds is correlated with the presence of 
the Rf gene. 
Example 5 
The Production of the First and Second Parent Plants of This Invention. 
First parent plants and second parent plants (i.e. maintainer plants) 
according to the invention are produced along the lines set out in FIG. 1. 
The male-sterile plants of step 1 are those produced in Example 1. The corn 
plants transformed with the color-linked restorer gene of step 2 are those 
produced in Example 4. 
A plant of Example 1 and a plant of Example 4 are crossed (Step 3) and the 
progeny plants with the genotype S/-, Rf/- are selected (Step 4), e.g. by 
demonstrating the presence of both the S and Rf genes in the nuclear 
genome (e.g. by means of PCR). 
The plants selected in Step 4 are then crossed with the male-sterile plants 
with genotype S/- (Step 5). The colored seeds (i.e. those containing the 
Rf gene) are selected, grown into plants, and examined for the presence of 
both the S and Rf genes (e.g. by PCR). The plants containing both the S 
and Rf genes are selfed and the seeds of each plant are examined on seed 
color (red or yellow). From the progeny of the selfings the non-colored 
seeds are grown into plants (Step 6). The progeny of the selfings in which 
all noncolored seeds grow into male-sterile plants are retained (Step 6). 
These male-sterile plants are all homozygous for the S gene and are 
crossed with their fertile siblings (of genotype S/S,Rf/Rf or S/S,Rf/-) 
(Step 7). For some crossings the seeds harvested from the male-sterile 
plants are 50% colored and 50% non-colored (step 7). The colored seeds all 
grow into fertile corn plants of genotype S/S,Rf/- which are the 
maintainer plants, or the second parent plants, of the present invention. 
The noncolored seeds all grow into male-sterile plants of the genotype 
S/S,-/- which are the first parent plants of this invention (Step 7). 
The first and second parent plants are crossed and the seeds harvested from 
the male-sterile plants are separated on the basis of color (Step 8). All 
colored seeds grow again in second parent plants and all noncolored seeds 
grow in first parent plants, thereby establishing an easy maintenance of a 
pure male-sterile line of corn. 
If the plant DNA that is flanking the s gene in the plants of Example 1 has 
been characterized, the progeny of the cross in Step 5 with genotypes 
S/S,-/- and S/S,R/- can be easily identified by means of PCR using probes 
corresponding to the flanking plant DNA. In this way Step 6 can be skipped 
because the plants of Step 5 which grow from colored seeds (genotype 
S/S,Rf/-) can be crossed directly to plants with genotype S/S,-/- (as in 
Step 7). 
All publications cited in this application are hereby incorporated by 
reference. 
Example 6 
Maintainer Slants Containing a Color-linked Restorer Gene Comprising the 
B-Peru Coding Region Under control of the Promoter of the C1-S Gene. 
Using conventional techniques a chimeric gene is inserted between the EcoRI 
and HindIII sites of the polylinker of plasmid pUC19. The chimeric gene 
comprises the following elements in sequence: 
i) the promoter region of the C1-S gene, i.e. the DNA fragment with the 
sequence of SEQ ID No. 1 from nucleotide positions 447 up to 1076 but 
containing at nucleotide positions 935-939 the sequence TTAG instead of 
TGCAG. 
ii) a single C nucleotide 
iii) the coding region and 3'untranslated region of the B-peru gene, i.e. 
the DNA fragment with the sequence of SEQ ID No. 7 from nucleotide 
positions 576 up to 4137. 
This plasmid (designated as pLH52), together with plasmid pCOL9S of Example 
2 (comprising a C1-S gene) and pTS256 of SEQ ID No. 2 (comprising the 
following chimeric genes: P35S-bar-3'nos and PTA29-barstar-3'nos), is used 
to transform corn essentially as described in Example 4. The transformed 
plants are then used to obtain second parent plants as described in 
Example 5. 
Example 7 
Maintainer Plants Containing a Color-linked Restorer Gene Comprising the 
B-Peru Coding Region Under Control of the 35S Promoter. 
Using conventional techniques a chimeric gene is inserted between the EcoRI 
and HindIII sites of the polylinker of plasmid pUC19. The chimeric gene 
comprises the following elements in sequence: 
i) The promoter region of the 35S promoter, i.e. the DNA fragment of pDE110 
which essentially has the sequence as described in SEQ ID No. 4 of WO 
92/09696 (which is incorporated herein by reference) from nucleotide 
positions 396 up to 1779 
ii) the coding region and 3'untranslated region of the B-peru gene, i.e. 
the DNA fragment with the sequence of SEQ ID No. 7 from nucleotide 
positions 576 up to 4137. 
This plasmid (designated as pP35S-Bp), together with plasmid pCOL9S of 
Example 2 (comprising a C1-S gene) and pTS256 of SEQ ID No. 2 (comprising 
the following chimeric genes: P35S-bar-3'nos and PTA-29-bar-3'nos), is 
used to transform corn essentially as described in Example 4. The 
transformed plants are then used to obtain second parent plants as 
described in Example 5. 
Alternatively plasmid p35SBperu as described in Goff et al, 1990, EMBO 
9:2517-2522 is used instead of pP35SBp. 
Example 8 
Maintainer Plants Containing a Color-linked Restorer Gene Comprising the 
Maize P Gene Coding Region Under the Controls the Promoter of the C1-S 
Gene. 
Using conventional techniques a chimeric gene is inserted in the EcoRI site 
of the polylinker of plasmid pUC19. The chimeric gene comprises the 
following elements in sequence; 
i) the promoter region of the C1-S gene, i.e. the DNA fragment with the 
sequence of SEQ ID No. 1 for nucleotide positions 447 up to 1076 but 
containing at nucleotide positions 935-939 the sequence TTAGG instead of 
TGCAG: 
ii) a single C nucleotide; 
iii) a DNA sequence comprising the coding region and 3'end untranslated 
region of the maize P gene as described by Grotewold et al in 1991, PNAS 
88:4587-4591 (nucleotides 320-1517). The maize P gene is an anthocyanin 
regulatory gene which specifies red phlobaphene pigmentation, a flavonoid 
pigment involved in the biosynthesis pathway of anthocyanin. In fact, the 
protein encoded by the P gene activates, among others, the A1 gene 
required for both anthocyanin and phlobaphene pigmentation. Two cDNA 
clones have been isolated and sequenced by Grotewold et al and are 
described in the publication referred to above. It is the longer cDNA 
which is of particular interest for construction of this chimeric gene. 
However, alternatively, the coding region of the shorter transcript can 
also be used in this chimeric gene, as well as the P gene leader sequence 
instead of the CI-S gene leader sequence. The P gene does not require a 
functional R or B gene to produce pigmentation. The visible pigment that 
is produced in the seeds of the maintainer plants is phlobaphene, a 
flavonoid pigment (like anthocyanin) directly involved in anthocyanin 
biosynthesis. 
iv) a DNA fragment containing the polyadenylation signal of the nopaline 
synthase gene of Agrobacterium tumefaciens, i.e. the DNA fragment with the 
sequence of SEQ ID. No. 2 from nucleotide position 1600 up to nucleotide 
position 2909. 
The resulting plasmid (designated as pPCS1-P), together with pTS256 of SEQ 
ID No. 2 is used to transform corn essentially as described in example 4. 
The transformed plants are then used to obtain second parent plants as 
described in example 5. 
Example 9 
Maintainer Plants Containing a Color-linked Restorer Gene Comprising the 
B-peru Coding Region Under the Control of the B-peru Promoter. 
Using conventional techniques a chimeric gene is inserted between the EcoR1 
and the HindIII sites of the polylinker of plasmid pUC19. The chimeric 
gene comprises the following elements in sequence: 
i) the promoter of the B-peru gene, i.e. a 1952 bp DNA sequence as 
disclosed in the EMBL databank under accession number X70791; 
ii) the coding region and 3'untranslated region of the B-peru gene, i.e. 
the DNA fragment with the sequence of SEQ ID No. 7 from nucleotide 
position 576 up to 4137. This plasmid (designated aspCOL11), together with 
plasmid pCOL 9S of example 2 (comprising a C1-S gene) and pTS256 of SEQ ID 
No. 2 (comprising the following chimeric genes: P35S-bar-3'nos and 
PTA29-barstar-3'nos) is used to transform corn essentially as described in 
example 4. The transformed plants are then used to obtain second parent 
plants as described in example 5. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4059 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: C1 gene of Zea mays 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 279..284 
(D) OTHER INFORMATION: /label=HpaI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 447..452 
(D) OTHER INFORMATION: /label=EcoRI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1735..1740 
(D) OTHER INFORMATION: /label=AatII 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1505..1510 
(D) OTHER INFORMATION: /label=EcoRI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2081..2086 
(D) OTHER INFORMATION: /label=XhoI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2418..2430 
(D) OTHER INFORMATION: /label=SfiI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2669..2674 
(D) OTHER INFORMATION: /label=SnaBI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2634..2639 
(D) OTHER INFORMATION: /label=SnaBI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 3008..3013 
(D) OTHER INFORMATION: /label=HpaI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1..1077 
(D) OTHER INFORMATION: /label=PC1 
/note= "region containing promoter of C1 gene" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1078..2134 
(D) OTHER INFORMATION: /label=C1 
/note= "coding region of C1 gene" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2135..2430 
(D) OTHER INFORMATION: /label=3'C1 
/note= "region containing polyadenylation signal 
of C1 gene" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1033..1038 
(D) OTHER INFORMATION: /label=TATA-Box 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1061..1062 
(D) OTHER INFORMATION: /label=transcript-init 
/note= "transcription initiation site" 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 1211..1299 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 1430..1575 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 935..939 
(D) OTHER INFORMATION: /label= C1- S 
/note= "TGCAG sequence (in C1 gene) which in the 
C1-S sequence is changed to TTAGG" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TATCAACCTCCTGTGTTATTTTTAGTGACGGTTTCTTAAAAAACACCACTAGAAATCGTA60 
TTTTTATAGGTGGTTCCTTAAGAAAACTGCATGCAGAAATCCATGACGGTTTTCTTAAGG120 
AACCGTATGTAGAAATACGATTTCTAGTGACGATCTTCTTAAGGAAACCACCACTAAAAA180 
TTATTTTTATCCTTAATTTTCGAGTTTTTCAAACGATCTCGTATGATGAAACCATCAAAA240 
TAAAAGTTGTACATCTCTAAAAGTTATGAAAATTTGTAGTTAACAACTTTTTTATTTGAA300 
CTCATTTTGGTTCTCAAAAATTGCATCTAAATTTGTCAAATTTAAAATTCAAATTTTCCA360 
AACGACCTCGGATGAAAAAAGTGTCAAAATGAAAGTTGTAGAACTTCAAAAGTTATTCAA420 
CTTTGTAGTCGACTATCTTTTTATTTGAATTCGCTTACGGTCTCAAACAAGCAATTTACA480 
CTCAGTTGGTTGTAATATGTGGACAATAAAACTACAAACTAGACACAAATCATACCATAG540 
ACGGAGTGGTAGCAGAGGGTACGCGCGAGGGTGAGATAGAGGATTCTCCTAAAATAAATG600 
CACTTTAGATGGGTAGGGTGGGGTGAGGCCTCTCCTAAAATGAAACTCGTTTAATGTTTC660 
TAAAAATAGTTTTCACTGGTGATCCTTAGTTACTGGCATGTAAAAATGATGATTTCTACT720 
GTCTCTCATATGGACGGTTATAAAAAATACCATTATATTGAAAATAGGTCTCTGCTGCTA780 
CACTCGCCCTCATAGCAGATCATGCATGCACGCATCATTCGATCAGTTTTCGTTCTGATG840 
CAGTTTTCGATAAATGCCAATTTTTTAACTGCATACGTTGCCCTTGCTCAGCACCAGCAC900 
AGCAGTGTCGTGTCGTCCATGCATGCACTTTAGGTGCAGTGCAGGGCCTCAACTCGGCCA960 
CGTAGTTAGCGCCACTGCTACAGATCGAGGCACCGGTCAGCCGGCCACGCACGTCGACCG1020 
CGCGCGTGCATTTAAATACGCCGACGACGGAGCTTGATCGACGAGAGAGCGAGCGCGATG1080 
GGGAGGAGGGCGTGTTGCGCGAAGGAAGGCGTTAAGAGAGGGGCGTGGACGAGCAAGGAG1140 
GACGATGCCTTGGCCGCCTACGTCAAGGCCCATGGCGAAGGCAAATGGAGGGAAGTGCCC1200 
CAGAAAGCCGGTAAAACTAGCTAGTCTTTTTATTTCATTTTGGGATCATATATATACCCC1260 
CGAGGCAAGACCGGAGGACGATCACGTGTGTGGGTGCAGGTTTGCGTCGGTGCGGCAAGA1320 
GCTGCCGGCTGCGGTGGCTGAACTACCTCCGGCCCAACATCAGGCGCGGCAACATCTCCT1380 
ACGACGAGGAGGATCTCATCATCCGCCTCCACAGGCTCCTCGGCAACAGGTCTGTGCAGT1440 
GGCCAGTGGTGGGCTAGCTTATTACACGAGCTGACGACGAGGCGATCGATCGAGCGTCTG1500 
CTGCGAATTCATCTGTTCCGGTGTCGGCCGTGTGAGAGTGAGCTCATTCATATGTACATG1560 
CGTGTTGGCGCGCAGGTGGTCGCTGATTGCAGGCAGGCTCCCTGGCCGAACAGACAATGA1620 
AATCAAGAACTACTGGAACAGCACGCTGGGCCGGAGGGCAGGCGCCGGCGCCGGCGCCGG1680 
CGGCAGCTGGGTCGTCGTCGCGCCGGACACCGGCTCGCACGCCACCCCGGCCGCGACGTC1740 
GGGCGCCTGCGAGACCGGCCAGAATAGCGCCGCTCATCGCGCGGACCCCGACTCAGCCGG1800 
GACGACGACGACCTCGGCGGCGGCGGTGTGGGCGCCCAAGGCCGTGCGGTGCACGGGCGG1860 
ACTCTTCTTCTTCCACCGGGACACGACGCCGGCGCACGCGGGCGAGACGGCGACGCCAAT1920 
GGCCGGTGGAGGTGGAGGAGGAGGAGGAGAAGCAGGGTCGTCGGACGACTGCAGCTCGGC1980 
GGCGTCGGTATCGCTTCGCGTCGGAAGCCACGACGAGCCGTGCTTCTCCGGCGACGGTGA2040 
CGGCGACTGGATGGACGACGTGAGGGCCCTGGCGTCGTTTCTCGAGTCCGACGAGGACTG2100 
GCTCCGCTGTCAGACGGCCGGGCAGCTTGCGTAGACAACAAGTACACGTATAGATGTCCA2160 
ATAAGCACGAGGCCCGCGAGCCCGGCACGAAGCCCGCTTTTTGGGCCCGGTCCGAGCCCG2220 
GCACGGCCCGGTTATATGCAGACCCGGGCCGGCCCGGCACGAATAAGCGGGCCGGGCTCG2280 
GACAGGAAATTAGGCACGGTGAGCTAGCCCGGCACGGCCCGTTTAGGTCTAAGCCCGTTA2340 
AGCCCGTTTTTTTACACTAAAACGTGCTTCTCGGCCCGCATAGCCCGCTTCTCGGCCCGC2400 
TTTTTTCGTGCTAAACGGGCCGGCCCGGCCCGGTTTAGGCCCGTTGCGGGCCGGGCTCGG2460 
ACAGGAAATTGAGCCCGCGTGCTTAGCCGGCCCGGCCCGGTTTTTTAATCGTGCCTGGCG2520 
GGCCAGGCCCAAAACGGGCCGGGCTTCACCGGGCCCGGGCCGGACCGGGCCGGGCGGCCC2580 
GTTTGGACATCTCTAAGTACACGTATGGAGGAGAATATATATATAGTCATGCGTACGTAT2640 
AGATTTTTTCATCCGATCCCAACAGAAATACGTATGAAAATGCTCTTCGTTCTTTTTCAT2700 
TTATCATATCTATACTATACTTAAAACACCAGTTTCAACGGTCGTCATGCGTCATTTTTT2760 
TACAAATAACCCCTCACAGCTATTTCAAATTAATCCGCTGCACGTCTATAGATGCCAAAC2820 
GACGCCCAACACGGGCTAGATGCACGCGGGCCACAACTATGGCACAGGCACGTCATGCCG2880 
GCCTGCTAACTGTGTCGGGCTAGCCCGTTAGCCCGTCGATCCATTTAATTAAATTAGCGT2940 
AACGACGCCCGACACGGGCTAGATGCACGTGGGCCACAACTATGGCACATGCACGTCATG3000 
CCGGCCTGTTAACTGTGTCGGGCCAGTCTGTTAGCCCATTGATCCATTTAATTAAATCAG3060 
CGTAAAATGTTAAAAACGGTGCAGGAGGTGGGGTTCGAACCCATACCCTGATGGAAGAAG3120 
GGCGGGAGACACTGGGTGAAACTGTCTAACCAGTAGAATATCTATCACGCTAAGATGTTT3180 
TTAATATTGAATATAAATTGTATATAAGCATATAAGTTTTTTTGTAAAATAAAAAATAAT3240 
CGTGTCGGGCCGGGCCATCACTACTGGCCGAGGCTACAACCCAAGCACGACACGACGTTC3300 
TTGGCTCTTGCAAGCATTAGGTCGTTTCTGAGACCATATTGGCGCAATGGACTACATGAT3360 
GTTTGGGGTTGCTGAATTGAATGGAGCAGCAATAATTTGTCACACTAACAGCAAAATGAA3420 
AGGTTATTTGTTGGTTTTAAACGTTAGTAATTGCTACGAAGTAGCATAATTTATATGGAG3480 
CGCATCCAGTTTTTATTGATGCCTGACTTTAGCAATCACTCCATATTTTGATCTATCTTT3540 
TTTATAAGTTTGACTTCATGGGACTTATTTTAGAACTTGATCTCACAAACTTTCTCTTAT3600 
TTTGTCTCTATATGATGAAATTGTGTCATTTTATAATCTTTGTTCATTCAGTCAATCGTT3660 
GTGAACTCTCTTCTAATCACTCACTTCATTAGTTGTGTTGTACCAAGACATATTTGCATA3720 
GAGTAAACAATAACATCAGTTAGCCAAATCAAAAAATATATTATACAGAGAGCGGAGACA3780 
ATCAAATAAAAAATCTTGAAATTTTTTTAATGGATAGTTTACGTGGGTATTGTTGTAAGC3840 
CGTCGCAACGCACGGGCAACCGACTAGTTTTAGTTTATAAATTAATAAACGTACGACAAA3900 
TATTAAGAACGCCACCTTTCCATGCCTACGCGCGCGTGAGACACGACCGGGGCACGTCAG3960 
ACGTGTGCCCCTGTTGTATAATTTATTTACTTTTTAATGACTATGTGCTGTTGGTTGCCG4020 
TTGGCTTCATCGTGTTCGTAGCCATGCATAAATCCAGCG4059 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4896 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: plasmid pTS256, linearized at HindIII 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: complement (39..317) 
(D) OTHER INFORMATION: /label=3'nos 
/note= "3'regulatory sequence containing the 
polyadenylation signal of the nopaline synthase 
gene of Agrobacterium T-DNA" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: complement (318..869) 
(D) OTHER INFORMATION: /label=bar 
/note= "coding region of bar gene of Streptomyces 
hygroscopicus" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: complement (870..1702) 
(D) OTHER INFORMATION: /label=P35S 
/note= "35S promoter of Cauliflower Mosaic Virus" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1740..2284 
(D) OTHER INFORMATION: /label=PTA29 
/note= "promoter of TA29 gene of Nicotiana 
tabacum" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2285..2557 
(D) OTHER INFORMATION: /label=barstar 
/note= "coding region of barstar gene of Bacillus 
amyloliquefaciens" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2558..2879 
(D) OTHER INFORMATION: /label=3'nos 
/note= "3'regulatory sequence containing the 
polyadenylation signal of the nopaline synthase 
gene of Agrobacterium T-DNA" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1..38 
(D) OTHER INFORMATION: /label=pUC19 
/note= "pUC19 derived sequence" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2880..4896 
(D) OTHER INFORMATION: /label=pUC19 
/note= "pUC19 derived sequence" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 3004..3009 
(D) OTHER INFORMATION: /label=EcoRI 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCTTCCCGATCTAGTAACATAGATGAC60 
ACCGCGCGCGATAATTTATCCTAGTTTGCGCGCTATATTTTGTTTTCTATCGCGTATTAA120 
ATGTTATAATGCGGGACTCTAATCATAAAAACCCATCTCATAAATAACGTCATGCATTAC180 
ATGTTAATTATTACATGCTTAACGTAATTCAACAGAAATTATATGATAATCATCGCAAGA240 
CCGGCAACAGGATTCAATCTTAAGAAACTTTATTGCCAAATGTTTGAACGATCTGCTTCG300 
GATCCTAGACGCGTGAGATCAGATCTCGGTGACGGGCAGGACCGGACGGGGCGGTACCGG360 
CAGGCTGAAGTCCAGCTGCCAGAAACCCACGTCATGCCAGTTCCCGTGCTTGAAGCCGGC420 
CGCCCGCAGCATGCCGCGGGGGGCATATCCGAGCGCCTCGTGCATGCGCACGCTCGGGTC480 
GTTGGGCAGCCCGATGACAGCGACCACGCTCTTGAAGCCCTGTGCCTCCAGGGACTTCAG540 
CAGGTGGGTGTAGAGCGTGGAGCCCAGTCCCGTCCGCTGGTGGCGGGGGGAGACGTACAC600 
GGTCGACTCGGCCGTCCAGTCGTAGGCGTTGCGTGCCTTCCAGGGGCCCGCGTAGGCGAT660 
GCCGGCGACCTCGCCGTCCACCTCGGCGACGAGCCAGGGATAGCGCTCCCGCAGACGGAC720 
GAGGTCGTCCGTCCACTCCTGCGGTTCCTGCGGCTCGGTACGGAAGTTGACCGTGCTTGT780 
CTCGATGTAGTGGTTGACGATGGTGCAGACCGCCGGCATGTCCGCCTCGGTGGCACGGCG840 
GATGTCGGCCGGGCGTCGTTCTGGGTCCATGGTTATAGAGAGAGAGATAGATTTATAGAG900 
AGAGACTGGTGATTTCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAA960 
GGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATGTCACAT1020 
CAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTG1080 
GGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAATGATAGCCTTTCCT1140 
TTATCGCAATGATGGCATTTGTAGGAGCCACCTTCCTTTTCTACTGTCCTTTCGATGAAG1200 
TGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGAAATTATCCTTTGTTGAAAA1260 
GTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGACATTTTTGGAGTAGACCAGA1320 
GTGTCGTGCTCCACCATGTTGACGAAGATTTTCTTCTTGTCATTGAGTCGTAAAAGACTC1380 
TGTATGAACTGTTCGCCAGTCTTCACGGCGAGTTCTGTTAGATCCTCGATTTGAATCTTA1440 
GACTCCATGCATGGCCTTAGATTCAGTAGGAACTACCTTTTTAGAGACTCCAATCTCTAT1500 
TACTTGCCTTGGTTTATGAAGCAAGCCTTGAATCGTCCATACTGGAATAGTACTTCTGAT1560 
CTTGAGAAATATGTCTTTCTCTGTGTTCTTGATGCAATTAGTCCTGAATCTTTTGACTGC1620 
ATCTTTAACCTTCTTGGGAAGGTATTTGATCTCCTGGAGATTGTTACTCGGGTAGATCGT1680 
CTTGATGAGACCTGCTGCGTAGGAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCC1740 
ATCTAGCTAAGTATAACTGGATAATTTGCATTAACAGATTGAATATAGTCGCAAACAAGA1800 
AGGGACAATTGACTTGTCACTTTATGAAAGATGATTCAAACATGATTTTTTATGTACTAA1860 
TATATACATCCTACTCGAATTAAAGCGACATAGGCTCGAAGTATGCACATTTAGCAATGT1920 
AAATTAAATCAGTTTTTGAATCAAGCTAAAAGCAGACTTGCATAAGGTGGGTGGCTGGAC1980 
TAGAATAAACATCTTCTCTAGCACAGCTTCATAATGTAATTTCCATAACTGAAATCAGGG2040 
TGAGACAAAATTTTGGTACTTTTTCCTCACACTAAGTCCATGTTTGCAACAAATTAATAC2100 
ATGAAACCTTAATGTTACCCTCAGATTAGCCTGCTACTCCCCATTTTCCTCGAAATGCTC2160 
CAACAAAAGTTAGTTTTGCAAGTTGTTGTGTATGTCTTGTGCTCTATATATGCCCTTGTG2220 
GTGCAAGTGTAACAGTACAACATCATCACTCAAATCAAAGTTTTTACTTAAAGAAATTAG2280 
CTACCATGAAAAAAGCAGTCATTAACGGGGAACAAATCAGAAGTATCAGCGACCTCCACC2340 
AGACATTGAAAAAGGAGCTTGCCCTTCCGGAATACTACGGTGAAAACCTGGACGCTTTAT2400 
GGGATTGTCTGACCGGATGGGTGGAGTACCCGCTCGTTTTGGAATGGAGGCAGTTTGAAC2460 
AAAGCAAGCAGCTGACTGAAAATGGCGCCGAGAGTGTGCTTCAGGTTTTCCGTGAAGCGA2520 
AAGCGGAAGGCTGCGACATCACCATCATACTTTCTTAATACGATCAATGGGAGATGAACA2580 
ATATGGAAACACAAACCCGCAAGCTTGGTCTAGAGGATCCGAAGCAGATCGTTCAAACAT2640 
TTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATA2700 
ATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTAT2760 
GAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAA2820 
AATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCG2880 
GGAAGATCCCCGGGTACCGAGCTCGAATTCTGATCAGGCCAACGCGCGGGGAGAGGCGGT2940 
TTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGG3000 
CTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGG3060 
GATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAG3120 
GCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA3180 
CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT3240 
GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCC3300 
TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCG3360 
GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGC3420 
TGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCA3480 
CTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAG3540 
TTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCT3600 
CTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACC3660 
ACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGA3720 
TCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCA3780 
CGTTAAGGGATTTTGGTCATGAGACTCGAGCCAAAAAGGATCTTCACCTAGATCCTTTTA3840 
AATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGT3900 
TACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATA3960 
GTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCC4020 
AGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAAC4080 
CAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAG4140 
TCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAAC4200 
GTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC4260 
AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCG4320 
GTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTC4380 
ATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCT4440 
GTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGC4500 
TCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTC4560 
ATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCC4620 
AGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC4680 
GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACA4740 
CGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGT4800 
TATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAATAAACAAATAGGGGTTC4860 
CGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCA4896 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3544 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: EcoRI-HindIII region of plasmid pTS200 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 3227..3504 
(D) OTHER INFORMATION: /label=3'nos 
/note= "3'regulatory sequence containing the 
polyadenylation signal of the nopaline synthase 
gene of Agrobacterium T-DNA" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2675..3226 
(D) OTHER INFORMATION: /label=bar 
/note= "coding region of bar gene of Streptomyces 
hygroscopicus" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1841..2674 
(D) OTHER INFORMATION: /label=P35S 
/note= "35S promoter of Cauliflower Mosaic Virus" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: complement (626..1803) 
(D) OTHER INFORMATION: /label=PCA55 
/note= "promoter of CA55 gene of Zea mays" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: complement (353..625) 
(D) OTHER INFORMATION: /label=barstar 
/note= "coding region of barstar gene of Bacillus 
amyloliquefaciens" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: complement (30..352) 
(D) OTHER INFORMATION: /label=3'nos 
/note= "3'regulatory sequence containing the 
polyadenylation signal of the nopaline synthase 
gene of Agrobacterium T-DNA" 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1..6 
(D) OTHER INFORMATION: /label=EcoRI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 3539..3544 
(D) OTHER INFORMATION: /label=HindIII 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GAATTCGAGCTCGGTACCCGGGGATCTTCCCGATCTAGTAACATAGATGACACCGCGCGC60 
GATAATTTATCCTAGTTTGCGCGCTATATTTTGTTTTCTATCGCGTATTAAATGTATAAT120 
TGCGGGACTCTAATCATAAAAACCCATCTCATAAATAACGTCATGCATTACATGTTAATT180 
ATTACATGCTTAACGTAATTCAACAGAAATTATATGATAATCATCGCAAGACCGGCAACA240 
GGATTCAATCTTAAGAAACTTTATTGCCAAATGTTTGAACGATCTGCTTCGGATCCTCTA300 
GACCAAGCTTGCGGGTTTGTGTTTCCATATTGTTCATCTCCCATTGATCGTATTAAGAAA360 
GTATGATGGTGATGTCGCAGCCTTCCGCTTTCGCTTCACGGAAAACCTGAAGCACACTCT420 
CGGCGCCATTTTCAGTCAGCTGCTTGCTTTGTTCAAACTGCCTCCATTCCAAAACGAGCG480 
GGTACTCCACCCATCCGGTCAGAGAATCCCATAAAGCGTCCAGGTTTTCACCGTAGTATT540 
CCGGAAGGGCAAGCTCCTTTTTCAATGTCTGGTGGAGGTCGCTGATACTTCTGATTTGTT600 
CCCCGTTAATGACTGCTTTTTTCATGGCTGCAGCTAGTTAGCTCGATGTATCTTCTGTAT660 
ATGCAGTGCAGCTTCTGCGTTTTGGCTGCTTTGAGCTGTGAAATCTCGCTTTCCAGTCCC720 
TGCGTGTTTTATAGTGCTGTACGTTCGTGATCGTGAGCAAACAGGGCGTGCCTCAACTAC780 
TGGTTTGGTTGGGTGACAGGCGCCAACTACGTGCTCGTAACCGATCGAGTGAGCGTAATG840 
CAACATTTTTTCTTCTTCTCTCGCATTGGTTTCATCCAGCCAGGAGACCCGAATCGAATT900 
GAAATCACAAATCTGAGGTACAGTATTTTTACAGTACCGTTCGTTCGAAGGTCTTCGACA960 
GGTCAAGGTAACAAAATCAGTTTTAAATTGTTGTTTCAGATCAAAGAAAATTGAGATGAT1020 
CTGAAGGACTTGGACCTTCGTCCAATGAAACACTTGGACTAATTAGAGGTGAATTGAAAG1080 
CAAGCAGATGCAACCGAAGGTGGTGAAAGTGGAGTTTCAGCATTGACGACGAAAACCTTC1140 
GAACGGTATAAAAAAGAAGCCGCAATTAAACGAAGATTTGCCAAAAAGATGCATCAACCA1200 
AGGGAAGACGTGCATACATGTTTGATGAAAACTCGTAAAAACTGAAGTACGATTCCCCAT1260 
TCCCCTCCTTTTCTCGTTTCTTTTAACTGAAGCAAAGAATTTGTATGTATTCCCTCCATT1320 
CCATATTCTAGGAGGTTTTGGCTTTTCATACCCTCCTCCATTTCAAATTATTTGTCATAC1380 
ATTGAAGATATACACCATTCTAATTTATACTAAATTACAGCTTTTAGATACATATATTTT1440 
ATTATACACTTAGATACGTATTATATAAAACACCTAATTTAAAATAAAAAATTATATAAA1500 
AAGTGTATCTAAAAAATCAAAATACGACATAATTTGAAACGGAGGGGTACTACTTATGCA1560 
AACCAATCGTGGTAACCCTAAACCCTATATGAATGAGGCCATGATTGTAATGCACCGTCT1620 
GATTAACCAAGATATCAATGGTCAAAGATATACATGATACATCCAAGTCACAGCGAAGGC1680 
AAATGTGACAACAGTTTTTTTTACCAGAGGGACAAGGGAGAATATCTATTCAGATGTCAA1740 
GTTCCCGTATCACACTGCCAGGTCCTTACTCCAGACCATCTTCCGGCTCTATTGATGCAT1800 
ACCAGGAATTGATCTAGAGTCGACCTGCAGGCATGCAAGCTCCTACGCAGCAGGTCTCAT1860 
CAAGACGATCTACCCGAGTAACAATCTCCAGGAGATCAAATACCTTCCCAAGAAGGTTAA1920 
AGATGCAGTCAAAAGATTCAGGACTAATTGCATCAAGAACACAGAGAAAGACATATTTCT1980 
CAAGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCATAAACCAAGGCA2040 
AGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCTACTGAATCTAAGGCCATGCATGG2100 
AGTCTAAGATTCAAATCGAGGATCTAACAGAACTCGCCGTGAAGACTGGCGAACAGTTCA2160 
TACAGAGTCTTTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACG2220 
ACACTCTGGTCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTG2280 
AGACTTTTCAACAAAGGATAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCT2340 
GTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCG2400 
ATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCC2460 
CACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGG2520 
ATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAG2580 
ACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGT2640 
CTCTCTCTATAAATCTATCTCTCTCTCTATAACCATGGACCCAGAACGACGCCCGGCCGA2700 
CATCCGCCGTGCCACCGAGGCGGACATGCCGGCGGTCTGCACCATCGTCAACCACTACAT2760 
CGAGACAAGCACGGTCAACTTCCGTACCGAGCCGCAGGAACCGCAGGAGTGGACGGACGA2820 
CCTCGTCCGTCTGCGGGAGCGCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGC2880 
CGGCATCGCCTACGCGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTC2940 
GACCGTGTACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACACCCA3000 
CCTGCTGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTGTCATCGGGCTGCC3060 
CAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCCCGCGGCATGCTGCG3120 
GGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTGGCAGCTGGACTTCAG3180 
CCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCACCGAGATCTGATCTCACGCGTCTAG3240 
GATCCGAAGCAGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTG3300 
CCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTA3360 
ACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTAT3420 
ACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCG3480 
CGGTGTCATCTATGTTACTAGATCGGGAAGATCCTCTAGAGTCGACCTGCAGGCATGCAA3540 
GCTT3544 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: oligonucleotide 1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CGTTTCTCGAATCCGACGAGG21 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4824 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: circular 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: plasmid pCOL9 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 396..401 
(D) OTHER INFORMATION: /label=EcoRI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2367..2379 
(D) OTHER INFORMATION: /label=SfiI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 884..888 
(D) OTHER INFORMATION: /label= C1- S 
/note= "TGCAG (in C1) which in C1-S allele is 
replaced with TTAGG" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA60 
CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG120 
TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC180 
ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCC240 
ATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTAT300 
TACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGT360 
TTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGCTTACGGTCTCAAACAAG420 
CAATTTACACTCAGTTGGTTGTAATATGTGGACAATAAAACTACAAACTAGACACAAATC480 
ATACCATAGACGGAGTGGTAGCAGAGGGTACGCGCGAGGGTGAGATAGAGGATTCTCCTA540 
AAATAAATGCACTTTAGATGGGTAGGGTGGGGTGAGGCCTCTCCTAAAATGAAACTCGTT600 
TAATGTTTCTAAAAATAGTTTTCACTGGTGATCCTTAGTTACTGGCATGTAAAAATGATG660 
ATTTCTACTGTCTCTCATATGGACGGTTATAAAAAATACCATTATATTGAAAATAGGTCT720 
CTGCTGCTACACTCGCCCTCATAGCAGATCATGCATGCACGCATCATTCGATCAGTTTTC780 
GTTCTGATGCAGTTTTCGATAAATGCCAATTTTTTAACTGCATACGTTGCCCTTGCTCAG840 
CACCAGCACAGCAGTGTCGTGTCGTCCATGCATGCACTTTAGGTGCAGTGCAGGGCCTCA900 
ACTCGGCCACGTAGTTAGCGCCACTGCTACAGATCGAGGCACCGGTCAGCCGGCCACGCA960 
CGTCGACCGCGCGCGTGCATTTAAATACGCCGACGACGGAGCTTGATCGACGAGAGAGCG1020 
AGCGCGATGGGGAGGAGGGCGTGTTGCGCGAAGGAAGGCGTTAAGAGAGGGGCGTGGACG1080 
AGCAAGGAGGACGATGCCTTGGCCGCCTACGTCAAGGCCCATGGCGAAGGCAAATGGAGG1140 
GAAGTGCCCCAGAAAGCCGGTAAAACTAGCTAGTCTTTTTATTTCATTTTGGGATCATAT1200 
ATATACCCCCGAGGCAAGACCGGAGGACGATCACGTGTGTGGGTGCAGGTTTGCGTCGGT1260 
GCGGCAAGAGCTGCCGGCTGCGGTGGCTGAACTACCTCCGGCCCAACATCAGGCGCGGCA1320 
ACATCTCCTACGACGAGGAGGATCTCATCATCCGCCTCCACAGGCTCCTCGGCAACAGGT1380 
CTGTGCAGTGGCCAGTGGTGGGCTAGCTTATTACACGAGCTGACGACGAGGCGATCGATC1440 
GAGCGTCTGCTGCGAATTCATCTGTTCCGGTGTCGGCCGTGTGAGAGTGAGCTCATTCAT1500 
ATGTACATGCGTGTTGGCGCGCAGGTGGTCGCTGATTGCAGGCAGGCTGCCTGGCCGAAC1560 
AGACAATGAAATCAAGAACTACTGGAACAGCACGCTGGGCCGGAGGGCAGGCGCCGGCGC1620 
CGGCGCCGGCGGCAGCTGGGTCGTCGTCGCGCCGGACACCGGCTCGCACGCCACCCCGGC1680 
CGCGACGTCGGGCGCCTGCGAGACCGGCCAGAATAGCGCCGCTCATCGCGCGGACCCCGA1740 
CTCAGCCGGGACGACGACGACCTCGGCGGCGGCGGTGTGGGCGCCCAAGGCCGTGCGGTG1800 
CACGGGCGGACTCTTCTTCTTCCACCGGGACACGACGCCGGCGCACGCGGGCGAGACGGC1860 
GACGCCAATGGCCGGTGGAGGTGGAGGAGGAGGAGGAGAAGCAGGGTCGTCGGACGACTG1920 
CAGCTCGGCGGCGTCGGTATCGCTTCGCGTCGGAAGCCACGACGAGCCGTGCTTCTCCGG1980 
CGACGGTGACGGCGACTGGATGGACGACGTGAGGGCCCTGGCGTCGTTTCTCGAGTCCGA2040 
CGAGGACTGGCTCCGCTGTCAGACGGCCGGGCAGCTTGCGTAGACAACAAGTACACGTAT2100 
AGATGTCCAATAAGCACGAGGCCCGCGAGCCCGGCACGAAGCCCGCTTTTTGGGCCCGGT2160 
CCGAGCCCGGCACGGCCCGGTTATATGCAGACCCGGGCCGGCCCGGCACGAATAAGCGGG2220 
CCGGGCTCGGACAGGAAATTAGGCACGGTGAGCTAGCCCGGCACGGCCCGTTTAGGTCTA2280 
AGCCCGTTAAGCCCGTTTTTTTACACTAAAACGTGCTTCTCGGCCCGCATAGCCCGCTTC2340 
TCGGCCCGCTTTTTTCGTGCTAAACGGGCCGGCCCGGCCCGGTTTAGGCCCGTTGCGGGC2400 
CGGGCTCGGACAGGAAATTGAGCCCGCGTGCTTAGCCGGCCCGGCCCGGTTTTTTAATCG2460 
TGCCTGGCGGGCCAGGCCCAAAACGGGCCGGGCTTCACCGGGCCCGGGCCGGACCGGGCC2520 
GGGCGGCCCGTTTGGACATCTCTAAGTACACGTATGGAGGAGAATATATATATAGTCATG2580 
CGTACAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCA2640 
CAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAG2700 
TGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGT2760 
CGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGC2820 
GCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGG2880 
TATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAA2940 
AGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG3000 
CGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGA3060 
GGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG3120 
TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGG3180 
GAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTC3240 
GCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCG3300 
GTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCA3360 
CTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGT3420 
GGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAG3480 
TTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG3540 
GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATC3600 
CTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTT3660 
TGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT3720 
TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCA3780 
GTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCG3840 
TCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAC3900 
CGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGG3960 
CCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCC4020 
GGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTA4080 
CAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAAC4140 
GATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTC4200 
CTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCAC4260 
TGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT4320 
CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAA4380 
TACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTT4440 
CTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCA4500 
CTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA4560 
AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATAC4620 
TCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCG4680 
GATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCC4740 
GAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATA4800 
GGCGTATCACGAGGCCCTTTCGTC4824 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3915 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: EcoRI-Hind III region of plasmid pCOL13 
(ix) FEATURE: 
(A) NAME/KEY: prim.sub.-- transcript 
(B) LOCATION: 188 
(ix) FEATURE: 
(A) NAME/KEY: exon 
(B) LOCATION: 188..212 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 213..556 
(ix) FEATURE: 
(A) NAME/KEY: exon 
(B) LOCATION: 557..718 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 719..1224 
(ix) FEATURE: 
(A) NAME/KEY: exon 
(B) LOCATION: 1225..2770 
(D) OTHER INFORMATION: /codon.sub.-- start= 2 
/note= "exon containing 3'end coding region of 
B-peru gene" 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 576..718 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1225..2770 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1268..2770 
(D) OTHER INFORMATION: /note= "3'end of B-peru coding 
region which is derived from cDNA" 
(ix) FEATURE: 
(A) NAME/KEY: 3'UTR 
(B) LOCATION: 2771..3272 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 3273..3891 
(D) OTHER INFORMATION: /label=3'region 
/note= "further 3'flanking region of B-peru gene. 
This region is only of approximate length and the 
sequence needs to be confirmed." 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1..6 
(D) OTHER INFORMATION: /label=EcoRI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 11..16 
(D) OTHER INFORMATION: /label=XbaI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 45..50 
(D) OTHER INFORMATION: /label=KpnI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 265..270 
(D) OTHER INFORMATION: /label=HindIII 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 329..334 
(D) OTHER INFORMATION: /label=XbaI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 835..840 
(D) OTHER INFORMATION: /label=BamHI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 1268..1273 
(D) OTHER INFORMATION: /label=MluI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2787..2792 
(D) OTHER INFORMATION: /label=HindIII 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2883..2888 
(D) OTHER INFORMATION: /label=MunI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 2827..2832 
(D) OTHER INFORMATION: /label=HindIII 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 3892..3897 
(D) OTHER INFORMATION: /label=SalI 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 3910..3915 
(D) OTHER INFORMATION: /label=HindIII 
(ix) FEATURE: 
(A) NAME/KEY: - 
(B) LOCATION: 3892..3915 
(D) OTHER INFORMATION: /label=polylinker 
/note= "part of polylinker of pUC19" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GAATTCAGGTTCTAGACTATTCTTGTGGCCTCGGGCGGATGGCGGGTACCCATGTCTTCG60 
TTAGGCTTATCTGACCGTGGAGATGAAATCTAACGGCTCATAGAAATTAAACTAACGTGG120 
ACACTCTGTCCTTGCTGTTTTGCTCCCTGCTCTTTATATATAGAATGCCTGCTTGCATTG180 
CACCCGTACGTACAGCGTAGCGCGGAGTGGAGGTGAGCTCCTCCTCCGATTCTTGCCTAA240 
TCTTTGGTCTTTGCACACGTACGAAAGCTTTTTGCATTGTTTCGTTGCTTCTGGATGATC300 
AGTACTCTTAGATATTAAGCGATACCGATCTAGAATCGAGTTGTTGTACTCTCTCTGTCC360 
CTTTTGTGCAGCTATAACTAGCTAGGTTCCTTCGCATAGAGCCTCTCTACAGAGTACAGA420 
CTAGCTAGCAGTGTCAGACACGAAATGGAAATGGTCACTTCCAAATTGCACGAGCTGGAA480 
TTATATACTCTTCTGATCTTCTTCACCGTCTCTTTATAGCGTGATATGCGTTTCTGGCTT540 
CTTGCTTACGTGAAGGATTATTAGTAAGGCGCGTGATGGCGCTCTCAGCTTCCCCGGCTC600 
AGGAAGAACTGCTGCAGCCTGCTGGGAGGCCGTTGAGGAAGCAGCTTGCTGCAGCCGCGA660 
GGAGCATCAACTGGAGCTATGCCCTCTTCTGGTCCATTTCAAGCACTCAACGACCTCGGT720 
AAATGGAAGTCCTGATAATCTATAATTTGTCTGGCAGTTTTCTACAACTCTGGTGAATGA780 
TCGTCACTTCGTTTGCCTGATACATACATACATACATATGAAATAAAGAAAGTCGGATCC840 
CGTGATGCGATTGTAGTTATCGCTTTTCCGCAAAATGGTTGCTTTTTGAATCTGCATTCG900 
TTTTTTTCCCACATCTTCTTCCTTCTCGCGAGTAACGACAACGCCACCCGCGCCGCCTGC960 
CGCCCATCGCCCCGCCTTGGCCGGCGAGAGCCTCAGCCTATTACACCAGCGGCGACCTCT1020 
TTTCCCCTTCCTCTCACCGCCCTCGTGGCCGTGCTCTCCCCCGCTCTAACCTGGTCTGGC1080 
CGCCTCCGCTGCCACCTGCTCCGGCGGCCTCACCCGCGTCTTTCTCGTCCCTACCCTCTC1140 
TGCCTCTGGGCGCATCATCATCTGATATTCTGATGCAAATAAAAAAGGTATACCATATAA1200 
GGACAACAGAAAATATGGTTGCAGGGTGCTGACGTGGACGGACGGGTTCTACAATGGCGA1260 
GGTGAAGACGCGTAAGATCTCCCACTCCGTGGAGCTGACAGCCGACCAGCTGCTCATGCA1320 
GAGGAGCGAGCAGCTCCGGGAGCTCTACGAGGCCCTCCGGTCCGGCGAGTGCGACCGCCG1380 
CGGCGCGCGGCCGGTGGGCTCGCTGTCGCCGGAGGACCTCGGGGACACCGAGTGGTACTA1440 
CGTGATCTGCATGACCTACGCCTTCCTGCCGGGCCAAGGCTTGCCCGGCAGGAGTTCCGC1500 
GAGCAACGAGCATGTCTGGCTGTGCAACGCGCACCTCGCCGGCAGCAAGGACTTCCCCCG1560 
CGCGCTCCTGGCCAAGAGCGCGTCCATTCAGACAATCGTCTGCATCCCGCTCATGGGTGG1620 
CGTGCTTGAGCTTGGTACTACTGATAAGGTGCCGGAGGACCCGGACTTGGTCAGCCGAGC1680 
AACCGTAGCATTCTGGGAGCCGCAATGTCCGACATACTCGAAAGAGCCGAGCTCCAACCC1740 
GTCAGCATACGAAACCGGGGAAGCCGCATACATAGTCGTGTTGGAGGACCTCGATCACAA1800 
TGCCATGGACATGGAGACGGTGACTGCCGCCGCCGGGAGACACGGAACCGGACAGGAGCT1860 
AGGAGAAGTCGAGAGCCCGTCAAATGCAAGCCTGGAGCACATCACCAAGGGGATCGACGA1920 
GTTCTACAGCCTCTGCGAGGAAATGGACGTGCAGCCGCTAGAGGATGCCTGGATAATGGA1980 
CGGGTCTAATTTCGAAGTCCCGTCGTCAGCGCTCCCGGTGGATGGCTCAAGCGCACCCGC2040 
TGATGGTTCTCGCGCGACAAGTTTCGTGGTTTGGACGAGGTCATCGCACTCCTGCTCGGG2100 
TGAAGCGGCGGTGCCGGTCATCGAAGAGCCGCAGAAATTGCTGAAGAAAGCGTTGGCCGG2160 
CGGCGGTGCTTGGGCGAACACGAACTGCGGTGGCGGGGGCACGACGGTAACAGCCCAGGA2220 
AAACGGCGCCAAGAACCACGTCATGTCAGAGCGAAAGCGCCGGGAGAAGCTCAACGAGAT2280 
GTTCCTCGTTCTCAAGTCGTTGGTTCCCTCCATTCACAAGGTGGACAAAGCATCCATCCT2340 
CGCCGAAACGATAGCCTATCTAAAGGAGCTTCAACGAAGGGTACAAGAACTGGAATCCAG2400 
GAGGCAAGGTGGCAGTGGGTGTGTCAGCAAGAAAGTCTGTGTGGGCTCCAACTCCAAGAG2460 
GAAGAGCCCAGAGTTCGCCGGTGGCGCGAAGGAGCACCCCTGGGTCCTCCCCATGGACGG2520 
CACCAGCAACGTCACCGTCACCGTCTCGGACACGAACGTGCTCCTGGAGGTGCAATGCCG2580 
GTGGGAGAAGCTCCTGATGACACGGGTGTTCGACGCCATCAAGAGCCTCCATTTGGACGC2640 
TCTCTCGGTTCAGGCTTCGGCACCAGATGGCTTCATGAGGCTCAAGATAGGAGCTCAGTT2700 
TGCAGGCTCCGGCGCCGTCGTGCCCGGAATGATCAGCCAATCTCTTCGTAAAGCTATAGG2760 
GAAGCGATGAAAGGGCGCTACATGTGAAGCTTAATTAATGGAAGCAAACTTGTATTTCTT2820 
GTGCAAAAGCTTACTATATATTTCTGCAAAACCTGGTGTGCCTTGTTTTGATTTTCAGTC2880 
GCCAATTGTGCCTTTGTTTTTATCAAGTGATGATCTACACATATATATAGGAATATTTGA2940 
AAAGAGCGATGTCATAGGGTTTTTTTATTACAAGGAACAAGTCTTTCACGTGCTGGCCTC3000 
ACAAATCCTAAGAGAAAATCTGCTCATTTTGATTGCGTTCCGCAACAACTCTGTAATCCA3060 
TATCCTATGTATCCGATCAACTAGTCGATAGCCTCCGTCCGCCACATCATCATATATCTA3120 
TCTATGTGTGTCATCTGACACATACTCCTCGCGTACTGTGCTGACATATGATACTGACAC3180 
AGCATATATGCATGCACATCGTCACACGACATATATCTCGCTACTACACAGATATTGGAT3240 
ACGATACTATATAGCATCATGCGTGCTGCGATNNNNNNNNNNNNNNNNNNNNNNNNNNNN3300 
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3360 
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3420 
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3480 
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3540 
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3600 
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3660 
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3720 
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3780 
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3840 
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTCGACCTG3900 
CAGGCATGCAAGCTT3915 
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