Transgenic plants including a transgene consisting of a hybrid nucleic acid sequence, comprising at least one unedited mitochondrial gene fragment from higher plants and process for producing them

Hybrid nucleic acid sequences including at least the coding region of an unedited mitochondrial gene of superior plants and controlling the male fertility of plants containing said sequences, transgenic plants having such sequences and methods of production of transgenic male-sterile plants and method of restoring male-fertile plants. The nuclei of the transgenic plants contain a hybrid sequence capable of being expressed (transgene), comprising at least one coding region of an unedited mitochondrial gene of superior plants and a sequence capable of transferring the protein expressed by said coding region, to the mitochondrion, said hybrid sequence being capable of modifying the male fertility of plants having incorporated said transgene, while leaving the other phenotype characteristics of said plants unaltered.

TECHNICAL FIELD 
The present invention relates to hybrid nucleic acid sequences, comprising 
at least the coding region of an unedited mitochondrial gene from higher 
plants and allowing the control of male fertility in plants containing the 
said sequences, to the transgenic plants having such sequences, as well as 
to a method for producing transgenic male-sterile plants and to a method 
for restoring male-fertile plants. 
BACKGROUND ART 
The control of male fertility in plants is one of the key problems for 
obtaining hybrids, and more particularly male-sterile lines which are of 
agronomic interest especially for controlling and improving seeds. Indeed, 
the large scale production of hybrid seeds with controlled characteristics 
is a real challenge since many crops have both male and female 
reproductive organs (stamens and pistils). This causes a high rate of 
self-pollination and makes difficult the control of crossings between 
lines for obtaining the desired hybrids. 
In order to allow non-inbred crossings to be obtained which make it 
possible to produce hybrid seeds having advantageous properties, the 
inventors have developed new transgenic male-sterile plants capable of 
being restored and which facilitate the development of hybrid crops. 
Cytoplasmic male sterility (MCS) is characterized by non-formation of the 
pollen after meiosis. 
In alloplasmic systems, MCS is due to a nucleus-cytoplasm incompatibility 
which may occur at several levels: replication of DNA, transcription of 
genes, maturation of transcripts, translation or assembly of multiprotein 
complexes. 
From the observations made on maize and petunia (Dewey R. E. et al., Cell, 
1986, 44, 439; Young E. G. et al., Cell. 1987, 50, 41), comes the 
hypothesis that MCS is due to a deficiency in the mitochondrial 
bioenergetic machinery. Indeed, MCS manifests itself by a reduction in the 
ATP and NADP levels. At the cellular level, this deficiency is correlated 
with degeneration of the cells of the anther lawn, while having no effect 
on the development of the plant. 
A number of methods have been proposed in the prior art for obtaining 
male-sterile plants. 
There may be mentioned especially the backcrossings which lead to the 
substitution of the nuclear genome of a species by another genome and 
this, in the cytoplasmic environment of the first species (alloplasmy); 
this substitution may also appear spontaneously in field crops. MCS can 
also be obtained by protoplast fusion (Lonsdale D. M., Genetic 
Engineering, 1987, 6, 47). 
In all these situations, the results are not reliable or reproducible; 
furthermore, in all cases, the manipulations are long, tedious and often 
difficult to control. 
Male-sterile plants have also been obtained by transgenosis, with the aid 
of a gene encoding an RNAse, under the control of an anther-specific 
promoter (Mariani C. et al., Nature, 1990, 347, 737). This transgene, when 
expressed, has a toxic effect on the cell insofar as the endogenos RNAs 
are degraded, thereby causing cell death. 
Another system, which also introduces a new artificial and destructive 
function, has been described by Worrall D. et al., (The Plant Cell, 1992, 
4, 759-771) (callase system) and has the same disadvantages as the RNAse 
system. 
Other methodologies have also been proposed for obtaining male-sterile 
plants; there may be mentioned especially the techniques which take 
advantage of the disruption of certain metabolic pathways (Van de Meer I. 
M. et al., The Plant Cell, 1992, 4, 253-262) (expression of a chalcone 
synthase antisense gene) or the techniques involving asymmetric somatic 
hybridization (Melchers C. et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 
6832-6836) to bring into contact, as in alloplasmic male-sterile lines, 
the cytoplasm of a donor individual and the nucleus of a recipient 
partner. The latter two processes have the major disadvantage of being 
highly unpredictable as regards the desired objective, namely the 
obtaining of male-sterile plants which makes it possible to control 
reproduction in these plants. 
The Applicant consequently set itself the objective of obtaining transgenic 
male-sterile plants in a controlled, reliable and reproducible manner 
which are capable of being used in agronomic programmes of seed 
improvement. 
SUMMARY OF THE INVENTION 
The subject of the present invention is transgenic plants having in their 
nuclei an expressible hybrid sequence (transgene) comprising at least one 
coding region of an unedited mitochondrial gene from higher plants and a 
sequence capable of transferring the protein expressed by the said coding 
region to the mitochondrion, which plants are characterized in that: 
the coding regions of the unedited mitochondrial genes are chosen from 
among the genes encoding a protein of the ATP synthase complex which are 
chosen from among the wheat ATP9 gene fragment, of the following formula 
I: 
__________________________________________________________________________ 
ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA 
ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC 
AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT 
AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC 
GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC 
TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA (SEQ ID NO: 7) 
__________________________________________________________________________ 
or the ATP6 gene, or from among the genes encoding a protein of the 
respiratory chain which are chosen from among the genes for subunits 1 to 
7 of NAD dehydrogenase, the gene for apocytochrome b and the genes for 
subunits I, II or III of cytochrome oxidase and 
the sequence capable of transferring the said expressed protein to the 
mitochondrion is selected from the group consisting of the fragments 
encoding yeast tryptophanyl tRNA synthetase (SCHMITZ, U. K. et al., 1989, 
The Plant Cell, 1, 783-791), and the .beta. subunit of Nicotiana 
plumbaginifolia ATPase (BOUTRY et al., 1987, Nature, 328:340-342), and the 
maize ATP/ADP translocator (BATHGATE et al., 1989, Eur. J. Biochem., 
183:303-310) or a 303 base pair EcoRI/KpnI fragment including codons 1 to 
62 of subunit IV of yeast cytochrome oxidase (MAARSE et al., 1984, EMBO 
J., 3, 2831-2837), 
which hybrid sequence is capable of modifying male fertility in plants 
having incorporated the said transgene while not modifying the other 
phenotypic characteristics of the said plants.

DETAILED DESCRIPTION OF THE INVENTION 
According to an advantageous embodiment of the said transgenic plants, the 
said hybrid nucleic acid sequence comprises the coding region of formula I 
of the gene encoding the unedited form of wheat ATP9, with which is 
associated as transfer sequence, codons 1 to 62 of the presequence of 
subunit IV of the yeast cytochrome oxidase (cox IV) (SEQ ID No. 1). 
According to another advantageous embodiment of the said transgenic plants, 
the said hybrid nucleic acid sequence comprises the fragment of the region 
encoding the unedited form of wheat ATP6, of the following formula II: 
__________________________________________________________________________ 
ATG GAT AAT TTT ATC CAG AAT CTG CCT GGT GCC TAC CCG GAA 
ACC CCA TTG GAT CAA TTT GCC ATT ATC CCA ATA ATT GAT CTT 
CAT GTG GGC AAC TTT TAT TTA TCA TTT ACA AAT GAA GTC TTG 
TAT ATG CTG CTC ACT GTC GTT TTG GTC GTT TTT CTT TTT TTT 
GTT GTT ACG AAA AAG GGA GGT GGA AAG TCA GTG CCA AAT GCA 
TGG CAA TCC TTG GTC GAG CTT ATT TAT GAT TTC GTG CTG AAC 
CTG GTA AAC GAA CAA ATA GGT GGT CTT TCC GGA AAT GTG AAA 
CAA AAG TTT TTC CCT CGC ATC TCG GTC ACT TTT ACT TTT TCG 
TTA TTT CGT AAT CCC CAG GGT ATG ATA CCC TTT AGC TTC ACA 
GTG ACA AGT CAT TTT CTC ATT ACT TTG GCT CTT TCA TTT TCC 
ATT TTT ATA GGC ATT ACG ATC GTT GGA TTT CAA AGA CAT GGG 
CTT CAT TTT TTT AGC TTC TTA TTA CCT GCG GGA GTC CCA CTG 
CCG TTA GCA CCT TTC TTA GTA CTC CTT GAG CTA ATC TCT TAT 
TGT TTT CGT GCA TTA AGC TTA GGA ATA CGT TTA TTT GCT AAT 
ATG ATG GCC GGT CAT AGT TTA GTA AAG ATT TTA AGT GGG TTT 
GCT TGG ACT ATG CTA TTT CTG AAT AAT ATT TTC TAT TTC ATA 
GGA GAT CTT GGT CCC TTA TTT ATA GTT CTA GCA TTA ACC GGT 
CTG GAA TTA GGT GTA GCT ATA TCA CAA GCT CAT GTT TCT ACG 
ATC TCA ATT TGT ATT TAC TTG AAT GAT GCT ACA AAT CTC CAT 
CAA AAT GAG TCA TTT CAT AAT TGA, (SEQ ID NO: 8) 
__________________________________________________________________________ 
with which is associated as transfer sequence, codons 1 to 62 of the 
presequence of subunit IV of yeast cytochrome oxidase (cox IV) (SEQ ID No. 
3). 
According to another advantageous embodiment of the said transgenic plants, 
the said hybrid nucleic acid sequence comprises the fragment of the region 
encoding the unedited form of cox II of the following formula III: 
__________________________________________________________________________ 
ATG ATT CTT CGT TCA TTA TCA TGT CGA TTC TTC ACA ATC GCT 
CTT TGT GAT GCT GCG GAA CCA TGG CAA TTA GGA TCT CAA GAC 
GCA GCA ACA CCT ATG ATG CAA GGA ATC ATT GAC TTA CAT CAC 
GAT ATC TTT TTC TTC CTC ATT CTT ATT TTG GTT TTC GTA TCA 
CGG ATG TTG GTT CGC GCT TTA TGG CAT TTC AAC GAG CAA ACT 
AAT CCA ATC CCA CAA AGG ATT GTT CAT GGA ACT ACT ATG GAA 
ATT ATT CGG ACC ATA TTT CCA AGT GTC ATT CTT TTG TTC ATT 
GCT ATA CCA TCG TTT GCT CTG TTA TAC TCA ATG GAC GGG GTA 
TTA GTA GAT CCA GCC ATT ACT ATC AAA GCT ATT GGA CAT CAA 
TGG TAT CGG ACT TAT GAG TAT TCG GAC TAT AAC AGT TCC GAT 
GAA CAG TCA CTC ACT TTT GAC AGT TAT ACG ATT CCA GAA GAT 
GAT CCA GAA TTG GGT CAA TCA CGT TTA TTA GAA GTT GAC AAT 
AGA GTG GTT GTA CCA GCC AAA ACT CAT CTA CGT ATG ATT GTA 
ACA CCC GCT GAT GTA CCT CAT AGT TGG GCT GTA CCT TCC TCA 
GGT GTC AAA TGT GAT GCT GTA CCT GGT CGT TCA AAT CTT ACC 
TTC ATC TCG GTA CAA CGA GAA GGA GTT TAC TAT GGT CAG TGC 
AGT GAG ATT CGT GGA ACT AAT CAT GCC TTT ACG CCT ATC GTC 
GTA GAA GCA GTG ACT TTG AAA GAT TAT GCG GAT TGG GTA TCC 
AAT GAA TTA ATC CTC CAA ACC AAC TAA, (SEQ ID NO: 9) 
__________________________________________________________________________ 
with which is associated as transfer sequence, codons 1 to 62 of the 
presequence of subunit IV of yeast cytochrome oxidase (cox IV) (SEQ ID No. 
5). 
The plants having incorporated the transgene in accordance with the 
invention (transgenic plants) are generally selected from plants which are 
of agronomic, medical or industrial interest. More precisely, any 
transformable and regenerable plant can constitute the raw material for 
obtaining a transgenic plant in accordance with the invention. 
For the purposes of the present invention, transformable is understood to 
mean any plant having the possibility of integrating a gene at the nuclear 
level in a manner which is stable and transmissible to its direct progeny. 
Also for the purposes of the present invention, regenerable is understood 
to mean any plant having the capacity to produce neoformed plants 
(neoformation or micropropagation). 
In a nonlimiting manner, the following plants can be subjected to 
transformation in accordance with the invention: 
tobacco, rape, sunflower, soya bean, tomato, potato, melon, carrot, pepper, 
chicory, clover, lupin, bean, pea, maize, wheat, rye, oat, barley, rice, 
millet, citrus, cotton. 
The plants, from which the unedited mitochondrial genes are obtained, are 
selected such that the changes in nucleotides (process called editing) 
between the unedited sequence and the edited sequence are substantial: at 
least 8 modified codons, and preferably at least 10 modified codons. 
Preferably, the unedited mitochondrial genes are obtained, in a nonlimiting 
manner, from wheat, tobacco, petunia or potato. 
Yeast presequences are in particular functional in the import of proteins 
into the mitochondrion in plants. 
In accordance with the invention, the plant from which the said unedited 
mitochondrial gene is obtained and the plant which incorporated the 
transgene may be identical or different. 
Surprisingly, the plants transformed by such a sequence have, in at least 
50% of them, a male-sterile phenotype, while having no other disruptions 
as regards the development of the plant. 
Also surprisingly, such transgenic plants make it possible to control, in a 
reliable and reproducible manner, the natural process of MCS, especially 
by avoiding self-pollination, without introducing new, artificial and 
destructive functions into the latter, as is the case especially in the 
systems described by Mariani et al. (RNAse system) or by WORRALL D. et al. 
(callase system). 
The subject of the present invention is also a hybrid nucleic acid 
sequence, comprising at least the coding region of an unedited 
mitochondrial gene from higher plants, with which is associated a sequence 
capable of transferring the protein expressed by the said coding region to 
the mitochondrion, characterized in that: 
the coding regions of the unedited mitochondrial genes are chosen from 
among the genes encoding a protein of the ATP synthase complex which are 
chosen from among the wheat ATP9 gene fragment, of the following formula 
I: 
__________________________________________________________________________ 
ATG TTA GAA GGT GCT AAA TCA ATA GGT GCC GGA GCT GCT ACA 
ATT GCT TTA GCC GGA GCT GCT GTC GGT ATT GGA AAC GTC CTC 
AGT TCT TTG ATT CAT TCC GTG GCG CGA AAT CCA TCA TTG GCT 
AAA CAA TCA TTT GGT TAT GCC ATT TTG GGC TTT GCT CTC ACC 
GAA GCT ATT GCA TTG TTT GCC CCA ATG ATG GCC TTT CTG ATC 
TCA TTC GTT TTC CGA TCG CAT AAA AAG TCA TGA (SEQ ID NO: 7) 
__________________________________________________________________________ 
or the ATP6 gene, or from among the genes encoding a protein of the 
respiratory chain, which are chosen from among the genes for subunits 1 to 
7 of NAD dehydrogenase, for apocytochrome b and for subunits I, II or III 
of cytochrome oxidase, and 
the nucleic sequence capable of transferring the said expressed protein to 
the mitochondrion is selected from the group consisting of the fragments 
encoding yeast tryptophanyl tRNA synthetase, the .beta. subunit of 
Nicotiana plumbaginifolia ATPase, the maize ATP/ADP translocator and a 303 
base pair EcoRI/KpnI fragment including codons 1 to 62 of subunit IV of 
yeast cytochrome oxidase, 
which hybrid sequence is capable of modifying male fertility in plants 
having incorporated it. 
According to an advantageous embodiment of the said hybrid nucleic acid 
sequence, it comprises the coding region of formula I of the gene encoding 
the unedited form of wheat ATP9, with which is associated as transfer 
sequence, codons 1 to 62 of the presequence of subunit IV of the yeast 
cytochrome oxidase (cox IV) (SEQ ID No. 1). 
According to another advantageous embodiment of the said hybrid nucleic 
acid sequence, it comprises the fragment of the region encoding the 
unedited form of wheat ATP6, of formula II above, with which is associated 
as transfer sequence, codons 1 to 62 of the presequence of subunit IV of 
yeast cytochrome oxidase (cox IV) (SEQ ID No. 3). 
According to another advantageous embodiment of the said hybrid nucleic 
acid sequence, it comprises the fragment of the region encoding the 
unedited form of cox II of formula III above, with which is associated as 
transfer sequence, codons 1 to 62 of the presequence of subunit IV of 
yeast cytochrome oxidase (cox IV) (SEQ ID No. 5). 
The subject of the present invention is also a plasmid, characterized in 
that it includes a hybrid nucleic acid sequence in accordance with the 
invention, associated with a promoter chosen from the promoters which are 
constitutively expressed and the promoters which are expressed in the 
anthers and with a suitable terminator. 
According to an advantageous embodiment of the said plasmid, it comprises 
the 35S promoter and the terminator of the CaMV VI gene. 
According to another advantageous embodiment of the said plasmid, it 
comprises in addition at least one marker gene, especially, and in a 
nonlimiting manner, a gene for resistance to an antibiotic, and preferably 
the gene for resistance to hygromycin. 
In accordance with the invention, the transgenic plants, as defined above, 
are capable of being obtained by means of a process for producing 
transgenic plants which comprises, for the transformation of the selective 
higher plant, the introduction of at least one copy of the hybrid nucleic 
sequence as defined above, into a recipient plant, by means of a plasmid 
containing the said sequence, as defined above. 
Such a transformation can advantageously be obtained by one of the 
following methods: protoplast transformation, agrotransformation, 
microinjection, biolistic. 
The subject of the present invention is also a process for inhibiting the 
production of pollen in higher plants, characterized in that it comprises 
the following steps: 
(a) inserting a hybrid nucleic acid sequence, as defined above, into the 
selected plants, by any appropriate means; 
(b) regenerating and culturing the transgenic plants obtained in (a); and 
(c) measuring the production and the viability of the pollen (test of 
germination in particular). 
Also surprisingly, the male function of the said transgenic male-sterile 
plants, in accordance with the invention, can be restored by crossing the 
said transgenic male-sterile plants with transgenic plants comprising in 
their nuclei a so-called antisense hybrid nucleic acid sequence, that is 
to say including at least the same coding region of unedited plant 
mitochrondrial gene as that included in the said transgenic male-sterile 
plants, in the reverse direction. 
The subject of the present invention is also a process for restoring 
male-fertile plants, from transgenic male-sterile plants, in accordance 
with the invention, characterized in that it comprises the following 
steps: 
(1) transforming the selected higher plant by introducing at least one copy 
of the hybrid nucleic sequence as defined above, into a recipient plant, 
by means of a plasmid containing the said sequence, in order to obtain 
transgenic male-sterile plants (TMSP); 
(2) transforming the same higher plant as in (1), by introducing at least 
one copy of an antisense hybrid nucleic sequence, including at least the 
same coding region of the unedited plant mitochondrial gene as that 
included in the said transgenic male-sterile plants obtained in (1), into 
a recipient plant, by means of a plasmid containing the said sequence, in 
order to obtain transgenic male-fertile plants (TMFP); 
(3) crossing the transgenic male-sterile plants obtained in (1) and the 
male-fertile plants obtained in (2), in order to obtain vigorous hybrids 
whose male fertility has been restored and which have preselected 
characteristics. 
The subject of the present invention is also plasmids including an 
antisense hybrid sequence, as defined above, associated with a promoter 
chosen from among the constitutive promoters and the promoters specific 
for the anthers and also associated with a suitable terminator. 
In addition to the preceding arrangements, the invention also comprises 
other arrangements, which will emerge from the description below, which 
refers to exemplary embodiments of the process which is the subject of the 
present invention. 
It should be understood, however, that these examples are given solely by 
way of illustration of the subject of the invention and do not in any 
manner constitute a limitation thereto. 
EXAMPLE 1 
Construction of a Chimeric Gene in Accordance with the Invention cox 
IV-ATP9 (SEQ ID No. 1). 
The sequences encoding ATP9 are obtained from a cDNA corresponding to the 
edited and unedited forms of wheat mitochondrial mRNA. 
ATP9 is fused with a 303 base pair EcoRI/KpnI fragment from a plasmid 
called 19.4 (MAARSE et al., EMBO J., 1984, 3, 2831-2837) , including 
codons 1 to 62 of subunit IV (cox IV) of yeast cytochrome oxidase. 
The resulting fragment, obtained after digestion with the enzyme HincII is 
ligated at the level of the SmaI restriction site of the plasmid pDH51 
(PIETRZAK et al., 1986, Nucleic Acids Res., 14:5857-5858). The hygromycin 
resistance gene is inserted at the level of the HindIII site of the 
plasmid pDH51, of the plasmid pEA903 (edited form of ATP9, FIG. 1) and of 
the plasmid pEA904 (unedited form of ATP9, FIG. 2) giving rise to the 
plasmids pH1 (FIG. 3), pH5 (FIG. 4) and pH2 (FIG. 5) respectively. The 
plasmid pH4 (FIG. 6) consists of the plasmid pEA904 in which the coding 
part cox IV/ATP9 is placed in reverse orientation compared with the 
plasmid pH2. 
The unedited cox IV-ATP9 and edited cox IV-ATP9 sequences are represented 
in FIGS. 12 and 13. 
All these genes are under the control of the CaMV 35S promoter and of the 
CaMV VI gene terminator. 
The sequences in accordance with the invention can be specifically 
amplified by means of the following oligonucleotide primers: 
(a) 5'-CACTACGTCAATCTATAAG-3' (SEQ ID No:10), extending from codon 3 to 
codon 9 of the presequence of subunit IV of yeast cytochrome oxidase and 
(b) 5'-TATGCTCAACACATGAGCG-3' (SEQ ID No:11), localized at the level of the 
CaMV VI gene terminator (45 base pairs upstream of the polyadenylation 
signal). 
The ATP9 mRNA in wheat undergoes C.fwdarw.U nucleotide changes (process 
called editing), at the level of 8 codons. The consequence of these 
modifications is the change of 5 amino acids in the corresponding protein 
(edited protein) and the loss, compared with the deduced sequence of the 
gene, of 6 residues from the C-terminal region, a loss which is caused by 
the creation of a stop codon. 
The unedited protein is more hydrophilic with 6 additional residues at the 
C-terminal level; furthermore, this selected unedited form of ATP9 
constitutes a particularly advantageous model of modified protein because 
it constitutes one element of the ATP synthase proton channel and, 
consequently, it is essential for the function of this complex; this 
fragment is also advantageous because of the small size of the coding 
sequence, which facilitates handling, and the fact that ATP9 may have a 
nuclear or mitochondrial localization. 
EXAMPLE 2 
Production of Transgenic Male-sterile Plants 
Both the plasmid constructs in accordance with the invention (see Example 
1, plasmid pH2) and the control constructs (plasmid pH1) and the 
constructs corresponding to the edited form of ATP9 (plasmid pH5) are used 
for the transformation of protoplasts of a Nicotiana tabacum cv. Petit 
Havana line, called SR1. 
*Transformation of the protoplasts: 
The protoplasts used for the transformation are isolated from the leaves of 
Nicotiana tabacum SR1 plants, cultivated under axenic conditions and one 
month old. The young leaves are removed, the central vein eliminated and 
the leaves are cut into thin slices. The fragments are then incubated in 
the dark at 26.degree. C., overnight, in an enzymatic solution consisting 
of K3 medium (NAGY and MALIGA, 1976) supplemented with R10 Onozuka 
cellulase (1.2%), R10 Onozuka macerozyme (0.4%) and Fluka driselase (0.1%) 
(pH 5.6). Before the harvest, the enzymatic solution is diluted with a 
0.6M sucrose solution, 0.1% (w/v) MES (pH 5.6) in the respective 
proportions 2v/1v. 
The protoplasts are separated from the undigested tissues by filtration 
through a 100 .mu.m sieve. The suspension is covered with a W5 solution 
(MENCZEL et al., Theor. Appl. Genetics, 1981, 59:191-195) being careful 
not to mix the liquid phases. After centrifuging at 600 rpm for 10 min, 
the protoplasts are assembled in the form of a band at the interface 
between the W5 solution and the enzymatic solution. They are carefully 
collected and washed twice with the W5 solution in order to remove traces 
of enzymes. The protoplasts are placed in a cold chamber at 4-6.degree. C. 
for 1-2 hours. After another centrifugation at 750 rpm for 5 min, they are 
resuspended in a mannitol/magnesium solution (0.5M Merck mannitol; 1.5 mM 
Prolabo MgCl.sub.2.6H.sub.2 O, 0.1% Sigma MES, pH 5.6) and their 
concentration is adjusted to 1.6.times.10.sup.6 protoplasts/ml. The 
protoplasts are subjected to a heat shock at 45.degree. C. for 5 minutes. 
After returning to room temperature, 300 .mu.l of protoplast suspension 
(5.times.10.sup.5 protoplasts) are distributed in a 12 ml conical tube. 
Next, 20 .mu.g of plasmid pH2 (or of plasmid pH4), depending on the 
transgenic plant which it is desired to obtain, 300 .mu.l of a solution of 
PEG 4000 40% (w/v) Merck PEG 4000; 0.4M Merck mannitol; Merck 
Ca(NO.sub.3).sub.2.4H.sub.2 O; pH 8 (solution sterilized by filtration on 
0.45 .mu.m)! and 60 .mu.g of calf thymus DNA as carrier DNA, are added to 
the protoplast suspension. The mixture is incubated at room temperature 
for 25-30 minutes and gently stirred from time to time. The transformation 
suspension is then gradually diluted by adding, in small portions, 10 ml 
of W5 over a period of 10 minutes. The protoplasts are recovered by 
centrifugation and taken up in 1 ml of K3 medium. 
* Culture of the protoplasts and regeneration of plants: 
The protoplasts are cultured in an amount of 5.times.10.sup.4 
protoplasts/ml, in 3 ml of a mixture of K3 and H medium (KAO and 
MICHAYLUK, 1975) in a 1:1 (v/v) proportion, solidified with agarose 
(0.8%). The resulting colonies are gradually cultured in the presence of 
hygromycin selection agent at 20 mg/l, in A50m medium (A medium containing 
50 g/l mannitol) (CABOCHE, 1980) for the first month, and then on A30m 
medium (the A medium containing 30 g/l mannitol) for the second month, and 
finally on A-m medium (A medium without mannitol), medium containing 40 
mg/ml of hygromycin, during the third month. For the regeneration, the 
calli are transferred onto the AR medium. The AR medium is the A medium 
containing only 20 g/l sucrose as carbohydrate source and 0.25 mg/l BAP as 
growth hormone. The plantlets derived from the calli are cultured on T 
medium (NITCH and NITCH, 1969). The MSoo medium is used for maintaining 
the plants. 
EXAMPLE 3 
Phenotypic Analysis of the Transgenic Plants Obtained 
The sizes of the 14-week old plants obtained in accordance with Example 2 
are specified in Table I below: 
TABLE I 
______________________________________ 
Fertility of the plant.sup.1 
Number 
of Number 
plants F F/S S Groove of Seeds.sup.2 
Lines 
tested (%) (%) (%) (cm) nodes (mg) 
______________________________________ 
SR1 1 100 0 0 87.0 24 109 .+-. 36 
H1 3 100 0 0 120 .+-. 6 
19 .+-. 1 
108 .+-. 14 
H2 16 50 -- -- -- -- 100 .+-. 32 
-- 19 -- 103 .+-. 26 
19 .+-. 2 
25 .+-. 17 
-- -- 31 -- -- 0 
H5 9 100 0 0 92 .+-. 23 
23 .+-. 5 
94 .+-. 28 
______________________________________ 
.sup.1 F = fertile, F/S = semifertile, S = malesterile 
.sup.2 mean value of production of seed per capsule after selfpollination 
H1 line = transgenic plants obtained with the plasmid pH1, 
H2 line = transgenic plants obtained with the plasmid pH2, 
H5 line = transgenic plants obtained with the plasmid pH5, 
Control line SR1 (nontransformed plant). 
The size of the plants is not significantly different from that of the 
nontransformed SR1 lines. The mean number of nodes is similar in the three 
different transgenic lines (19 to 24 nodes per plant). 
Apparently, there is no change in the function of the vegetative meristems 
in the differentiation of the nodes and of the leaves of the transgenic 
plants. 
Flowering in the H1, H2 and H5 lines is induced 7 to 14 weeks after 
transplantation. The flowers from the transgenic plants are similar in 
shape and in colour to those of the SR1 flowers (red-pink petals and 
anthers in each flower). The male-sterile plants have white anthers 
containing few or no pollen grains (FIGS. 7A1 and 7B), whereas the fertile 
plants have yellow-white anthers with normal pollen grains (FIGS. 7A2 and 
7C). There is no difference in the shape and in the colour of the pistil 
between the male-sterile and male-fertile plants. 
EXAMPLE 4 
Analysis of the Fertility of the Transgenic Plants 
The transformants H1 and H5 produce fertile plants, whereas the 
transformants H2 have fertility, semi-fertility or sterility 
characteristics which are defined on the basis of germination of the 
pollen or by the reaction with fluorescein diacetate. 
In the transgenic fertile plants, the viability of the pollen is between 31 
and 75%, close to the values found in the SR1 control line; in the 
semifertile plants, the viability of the pollen is about 10 to 20%; in the 
male-sterile plants, the viability is generally less than 2%. 
The fertility of the plants is also determined by the production of seeds 
after self-pollination or backcrossing. The results are also illustrated 
in Table I above. 
The H1 and H5 lines have a mean seed production of 100 mg/capsule, 
comparable with that of the SR1 control lines (110 mg/capsule). The H2 
lines which correspond to sterile plants produce no seed, the semifertile 
plants produce between 12 and 50 mg/capsule, the fertile plants produce on 
average 100 mg/capsule. These values correlate well with the pollen 
viability. 
The female fertility characteristic, for the sterile and semifertile 
plants, is determined by backcrossing with the SR1 lines as male parent. 
All the male-sterile plants are fertile females and produce a normal 
quantity of viable seeds (63 to 92 mg/capsule), with a seed viability 
value greater than 77%. Thus, the sterile or semifertile character in 50% 
of the H2 lines is due to the absence or to the very low production of 
viable pollen. 
The transmission of the transgenes is analysed through the genetic 
segregation of the hygromycin phosphotransferase (hpt) gene in the 
descendants (between 200 and 500 descendants analysed. After 
self-pollination (fertile and/or semifertile plants), the resistance to 
hygromycin is transmitted in most of the cases as a mendelian (mono- or 
digenic) character. 
After backcrossing with the SR1 parent (sterile plant), four of the five 
male-sterile plants inherit the character for hygromycin resistance as a 
digenic mendelian character, this expressing two active loci. 
These analyses show that the sterile plants are only affected in relation 
to the production of pollen, since they are fertile females and produce a 
quantity of seeds per fruit (100 to 150 mg) comparable or even greater 
than that of the controls. 
EXAMPLE 5 
Molecular Analysis of the Transformants 
In order to demonstrate the presence and the transcription of the ATP9 
transgene, the analysis of the transcription products is performed by 
Southern and Northern type hybridization. The total DNA is isolated from 
the (sterile, semifertile and fertile) H2 lines and the H5 lines. 
Moreover, the chimeric gene is analysed by PCR amplification. 
* Methods used: 
The total DNA is isolated from 10 g of leaf tissue essentially as described 
in SAGHAL-MAROOF M. A. et al., 1984. Proc. Natl. Acad. Sci. USA, 81, 
8014-8018. 1 .mu.g of DNA is amplified in a final volume of 100 .mu.l, 
using 0.5 unit of Taq polymerase, 0.18 mM dNTPs and 100 pmol of each of 
the primers. The primers used are those specified in Example 1. The use of 
these primers excludes the amplification of the endogenous ATP9 (see FIG. 
8C). 
The denaturation step is performed at 95.degree. C. for 1 min, the 
hybridization step is performed for 2 min at 52.degree. C. and the 
polymerization step is performed for 1 min at 72.degree. C. 
25 cycles are performed, the samples are subjected to electrophoresis on a 
1.5% agarose gel and transferred onto a Hybond-N.sup.+ membrane 
(Amersham), as described in SAGHAL-MAROOF M. A. al. (reference cited). The 
filters are prehybridized at 42.degree. C. in 50% deionized formamide, 
5.times.SSC, 8.times.Denhardt and 0.5% SDS. The filters are hybridized 
with the 300 base pair coding sequence of ATP9, a .sup.32 P-labelled 
EcoRI/HindIII fragment. 
A band (corresponding to a product comprising 700 base pairs) is observed 
in most of the H2 and H5 lines as expected. FIG. 8A shows the results 
obtained with the H2.2 and H2.16 DNA derived from male-sterile plants 
(lanes 1 and 2) and with fertile plants (H5.6 and H5.15 DNA, lanes 3 and 
4). The DNA derived from nontransformed plants SR1 gives no signal (lane 
5). 
The total RNA from the SR1, H2 and H5 lines is extracted, from the leaves, 
as follows: 5 g of leaves are cryo ground; then a first extraction is 
performed using the frozen powder, with 5 ml of a phenol; chloroform; 
isoamyl alcohol mixture (25:24:1; v:v:v) and 5 ml of TNES+DTT (0.1 M NaCl, 
10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% SDS and 2 mM dithiothreitol); a 
second extraction is then performed, using the aqueous phase, twice with 
an equal volume of chloroform and isoamyl alcohol (24:1; v:v) and the RNA 
is precipitated with an equal volume of 4 M lithium chloride at 0.degree. 
C. overnight. 
The RNAs are dissolved in DEPC-treated water. The RNA concentration is 
measured by the optical density (OD) at 260 nm. The poly(A).sup.+ RNAs 
are purified by oligo(dT)-cellulose affinity chromatography. 20 .mu.g of 
total RNA and 1 .mu.g of poly(A).sup.+ RNA are subjected to 
electrophoresis on 1.5% agarose gel, formaldehyde/formamide buffer, and 
then transferred onto Hybond-N.sup.+ nylon membranes. The hybridizations 
with the ATP9 probe are performed as described above. 
A 0.48 kb band is obtained with the SR1 control lines (FIG. 8B, lane 1). 
This band is present in all the lines and corresponds to the mitochondrial 
endogenous mRNA. 
An additional transcript, corresponding to a 0.98 kb band is present only 
in the transformed plants. As illustrated in FIG. 8B, these molecules can 
be separated from the endogenous mRNA by oligo(dT)-cellulose 
chromatography, confirming its cytoplasmic origin. 
FIG. 8B, (lanes 3 and 4) , shows the results obtained with the male-sterile 
plants H2.2 and H2.16 and with the fertile plants H5.6 and H5.15, (lanes 5 
and 6). The 0.98 kb transcript is absent from the nontransformed controls 
(lane 2). 
In parallel, by the PCR technique for cDNA, it is possible to obtain 
transcripts derived from the transgene by virtue of the sequences added 
during the in vitro manipulation such as the presequence regions obtained 
from the yeast (cox IV) and the CaMV termination region. Furthermore, only 
the 0.98 kb transcript hybridizes with a probe obtained from the cox IV 
sequence fused with ATP9. 
EXAMPLE 6 
Analysis of the Production of the Chimeric Protein 
In order to understand if the transgenes affect the expression of the 
endogenous mitochondrial ATP9 gene, the total RNA from the transformed 
plants H2 and H5 as well as from the control plants was hybridized with a 
specific mitochondrial probe. 
As shown in FIG. 9, no substantial difference is observed when the 
transgene is edited or unedited and the labelling is similar to that of 
the control. 
The production of the transgenic protein is analysed by immunoblotting of 
the mitochondrial and cytosolic extracts. Antibodies directed against 
fragments 21 to 54 of the presequence part of yeast cox IV, which are part 
of the transgene, are obtained in rabbits. 
The procedure is carried out as follows: a XbaI/KpnI fragment containing 
codons 21 to 54 of yeast cox IV is isolated from the abovementioned 
plasmid 19.4. 
This fragment is ligated to the plasmid pGEX-A (FIG. 10) in phase with the 
coding sequence of glutathione S-transferase, under the control of the 
.beta.-galactosidase promoter. 
The fusion protein is induced after transformation of E. coli DH5A cells by 
IPTG. These cells produce about 80 mg of protein per liter of culture. 
The fused protein is purified from an E. coli extract by affinity 
chromatography on a glutathione agarose column. The protein eluted by 
glutathione is obtained with a purity level of the order of 95%. The 
fusion protein is used as antigen to produce anti-cox IV antibodies in 
rabbits. 
Greenhouse plant leaves are used for cell fractionation. 100 .mu.g of 
cytosolic and mitochondrial proteins are fractionated by urea/SDS-PAGE. 
The immunoreaction is performed using an anti-cox IV antiserum diluted 
1/500th according to the DARLEY-USMAR et al. method 1987, Mitochondria, a 
practical approach, eds DARLEY-USMAR, (IRL Press Ltd.) pp. 113-152!. The 
proteins from transgenic plants carrying the male-sterile phenotype are 
revealed by peroxidase-conjugated anti-rabbit IgG antibodies. 
No signal is observed either with the mitochondrial fraction (FIG. 11B, 
lane 1), or with the cytosolic fraction from the nontransformed SR1 line. 
The mitochondrial fraction of the H2.2 male-sterile and H5.15 fertile 
plants (FIG. 11B, lanes 2 and 4 respectively) show a 12 kDa band 
corresponding to the expected size for the protein (see FIG. 11A, which 
specifies the structure of the 15 kDa precursor and the 12 kDa imported 
protein). 
The cytosolic proteins from these lines (FIG. 11B, lanes 3 and 5) show two 
bands, one at 15 kDa, the expected size for the chimeric precursor 
polypeptide, and the other at 14 kDa. The nature of this latter 
polypeptide remains to be determined; it is probably a degradation product 
of the 15 kDa precursor. 
The protein associated with the mitochondrial fraction of the H5.15 line 
(FIG. 11, lane 4) migrates roughly to the same position as the 
mitochondrial protein H2.2, but slightly downstream. This difference is 
due to the fact that the chimeric genes differ in the position of their 
stop codon. Indeed, as already specified above, the edited protein has 6 
residues less than the unedited protein due to the generation of a stop 
codon during the editing of the RNA. 
EXAMPLE 7 
Study of the Respiration of the Mitochondria from the Transgenic Plants 
The effect of the transgene at the subcellular level should result in a 
dysfunction of the respiratory function of the mitochondrion. Analysis of 
the respiration of the nonchlorophyllian plants of the transgenic plants 
was performed. 
The determination of the respiration rates of the nonchlorophyllian organs 
(roots), in the presence or in the absence of decouplers, is carried out 
by analysing the consumption of oxygen by means of a Clark electrode. More 
detailed studies were performed on mitochondria purified by differential 
centrifugation and on a Ficoll gradient. The effect of decouplers on 
respiration and the ADP/O ratios were determined on mitochondria derived 
from male-sterile lines and compared with the transformed or wild-type 
control plants. 
These different measurements show that the mitochondrial function is 
reduced in the male-sterile plants compared with the nontransformed or 
transformed control with the plasmid pH5. This situation is similar to 
that encountered in the natural male-sterile plants. 
It stems from the above that the expression in the transgenic tobacco 
plants of a DNA sequence encoding unedited wheat mitochondrial ATP9 has no 
effect on most of the phenotypic characters of the transformed plants, 
except for the appearance of male sterility. 
Indeed, the size, the growth rate, the number of nodes, the shape and the 
size of the leaves and of the flowers are similar in the transgenic plants 
and in the control plants. However, significant effects are observed in 
the male reproductive organs when the wheat ATP9 sequence, in its unedited 
form, is expressed in tobacco plants. 
Indeed, the transformation experiments performed with the plasmid pH2 lead 
to the production of many plants (50%) modified in relation to their 
fertility. Approximately 19% are semifertile and 31% are completely 
sterile. 
All the semifertile and sterile H2 lines express the transgene in the 
polyadenylated mRNA form. The fertile H2 lines do not have the 0.98 kb 
transcript, even when the transgene is detected after PCR amplification, 
thereby indicating that the transgene is inactive in this latter case. 
Some results also show, unexpectedly, that the male-sterile phenotype is 
correlated only with the presence of unedited ATP9 sequence whereas the 
transformants obtained with the edited ATP9 form are all fertile. 
In all cases, the sterile plants are only male-sterile plants and can be 
pollenated with a foreign pollen, thereby reflecting a normal female 
fertility. 
EXAMPLE 8 
Production of Transgenic Plants Having an Antisense Hybrid Sequence in 
Accordance with the Invention 
The procedure is carried out as in Example 2, the transformation of 
protoplasts being however performed by means of the plasmids pH4. 
By crossing these male-fertile plants with the male-sterile transgenic 
plants in accordance with the invention, noninbred male-fertile hybrids 
are obtained. 
EXAMPLE 9 
Construction of a Chimeric Gene in Accordance with the Invention cox 
IV-ATP6 (SEQ ID No. 3) 
The sequences encoding ATP6 are obtained from a cDNA corresponding to the 
edited and unedited forms of wheat mitochondrial mRNA. 
The unedited ATP6 fragment selected has the sequence of formula II defined 
above and is fused with the yeast transfer sequence cox IV as defined 
above. 
The resulting fragment is similar to that obtained in Example 1. 
The ATP6 mRNA in wheat undergoes nucleotide changes (editing) at the level 
of 12 codons. The consequence of the modifications is the change of 11 
amino acids and the loss, compared with the deduced sequence of the gene, 
of 7 residues, from the C-terminal region, a loss caused by the creation 
of a stop codon. 
EXAMPLE 10 
Construction of a Chimeric Gene in Accordance with the Invention cox IV-cox 
II (SEQ ID No.5) 
The sequences encoding cox II are obtained from a cDNA corresponding to the 
edited and unedited forms of wheat mitochondrial mRNA. 
The fragment of the unedited cox II gene has the sequence of formula III 
defined above and is fused with the yeast transfer sequence cox IV as 
defined above. 
The resulting fragment is similar to that obtained in Example 1. 
The mRNA in wheat undergoes nucleotide changes (editing) in 16 codons. The 
consequence of the modifications is the change of 16 amino acids compared 
with the deduced sequence of the cox II gene. 
As evident from the above, the invention is not in the least limited to the 
implementations, embodiments and applications which have just been 
described more explicitly; on the contrary, it embraces all the variants 
which may occur to a specialist in this field without departing from the 
framework or the scope of the present invention. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 13 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 568 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 99..524 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- GTCAACGTAT TCTTCTCCCT GAAGAAACAG TATACTAACA ATACTCACCC AT - #TTCGATTT 
60 
#CTA CGT 113ATAG ATAACAAGCA CAAGCACA ATG CTT TCA 
# Met Leu Ser Leu Arg 
# 5 1 
- CAA TCT ATA AGA TTT TTC AAG CCA GCC ACA AG - #A ACT TTG TGT AGC TCT 
161 
Gln Ser Ile Arg Phe Phe Lys Pro Ala Thr Ar - #g Thr Leu Cys Ser Ser 
# 20 
- AGA TAT CTG CTT CAG CAA AAA CCC GTG GTG AA - #A ACT GCC CAA AAC TTA 
209 
Arg Tyr Leu Leu Gln Gln Lys Pro Val Val Ly - #s Thr Ala Gln Asn Leu 
# 35 
- GCA GAA GTT AAT GGT CCA GAA ACT TTG ATT GG - #T CCT GGT GCT AAA GAG 
257 
Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gl - #y Pro Gly Ala Lys Glu 
# 50 
- GGT ACC CGG GGA TCC TCT AGA GTC GAG ATG TT - #A GAA GGT GCT AAA TCA 
305 
Gly Thr Arg Gly Ser Ser Arg Val Glu Met Le - #u Glu Gly Ala Lys Ser 
# 65 
- ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GC - #C GGA GCT GCT GTC GGT 
353 
Ile Gly Ala Gly Ala Ala Thr Ile Ala Leu Al - #a Gly Ala Ala Val Gly 
# 85 
- ATT GGA AAC GTC CTC AGT TCT TTG ATT CAT TC - #C GTG GCG CGA AAT CCA 
401 
Ile Gly Asn Val Leu Ser Ser Leu Ile His Se - #r Val Ala Arg Asn Pro 
# 100 
- TCA TTG GCT AAA CAA TCA TTT GGT TAT GCC AT - #T TTG GGC TTT GCT CTC 
449 
Ser Leu Ala Lys Gln Ser Phe Gly Tyr Ala Il - #e Leu Gly Phe Ala Leu 
# 115 
- ACC GAA GCT ATT GCA TTG TTT GCC CCA ATG AT - #G GCC TTT CTG ATC TCA 
497 
Thr Glu Ala Ile Ala Leu Phe Ala Pro Met Me - #t Ala Phe Leu Ile Ser 
# 130 
- TTC GTT TTC CGA TCG CAT AAA AAG TCA TGAGATCAA - #A AAAGAAATGT 
544 
Phe Val Phe Arg Ser His Lys Lys Ser 
# 140 
# 568ATGT CGAC 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 142 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Ph - #e Lys Pro Ala Thr Arg 
# 15 
- Thr Leu Cys Ser Ser Arg Tyr Leu Leu Gln Gl - #n Lys Pro Val Val Lys 
# 30 
- Thr Ala Gln Asn Leu Ala Glu Val Asn Gly Pr - #o Glu Thr Leu Ile Gly 
# 45 
- Pro Gly Ala Lys Glu Gly Thr Arg Gly Ser Se - #r Arg Val Glu Met Leu 
# 60 
- Glu Gly Ala Lys Ser Ile Gly Ala Gly Ala Al - #a Thr Ile Ala Leu Ala 
# 80 
- Gly Ala Ala Val Gly Ile Gly Asn Val Leu Se - #r Ser Leu Ile His Ser 
# 95 
- Val Ala Arg Asn Pro Ser Leu Ala Lys Gln Se - #r Phe Gly Tyr Ala Ile 
# 110 
- Leu Gly Phe Ala Leu Thr Glu Ala Ile Ala Le - #u Phe Ala Pro Met Met 
# 125 
- Ala Phe Leu Ile Ser Phe Val Phe Arg Ser Hi - #s Lys Lys Ser 
# 140 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 1106 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 99..1103 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- GTCAACGTAT TCTTCTCCCT GAAGAAACAG TATACTAACA ATACTCACCC AT - #TTCGATTT 
60 
#CTA CGT 113ATAG ATAACAAGCA CAAGCACA ATG CTT TCA 
# Met Leu Ser Leu Arg 
# 145 
- CAA TCT ATA AGA TTT TTC AAG CCA GCC ACA AG - #A ACT TTG TGT AGC TCT 
161 
Gln Ser Ile Arg Phe Phe Lys Pro Ala Thr Ar - #g Thr Leu Cys Ser Ser 
# 160 
- AGA TAT CTG CTT CAG CAA AAA CCC GTG GTG AA - #A ACT GCC CAA AAC TTA 
209 
Arg Tyr Leu Leu Gln Gln Lys Pro Val Val Ly - #s Thr Ala Gln Asn Leu 
# 175 
- GCA GAA GTT AAT GGT CCA GAA ACT TTG ATT GG - #T CCT GGT GCT AAA GAG 
257 
Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gl - #y Pro Gly Ala Lys Glu 
180 1 - #85 1 - #90 1 - 
#95 
- GGT ACC CGG GGA TCC TCT AGA GTC GAG ATG GA - #T AAT TTT ATC CAG AAT 
305 
Gly Thr Arg Gly Ser Ser Arg Val Glu Met As - #p Asn Phe Ile Gln Asn 
# 210 
- CTG CCT GGT GCC TAC CCG GAA ACC CCA TTG GA - #T CAA TTT GCC ATT ATC 
353 
Leu Pro Gly Ala Tyr Pro Glu Thr Pro Leu As - #p Gln Phe Ala Ile Ile 
# 225 
- CCA ATA ATT GAT CTT CAT GTG GGC AAC TTT TA - #T TTA TCA TTT ACA AAT 
401 
Pro Ile Ile Asp Leu His Val Gly Asn Phe Ty - #r Leu Ser Phe Thr Asn 
# 240 
- GAA GTC TTG TAT ATG CTG CTC ACT GTC GTT TT - #G GTC GTT TTT CTT TTT 
449 
Glu Val Leu Tyr Met Leu Leu Thr Val Val Le - #u Val Val Phe Leu Phe 
# 255 
- TTT GTT GTT ACG AAA AAG GGA GGT GGA AAG TC - #A GTG CCA AAT GCA TGG 
497 
Phe Val Val Thr Lys Lys Gly Gly Gly Lys Se - #r Val Pro Asn Ala Trp 
260 2 - #65 2 - #70 2 - 
#75 
- CAA TCC TTG GTC GAG CTT ATT TAT GAT TTC GT - #G CTG AAC CTG GTA AAC 
545 
Gln Ser Leu Val Glu Leu Ile Tyr Asp Phe Va - #l Leu Asn Leu Val Asn 
# 290 
- GAA CAA ATA GGT GGT CTT TCC GGA AAT GTG AA - #A CAA AAG TTT TTC CCT 
593 
Glu Gln Ile Gly Gly Leu Ser Gly Asn Val Ly - #s Gln Lys Phe Phe Pro 
# 305 
- CGC ATC TCG GTC ACT TTT ACT TTT TCG TTA TT - #T CGT AAT CCC CAG GGT 
641 
Arg Ile Ser Val Thr Phe Thr Phe Ser Leu Ph - #e Arg Asn Pro Gln Gly 
# 320 
- ATG ATA CCC TTT AGC TTC ACA GTG ACA AGT CA - #T TTT CTC ATT ACT TTG 
689 
Met Ile Pro Phe Ser Phe Thr Val Thr Ser Hi - #s Phe Leu Ile Thr Leu 
# 335 
- GCT CTT TCA TTT TCC ATT TTT ATA GGC ATT AC - #G ATC GTT GGA TTT CAA 
737 
Ala Leu Ser Phe Ser Ile Phe Ile Gly Ile Th - #r Ile Val Gly Phe Gln 
340 3 - #45 3 - #50 3 - 
#55 
- AGA CAT GGG CTT CAT TTT TTT AGC TTC TTA TT - #A CCT GCG GGA GTC CCA 
785 
Arg His Gly Leu His Phe Phe Ser Phe Leu Le - #u Pro Ala Gly Val Pro 
# 370 
- CTG CCG TTA GCA CCT TTC TTA GTA CTC CTT GA - #G CTA ATC TCT TAT TGT 
833 
Leu Pro Leu Ala Pro Phe Leu Val Leu Leu Gl - #u Leu Ile Ser Tyr Cys 
# 385 
- TTT CGT GCA TTA AGC TTA GGA ATA CGT TTA TT - #T GCT AAT ATG ATG GCC 
881 
Phe Arg Ala Leu Ser Leu Gly Ile Arg Leu Ph - #e Ala Asn Met Met Ala 
# 400 
- GGT CAT AGT TTA GTA AAG ATT TTA AGT GGG TT - #T GCT TGG ACT ATG CTA 
929 
Gly His Ser Leu Val Lys Ile Leu Ser Gly Ph - #e Ala Trp Thr Met Leu 
# 415 
- TTT CTG AAT AAT ATT TTC TAT TTC ATA GGA GA - #T CTT GGT CCC TTA TTT 
977 
Phe Leu Asn Asn Ile Phe Tyr Phe Ile Gly As - #p Leu Gly Pro Leu Phe 
420 4 - #25 4 - #30 4 - 
#35 
- ATA GTT CTA GCA TTA ACC GGT CTG GAA TTA GG - #T GTA GCT ATA TCA CAA 
1025 
Ile Val Leu Ala Leu Thr Gly Leu Glu Leu Gl - #y Val Ala Ile Ser Gln 
# 450 
- GCT CAT GTT TCT ACG ATC TCA ATT TGT ATT TA - #C TTG AAT GAT GCT ACA 
1073 
Ala His Val Ser Thr Ile Ser Ile Cys Ile Ty - #r Leu Asn Asp Ala Thr 
# 465 
# 1106T CAA AAT GAG TCA TTT CAT AAT TG - #A 
Asn Leu His Gln Asn Glu Ser Phe His Asn 
# 475 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 335 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Ph - #e Lys Pro Ala Thr Arg 
# 15 
- Thr Leu Cys Ser Ser Arg Tyr Leu Leu Gln Gl - #n Lys Pro Val Val Lys 
# 30 
- Thr Ala Gln Asn Leu Ala Glu Val Asn Gly Pr - #o Glu Thr Leu Ile Gly 
# 45 
- Pro Gly Ala Lys Glu Gly Thr Arg Gly Ser Se - #r Arg Val Glu Met Asp 
# 60 
- Asn Phe Ile Gln Asn Leu Pro Gly Ala Tyr Pr - #o Glu Thr Pro Leu Asp 
# 80 
- Gln Phe Ala Ile Ile Pro Ile Ile Asp Leu Hi - #s Val Gly Asn Phe Tyr 
# 95 
- Leu Ser Phe Thr Asn Glu Val Leu Tyr Met Le - #u Leu Thr Val Val Leu 
# 110 
- Val Val Phe Leu Phe Phe Val Val Thr Lys Ly - #s Gly Gly Gly Lys Ser 
# 125 
- Val Pro Asn Ala Trp Gln Ser Leu Val Glu Le - #u Ile Tyr Asp Phe Val 
# 140 
- Leu Asn Leu Val Asn Glu Gln Ile Gly Gly Le - #u Ser Gly Asn Val Lys 
145 1 - #50 1 - #55 1 - 
#60 
- Gln Lys Phe Phe Pro Arg Ile Ser Val Thr Ph - #e Thr Phe Ser Leu Phe 
# 175 
- Arg Asn Pro Gln Gly Met Ile Pro Phe Ser Ph - #e Thr Val Thr Ser His 
# 190 
- Phe Leu Ile Thr Leu Ala Leu Ser Phe Ser Il - #e Phe Ile Gly Ile Thr 
# 205 
- Ile Val Gly Phe Gln Arg His Gly Leu His Ph - #e Phe Ser Phe Leu Leu 
# 220 
- Pro Ala Gly Val Pro Leu Pro Leu Ala Pro Ph - #e Leu Val Leu Leu Glu 
225 2 - #30 2 - #35 2 - 
#40 
- Leu Ile Ser Tyr Cys Phe Arg Ala Leu Ser Le - #u Gly Ile Arg Leu Phe 
# 255 
- Ala Asn Met Met Ala Gly His Ser Leu Val Ly - #s Ile Leu Ser Gly Phe 
# 270 
- Ala Trp Thr Met Leu Phe Leu Asn Asn Ile Ph - #e Tyr Phe Ile Gly Asp 
# 285 
- Leu Gly Pro Leu Phe Ile Val Leu Ala Leu Th - #r Gly Leu Glu Leu Gly 
# 300 
- Val Ala Ile Ser Gln Ala His Val Ser Thr Il - #e Ser Ile Cys Ile Tyr 
305 3 - #10 3 - #15 3 - 
#20 
- Leu Asn Asp Ala Thr Asn Leu His Gln Asn Gl - #u Ser Phe His Asn 
# 335 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 1067 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 99..1064 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- GTCAACGTAT TCTTCTCCCT GAAGAAACAG TATACTAACA ATACTCACCC AT - #TTCGATTT 
60 
#CTA CGT 113ATAG ATAACAAGCA CAAGCACA ATG CTT TCA 
# Met Leu Ser Leu Arg 
# 340 
- CAA TCT ATA AGA TTT TTC AAG CCA GCC ACA AG - #A ACT TTG TGT AGC TCT 
161 
Gln Ser Ile Arg Phe Phe Lys Pro Ala Thr Ar - #g Thr Leu Cys Ser Ser 
# 355 
- AGA TAT CTG CTT CAG CAA AAA CCC GTG GTG AA - #A ACT GCC CAA AAC TTA 
209 
Arg Tyr Leu Leu Gln Gln Lys Pro Val Val Ly - #s Thr Ala Gln Asn Leu 
# 370 
- GCA GAA GTT AAT GGT CCA GAA ACT TTG ATT GG - #T CCT GGT GCT AAA GAG 
257 
Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gl - #y Pro Gly Ala Lys Glu 
# 385 
- GGT ACC CGG GGA TCC TCT AGA GTC GAG ATG AT - #T CTT CGT TCA TTA TCA 
305 
Gly Thr Arg Gly Ser Ser Arg Val Glu Met Il - #e Leu Arg Ser Leu Ser 
# 400 
- TGT CGA TTC TTC ACA ATC GCT CTT TGT GAT GC - #T GCG GAA CCA TGG CAA 
353 
Cys Arg Phe Phe Thr Ile Ala Leu Cys Asp Al - #a Ala Glu Pro Trp Gln 
405 4 - #10 4 - #15 4 - 
#20 
- TTA GGA TCT CAA GAC GCA GCA ACA CCT ATG AT - #G CAA GGA ATC ATT GAC 
401 
Leu Gly Ser Gln Asp Ala Ala Thr Pro Met Me - #t Gln Gly Ile Ile Asp 
# 435 
- TTA CAT CAC GAT ATC TTT TTC TTC CTC ATT CT - #T ATT TTG GTT TTC GTA 
449 
Leu His His Asp Ile Phe Phe Phe Leu Ile Le - #u Ile Leu Val Phe Val 
# 450 
- TCA CGG ATG TTG GTT CGC GCT TTA TGG CAT TT - #C AAC GAG CAA ACT AAT 
497 
Ser Arg Met Leu Val Arg Ala Leu Trp His Ph - #e Asn Glu Gln Thr Asn 
# 465 
- CCA ATC CCA CAA AGG ATT GTT CAT GGA ACT AC - #T ATG GAA ATT ATT CGG 
545 
Pro Ile Pro Gln Arg Ile Val His Gly Thr Th - #r Met Glu Ile Ile Arg 
# 480 
- ACC ATA TTT CCA AGT GTC ATT CTT TTG TTC AT - #T GCT ATA CCA TCG TTT 
593 
Thr Ile Phe Pro Ser Val Ile Leu Leu Phe Il - #e Ala Ile Pro Ser Phe 
485 4 - #90 4 - #95 5 - 
#00 
- GCT CTG TTA TAC TCA ATG GAC GGG GTA TTA GT - #A GAT CCA GCC ATT ACT 
641 
Ala Leu Leu Tyr Ser Met Asp Gly Val Leu Va - #l Asp Pro Ala Ile Thr 
# 515 
- ATC AAA GCT ATT GGA CAT CAA TGG TAT CGG AC - #T TAT GAG TAT TCG GAC 
689 
Ile Lys Ala Ile Gly His Gln Trp Tyr Arg Th - #r Tyr Glu Tyr Ser Asp 
# 530 
- TAT AAC AGT TCC GAT GAA CAG TCA CTC ACT TT - #T GAC AGT TAT ACG ATT 
737 
Tyr Asn Ser Ser Asp Glu Gln Ser Leu Thr Ph - #e Asp Ser Tyr Thr Ile 
# 545 
- CCA GAA GAT GAT CCA GAA TTG GGT CAA TCA CG - #T TTA TTA GAA GTT GAC 
785 
Pro Glu Asp Asp Pro Glu Leu Gly Gln Ser Ar - #g Leu Leu Glu Val Asp 
# 560 
- AAT AGA GTG GTT GTA CCA GCC AAA ACT CAT CT - #A CGT ATG ATT GTA ACA 
833 
Asn Arg Val Val Val Pro Ala Lys Thr His Le - #u Arg Met Ile Val Thr 
565 5 - #70 5 - #75 5 - 
#80 
- CCC GCT GAT GTA CCT CAT AGT TGG GCT GTA CC - #T TCC TCA GGT GTC AAA 
881 
Pro Ala Asp Val Pro His Ser Trp Ala Val Pr - #o Ser Ser Gly Val Lys 
# 595 
- TGT GAT GCT GTA CCT GGT CGT TCA AAT CTT AC - #C TTC ATC TCG GTA CAA 
929 
Cys Asp Ala Val Pro Gly Arg Ser Asn Leu Th - #r Phe Ile Ser Val Gln 
# 610 
- CGA GAA GGA GTT TAC TAT GGT CAG TGC AGT GA - #G ATT CGT GGA ACT AAT 
977 
Arg Glu Gly Val Tyr Tyr Gly Gln Cys Ser Gl - #u Ile Arg Gly Thr Asn 
# 625 
- CAT GCC TTT ACG CCT ATC GTC GTA GAA GCA GT - #G ACT TTG AAA GAT TAT 
1025 
His Ala Phe Thr Pro Ile Val Val Glu Ala Va - #l Thr Leu Lys Asp Tyr 
# 640 
- GCG GAT TGG GTA TCC AAT CAA TTA ATC CTC CA - #A ACC AAC TAA 
#1067 
Ala Asp Trp Val Ser Asn Gln Leu Ile Leu Gl - #n Thr Asn 
645 6 - #50 6 - #55 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 322 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Ph - #e Lys Pro Ala Thr Arg 
# 15 
- Thr Leu Cys Ser Ser Arg Tyr Leu Leu Gln Gl - #n Lys Pro Val Val Lys 
# 30 
- Thr Ala Gln Asn Leu Ala Glu Val Asn Gly Pr - #o Glu Thr Leu Ile Gly 
# 45 
- Pro Gly Ala Lys Glu Gly Thr Arg Gly Ser Se - #r Arg Val Glu Met Ile 
# 60 
- Leu Arg Ser Leu Ser Cys Arg Phe Phe Thr Il - #e Ala Leu Cys Asp Ala 
# 80 
- Ala Glu Pro Trp Gln Leu Gly Ser Gln Asp Al - #a Ala Thr Pro Met Met 
# 95 
- Gln Gly Ile Ile Asp Leu His His Asp Ile Ph - #e Phe Phe Leu Ile Leu 
# 110 
- Ile Leu Val Phe Val Ser Arg Met Leu Val Ar - #g Ala Leu Trp His Phe 
# 125 
- Asn Glu Gln Thr Asn Pro Ile Pro Gln Arg Il - #e Val His Gly Thr Thr 
# 140 
- Met Glu Ile Ile Arg Thr Ile Phe Pro Ser Va - #l Ile Leu Leu Phe Ile 
145 1 - #50 1 - #55 1 - 
#60 
- Ala Ile Pro Ser Phe Ala Leu Leu Tyr Ser Me - #t Asp Gly Val Leu Val 
# 175 
- Asp Pro Ala Ile Thr Ile Lys Ala Ile Gly Hi - #s Gln Trp Tyr Arg Thr 
# 190 
- Tyr Glu Tyr Ser Asp Tyr Asn Ser Ser Asp Gl - #u Gln Ser Leu Thr Phe 
# 205 
- Asp Ser Tyr Thr Ile Pro Glu Asp Asp Pro Gl - #u Leu Gly Gln Ser Arg 
# 220 
- Leu Leu Glu Val Asp Asn Arg Val Val Val Pr - #o Ala Lys Thr His Leu 
225 2 - #30 2 - #35 2 - 
#40 
- Arg Met Ile Val Thr Pro Ala Asp Val Pro Hi - #s Ser Trp Ala Val Pro 
# 255 
- Ser Ser Gly Val Lys Cys Asp Ala Val Pro Gl - #y Arg Ser Asn Leu Thr 
# 270 
- Phe Ile Ser Val Gln Arg Glu Gly Val Tyr Ty - #r Gly Gln Cys Ser Glu 
# 285 
- Ile Arg Gly Thr Asn His Ala Phe Thr Pro Il - #e Val Val Glu Ala Val 
# 300 
- Thr Leu Lys Asp Tyr Ala Asp Trp Val Ser As - #n Gln Leu Ile Leu Gln 
305 3 - #10 3 - #15 3 - 
#20 
- Thr Asn 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 243 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- ATGTTAGAAG GTGCTAAATC AATAGGTGCC GGAGCTGCTA CAATTGCTTT AG - #CCGGAGCT 
60 
- GCTGTCGGTA TTGGAAACGT CCTCAGTTCT TTGATTCATT CCGTGGCGCG AA - #ATCCATCA 
120 
- TTGGCTAAAC AATCATTTGG TTATGCCATT TTGGGCTTTG CTCTCACCGA AG - #CTATTGCA 
180 
- TTGTTTGCCC CAATGATGGC CTTTCTGATC TCATTCGTTT TCCGATCGCA TA - #AAAAGTCA 
240 
# 243 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 822 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- ATGGATAATT TTATCCAGAA TCTGCCTGGT GCCTACCCGG AAACCCCATT GG - #ATCAATTT 
60 
- GCCATTATCC CAATAATTGA TCTTCATGTG GGCAACTTTT ATTTATCATT TA - #CAAATGAA 
120 
- GTCTTGTATA TGCTGCTCAC TGTCGTTTTG GTCGTTTTTC TTTTTTTTGT TG - #TTACGAAA 
180 
- AAGGGAGGTG GAAAGTCAGT GCCAAATGCA TGGCAATCCT TGGTCGAGCT TA - #TTTATGAT 
240 
- TTCGTGCTGA ACCTGGTAAA CGAACAAATA GGTGGTCTTT CCGGAAATGT GA - #AACAAAAG 
300 
- TTTTTCCCTC GCATCTCGGT CACTTTTACT TTTTCGTTAT TTCGTAATCC CC - #AGGGTATG 
360 
- ATACCCTTTA GCTTCACAGT GACAAGTCAT TTTCTCATTA CTTTGGCTCT TT - #CATTTTCC 
420 
- ATTTTTATAG GCATTACGAT CGTTGGATTT CAAAGACATG GGCTTCATTT TT - #TTAGCTTC 
480 
- TTATTACCTG CGGGAGTCCC ACTGCCGTTA GCACCTTTCT TAGTACTCCT TG - #AGCTAATC 
540 
- TCTTATTGTT TTCGTGCATT AAGCTTAGGA ATACGTTTAT TTGCTAATAT GA - #TGGCCGGT 
600 
- CATAGTTTAG TAAAGATTTT AAGTGGGTTT GCTTGGACTA TGCTATTTCT GA - #ATAATATT 
660 
- TTCTATTTCA TAGGAGATCT TGGTCCCTTA TTTATAGTTC TAGCATTAAC CG - #GTCTGGAA 
720 
- TTAGGTGTAG CTATATCACA AGCTCATGTT TCTACGATCT CAATTTGTAT TT - #ACTTGAAT 
780 
# 822 ATCA AAATGAGTCA TTTCATAATT GA 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 783 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- ATGATTCTTC GTTCATTATC ATGTCGATTC TTCACAATCG CTCTTTGTGA TG - #CTGCGGAA 
60 
- CCATGGCAAT TAGGATCTCA AGACGCAGCA ACACCTATGA TGCAAGGAAT CA - #TTGACTTA 
120 
- CATCACGATA TCTTTTTCTT CCTCATTCTT ATTTTGGTTT TCGTATCACG GA - #TGTTGGTT 
180 
- CGCGCTTTAT GGCATTTCAA CGAGCAAACT AATCCAATCC CACAAAGGAT TG - #TTCATGGA 
240 
- ACTACTATGG AAATTATTCG GACCATATTT CCAAGTGTCA TTCTTTTGTT CA - #TTGCTATA 
300 
- CCATCGTTTG CTCTGTTATA CTCAATGGAC GGGGTATTAG TAGATCCAGC CA - #TTACTATC 
360 
- AAAGCTATTG GACATCAATG GTATCGGACT TATGAGTATT CGGACTATAA CA - #GTTCCGAT 
420 
- GAACAGTCAC TCACTTTTGA CAGTTATACG ATTCCAGAAG ATGATCCAGA AT - #TGGGTCAA 
480 
- TCACGTTTAT TAGAAGTTGA CAATAGAGTG GTTGTACCAG CCAAAACTCA TC - #TACGTATG 
540 
- ATTGTAACAC CCGCTGATGT ACCTCATAGT TGGGCTGTAC CTTCCTCAGG TG - #TCAAATGT 
600 
- GATGCTGTAC CTGGTCGTTC AAATCTTACC TTCATCTCGG TACAACGAGA AG - #GAGTTTAC 
660 
- TATGGTCAGT GCAGTGAGAT TCGTGGAACT AATCATGCCT TTACGCCTAT CG - #TCGTAGAA 
720 
- GCAGTGACTT TGAAAGATTA TGCGGATTGG GTATCCAATC AATTAATCCT CC - #AAACCAAC 
780 
# 783 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 19 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
# 19 AAG 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 19 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
# 19 GCG 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 568 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (v) FRAGMENT TYPE: linear 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 99..506 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- GTCAACGTAT TCTTCTCCCT GAAGAAACAG TATACTAACA ATACTCACCC AT - #TTCGATTT 
60 
#CTA CGT 113ATAG ATAACAAGCA CAAGCACA ATG CTT TCA 
# Met Leu Ser Leu Arg 
# 325 
- CAA TCT ATA AGA TTT TTC AAG CCA GCC ACA AG - #A ACT TTG TGT AGC TCT 
161 
Gln Ser Ile Arg Phe Phe Lys Pro Ala Thr Ar - #g Thr Leu Cys Ser Ser 
# 340 
- AGA TAT CTG CTT CAG CAA AAA CCC GTG GTG AA - #A ACT GCC CAA AAC TTA 
209 
Arg Tyr Leu Leu Gln Gln Lys Pro Val Val Ly - #s Thr Ala Gln Asn Leu 
# 355 
- GCA GAA GTT AAT GGT CCA GAA ACT TTG ATT GG - #T CCT GGT GCT AAA GAG 
257 
Ala Glu Val Asn Gly Pro Glu Thr Leu Ile Gl - #y Pro Gly Ala Lys Glu 
360 3 - #65 3 - #70 3 - 
#75 
- GGT ACC CGG GGA TCC TCT AGA GTC GAG ATG TT - #A GAA GGT GCT AAA TTA 
305 
Gly Thr Arg Gly Ser Ser Arg Val Glu Met Le - #u Glu Gly Ala Lys Leu 
# 390 
- ATA GGT GCC GGA GCT GCT ACA ATT GCT TTA GC - #C GGA GCT GCT GTC GGT 
353 
Ile Gly Ala Gly Ala Ala Thr Ile Ala Leu Al - #a Gly Ala Ala Val Gly 
# 405 
- ATT GGA AAC GTT TTC AGT TCT TTG ATT CAT TC - #C GTG GCG CGA AAT CCA 
401 
Ile Gly Asn Val Phe Ser Ser Leu Ile His Se - #r Val Ala Arg Asn Pro 
# 420 
- TCA TTG GCT AAA CAA TTA TTT GGT TAT GCC AT - #T TTG GGC TTT GCT CTC 
449 
Ser Leu Ala Lys Gln Leu Phe Gly Tyr Ala Il - #e Leu Gly Phe Ala Leu 
# 435 
- ACC GAA GCT ATT GCA TTG TTT GCC CTA ATG AT - #G GCC TTT TTG ATC TTA 
497 
Thr Glu Ala Ile Ala Leu Phe Ala Leu Met Me - #t Ala Phe Leu Ile Leu 
440 4 - #45 4 - #50 4 - 
#55 
- TTC GTT TTC TGATCGCATA AAAAGTCATG AGATCAAAAA AGAAATGTG - #T 
546 
Phe Val Phe 
# 568TCG AC 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 136 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Ph - #e Lys Pro Ala Thr Arg 
# 15 
- Thr Leu Cys Ser Ser Arg Tyr Leu Leu Gln Gl - #n Lys Pro Val Val Lys 
# 30 
- Thr Ala Gln Asn Leu Ala Glu Val Asn Gly Pr - #o Glu Thr Leu Ile Gly 
# 45 
- Pro Gly Ala Lys Glu Gly Thr Arg Gly Ser Se - #r Arg Val Glu Met Leu 
# 60 
- Glu Gly Ala Lys Leu Ile Gly Ala Gly Ala Al - #a Thr Ile Ala Leu Ala 
# 80 
- Gly Ala Ala Val Gly Ile Gly Asn Val Phe Se - #r Ser Leu Ile His Ser 
# 95 
- Val Ala Arg Asn Pro Ser Leu Ala Lys Gln Le - #u Phe Gly Tyr Ala Ile 
# 110 
- Leu Gly Phe Ala Leu Thr Glu Ala Ile Ala Le - #u Phe Ala Leu Met Met 
# 125 
- Ala Phe Leu Ile Leu Phe Val Phe 
# 135 
__________________________________________________________________________