DNA sequence imparting cytoplasmic male sterility, mitochondrial genome, nuclear genome, mitochondria and plant containing said sequence and process for the preparation of hybrids

Ogura sterility DNA is used to develop agronomically valuable hybrids that lack the undesirable features of plants having Ogura DNA (chlorosis at low temperature; poor fertile fertility; aberrant flower morphology) but still impart efficient male sterility (which can be easily restored). Ogura sterility is carried by a DNA sequence defined by nucleotides 928-2273 of a 2428 base sequence reproduced in the specification. When present in mitochondrial or nuclear genome of a plant it confers cytoplasmic male sterility. Also disclosed are recombinant plant nuclear and mitochondrial genomes containing a DNA sequence defined by nucleotides 928-1569 of the specified sequence (or its homologous); cytoplasm containing such a mitochondrial genome; Brassica plants or hybrids containing the sterility DNA; and, DNA probes of at least 10 bases from the 928-1569 sequences. DNA probes are used to detect male sterility and for selecting clones without the need for lengthy agronomic/rehybridization steps.

The present invention relates to a biological material possessing a male 
sterility which is useful for the development of hybrid varieties of 
species of agronomic importance. 
It relates especially to a plant belonging to the Cruciferae family, in 
which the cytoplasm of the cells contains organelles possessing nucleotide 
sequences conferring a male sterility and good agronomic characteristics. 
The development of hybrid varieties can be facilitated or made possible by 
the use of a cytoplasmic male sterility system. The hybrids are obtained 
by cross-fertilisation between two parent populations, one performing the 
role of male, the other of female. One of the stumbling blocks encountered 
when it is desired to obtain hybrid varieties of uniform quality by sexual 
crossing on self-fertile species is the capacity of the plant to 
self-pollinate. Male sterility systems enable female plants to be obtained 
which are incapable of self-fertilisation and from which, after 
pollination, the seeds, which are all hybrids, can be harvested directly 
without resorting to laborious techniques such as castration of the 
flowers. 
The genetic determinants of male sterility include those which are carried 
by the cytoplasm. At each sexually produced generation, they are 
transmitted exclusively by the mother. 100% of male-sterile offspring are 
thereby produced at each generation with a cytoplasmic male sterility 
(CMS) system. These genetic determinants are carried by the genome of the 
mitochondria. 
A suitable cytoplasmic male sterility system in Cruciferae is defined by 
the following characteristics: 
1) The male sterility must be total, that is to say there must be no pollen 
production regardless of the culture conditions and regardless of the line 
which it is desired to use as a female parent. Were this not the case, the 
seeds harvested from these female plants would be derived in part from 
self-fertilisation and would hence not be of the F1 hybrid type. 
2) The production of these seeds must be carried out taking advantage of 
the natural pollen vectors, that is to say, in the case of these species, 
Hymenoptera, Diptera and wind. The pollen must be transported from the 
pollinating plants to the male-sterile (female) plants. In fact, only 
insects can effect this transport at a distance in Cruciferae. 
The female plants must consequently be sufficiently attractive to the 
insects which come and collect the nectar in them. The morphology of the 
flowers must force the insect to perform this task via the top of the 
flower so that its thorax, principally, comes into contact with the 
stigma. In practice, this amounts to a situation where the base of the 
petals has to form a kind of tube around the base of pistil. 
3) The morphology of the female organs (pistil) must be identical to that 
of a fertile plant, especially one single pistil per flower and 
rectilinear in shape. In effect, male sterilities often result also in a 
feminisation of the anthers, which are transformed into pseudopistils, and 
even in transformation of the nectaries into complete flowers. Sometimes 
the pistil or pistils thereby produced are also deformed. All these 
abberations do not permit good seed production, and this may be regarded 
as equivalent to some degree of female sterility. 
4) For the production of F1 hybrid varieties in the species from which the 
seeds are harvested, such as colza or mustards, it is essential that the 
male parent of the hybrid completely negates the effect of the 
male-sterile cytoplasm, so that the hybrid plants are readily pollinated. 
In Cruciferae, the first case of male-sterile cytoplasm or CMS was 
described by Ogura (1968) in the radish, Raphanus sativus. Bannerot (1974, 
1977) transferred the Ogura cytoplasm in Brassicae, thereby obtaining 
plants which possess a cytoplasmic male sterility. These same plants did 
not possess satisfactory agronomic characteristics (chlorosis on lowering 
temperatures, poor female fertility), resulting in a poor yield making 
them unsuitable for commercial use. 
In Cruciferae, to avoid this chlorosis, the nuclear and chloroplast genomes 
of one and the same genus should be combined in the same cell. Thus, 
Brassica plants possessing one of the chloroplast genomes of Brassicae no 
longer exhibit chlorosis. If they possess the whole of the Ogura 
mitochondrial genome, they exhibit total cytoplasmic male sterility but, 
however, the flowers will have an aberrant morphology making it impossible 
for them to be pollinated by the natural vectors. 
In addition, for species in which the interest lies in the seeds, it is 
appropriate to restore the male fertility of hybrid varieties which are 
marketed by means of nuclear genes known as restorer genes. 
It is difficult to restore the male fertility of a plant possessing the 
whole of the Ogura mitochondrial genome, since it is necessary to bring 
about the simultaneous participation of several restorer genes. 
We set out to obtain a suitable male sterility system by removing the genes 
responsible for the undesirable characters of the Ogura cytoplasm while 
retaining a male sterility which is effective and easy to restore. 
Thus, the present invention relates to a DNA sequence, which we shall refer 
to as Ogura sterility DNA sequence, characterised in that: 
a) it is carried by a DNA sequence bounded by nucleotides 928 and 2273 in 
FIG. 1 (SEQ ID NO: 1), or 
b) it possesses an at least 50% homology with the said sequence mentioned 
in a), 
and confers, when it is present in the mitochondrial genome of a plant, a 
cytoplasmic male sterility on the said plant. 
In particular, the subject of the present invention is an Ogura sterility 
DNA sequence, characterised in that: 
c) it is carried by the sequence bounded by nucleotides 928 and 1569 in 
FIG. 1 (SEQ ID NO: 1), or 
d) it possesses an at least 50% homology with the said sequence mentioned 
in c), 
and in that it is transcribed to RNA in the mitochondria of male-sterile 
plants.

EXAMPLE 1 
DEMONSTRATION OF THE DNA SEQUENCE RESPONSIBLE FOR OGURA CYTOPLASMIC MALE 
STERILITY 
1. Plant 
"Cybrid" denotes forms obtained by the fusion of isolated protoplasts 
followed by regeneration of the whole plant. This method of production 
enables cytoplasmic information originating from both parents to be mixed 
in the cell. Cybrid No. 13 was obtained from among 820 plants regenerated 
by protoplast fusions between an Ogura-cms, triazine-resistant B. napus 
cybrid (progeny of cybrid 77 described in Pelletier et al., 1983 and 
Chetrit et al., 1985) and the triazine-sensitive and fertile variety of 
Brutor origin. A triazine resistance test (Ducruet and Gasquez, 1978) 
carried out on a leaf sample of each regenerant enabled the type of 
chloroplast (triazine-resistant chloroplasts originating from the parent 
77 or triazine-sensitive chloroplasts originating from the Brutor line) to 
be determined. The plants were cultivated and the flowering stage was 
observed. Plants exhibiting non-parental combinations (either 
sensitive/male-sterile or resistant/male-fertile) were selected as 
cybrids. Cybrid No. 13 was of the sensitive/male-sterile type. Cybrid 1 
was of the resistant/male-fertile type. 
2. Nucleic acid isolation 
The total DNA was isolated from leaves originating from 4-week-old plants 
according to the method described by Dellaporta (1983). The mitochondrial 
DNA was extracted from leaves of 8-week-old plants as has been described 
by Vedel and Mathieu, 1982, with the following variants: 
the mitochondria were not purified on a sucrose gradient before lysis, and 
lysis was carried out in 4% sarcosyl with 0.5 mg/ml proteinase K 
(Boehringer Mannheim GmbH) in 50 mM Tris pH 8, 20 mM EDTA. After 
precipitation, the mitochondrial DNA was purified by centrifugation on an 
ethidium bromide/caesium chloride gradient (method 1--Vedel and Mathieu, 
1982) in polyallomer centrifuge tubes. 
The total RNA was isolated from leaves or floral buds according to Logemann 
et al., 1987. 
The mitochondrial RNA was extracted from 8-week-old cauliflowers according 
to the technique of Stern and Newton, 1986. 
3. Restriction analyses of the mitochondrial DNA and agarose gel 
electrophoresis 
These were carried out as described in Pelletier et al., 1983. The total or 
mitochondrial RNA was loaded onto electrophoresis gels containing 
formaldehyde, as has been described by Sambrook et al., 1989. 
4. Hybridisation 
Transfer of DNA or RNA onto nylon filters (Hybond-N, Amersham) was carried 
out by capillary absorption with 6.times.SSC or 10.times.SSPE, 
respectively, according to the manufacturer's instructions. 
Prehybridisation and hybridisation were performed according to Amersham, 
using probes labelled by the multiprimer DNA labelling system (Amersham) 
after purification on Sephadex G-50 columns (Sambrook et al., 1989). 
5. Cloning of the mitochondrial DNA 
Two genomic libraries of male-sterile (13-7) and revertant (13-6) cybrid 
lines were constructed in a phage lambda EMBL3 vector cultured on the 
restrictive E. coli strain Nm539 (Frischauf et al., 1983). Approximately 
2.5.times.10.sup.4 clones per .mu.g of mitochondrial DNA were obtained. 
The mitochondrial DNA libraries were assayed and plated out in order to 
isolate the plaques, which were transferred onto nylon filters as 
described in Sambrook et al., 1989. The hybridisation probe used to screen 
the two libraries of mitochondrial DNA was prepared as follows: the 
mitochondrial DNA fragment specific for the cms was eluted using the Gene 
cleans procedure (BIO 101 INC.) from a mitochondrial DNA digestion product 
loaded onto a preparative agarose gel. The eluted DNA was then labelled as 
has been described. 
The extraction of lambda DNA, subcloning of the 2.5 NcoI fragment into the 
NcoI site of pTrc99A (Amann et al., 1988) and extractions of plasmid DNA 
were performed according to the protocols of Sambrook et al., 1989. The 
recombinant plasmids were introduced into an E. coli strain NM522 (Gough 
and Murray, 1983). 
6. Genetic study of cybrid 13 and its progeny 
In the first generation of progeny obtained by pollination of cybrid 13 
with Brutor, composed of 13 plants, 5 are totally male-sterile (including 
plants 13-2 and 13-7), one is male-fertile (No. 13-6) and 7 are almost 
completely sterile with a few male-fertile flowers. 
The fertile plant 13-6 was self-pollinated and crossed with Brutor. In both 
cases, only fertile plants (43 and 42, respectively) are obtained. 
In the crosses between the male-sterile plant No. 13-7 and Brutor, 24 
progeny are completely sterile and 6 possess a few fertile flowers, a 
similar result to that observed with the cybrid itself. The plant 13-2 was 
crossed with the restorer line RF, which is heterozygotic for the specific 
restorer genes for Ogura male sterility (Chetrit et al., 1985). The 
progeny of this cross is composed of 53 male-sterile plants, 37 
male-fertile plants and 9 plants which are almost completely sterile 
although they possess a few fertile flowers. These results suggest that 
the male-sterile plants of the cybrid family 13 contain the Ogura-cms 
determinant, like the other cybrids studied beforehand with a simpler 
restoration profile (Chetrit et al., 1985). 
At this stage of the study, two possibilities may be envisaged: either 
cybrid 13 contains a mixture of "male-fertile" and "male-sterile" 
mitochondrial genomes, and it is possible to select further for both 
phenotypes, or cybrid 13 contains a recombinant mitochondrial genome of 
unstable structure which reverts to a more stable "fertile" configuration, 
and it will be impossible to maintain a homogeneous male-sterile phenotype 
among subsequent generations. 
Male-sterile plants obtained from the progeny of the male-sterile plant No. 
13-7 were developed both by taking cuttings and by sexual crossing with 
Brutor. After a variable number of generations (1 to 5) by both methods, 
all the families give fertile plants. In contrast, the completely fertile 
plants thereby obtained never give rise again to sterile plants. 
In the light of these results, it may be considered that the second 
explanation proposed above, that is to say that cybrid 13 carries an 
unstable mitochondrial genome which loses the Ogura-cms determinant during 
the process leading to a "fertile" configuration, without the possibility 
of a return to a sterile phenotype, is the correct one. 
7. Comparison between the mitochondrial DNAs of male-sterile and fertile 
revertant offspring. Isolation of a fragment specific to the male-sterile 
plants 
The mitochondrial DNA was extracted from the leaves of male-sterile 13-7 
progeny and of fertile revertants (13-6 or 13-7 progeny), and digested 
with several restriction enzymes in order to compare their restriction 
profiles. The mitochondrial genomes of the two types are very similar, 
since no difference can be observed between the restriction profile of the 
male-sterile mitochondria and fertile revertants using various enzymes. 
However, a 6.8-kb restriction fragment was detected in the restriction 
profile of the mitochondrial DNA of the male-sterile plants digested with 
NruI, and was never observed in the corresponding profiles of the fertile 
revertants. 
The fragment (referred to as N6.8) was eluted from an agarose gel, labelled 
and used as a probe on NruI mitochondrial DNA restriction profiles: a 
large signal at 6.8 kb was observed in all the male-sterile progeny of 
cybrid 13, whereas no fragment of this size hybridised with the probe in 
the genomes of mitochondria of fertile revertants. In addition, the probe 
N6.8 hybridises with a 6.8-kb fragment in the Ogura mitochondrial DNA 
digested with NruI, but not in B. napus cv Brutor, showing the Ogura 
origin of this fragment. 
A lambda library containing mitochondrial DNA extracts originating from 
male-sterile plants (13-7) was tested with the eluted labelled fragment, 
and out of 8 clones hybridising, 2 recombinant phages were isolated, 
containing the whole N6.8 fragment and adjacent sequences. A detailed 
restriction map of this region was obtained. Hybridisation of the 
restriction profiles of the mitochondrial DNA originating from fertile and 
sterile progeny of cybrid 13 with N6.8 as a probe enabled the region 
specific for the male-sterile genotype to be limited to a 2.5-kb NcoI 
fragment. 
The 2.5-kb NcoI fragment was labelled and used as a probe with respect to 
mitochondrial DNA originating from 13-7 and 13-6 progeny digested with 
NcoI. Apart from the signal at 2.5 kb which is specific for the 
male-sterile profile, several NcoI fragments hybridise in both the fertile 
revertant and male-sterile profiles; these fragments are at 2.2, 10 and 14 
kb. A 2.7-kb NcoI fragment hybridises strongly in the mitochondrial genome 
of the fertile progeny and not in that of the sterile progeny. Analysis of 
this hybridisation profile leads to the conclusion that the 2.5-kb NcoI 
fragment, although specific for the male-sterile mitochondrial DNA, 
contains sequences which are repeated elsewhere in the mitochondrial 
genome (on 2.2-, 10- and 14-kb fragments after NcoI), and these repeated 
sequences are also present in the mitochondrial DNA of fertile revertants 
apart from the specific 2.7-kb fragment. 
The total RNA is extracted from leaves or buds of progeny of cybrids 13, or 
of male-sterile or fertile cybrids (originating from other fusion 
experiments) and of Brutor line. Northern blotting was carried out and the 
blots were hybridised with a probe corresponding to the insert of the 
lambda clone containing N6.8 described in Example 3. A major 1.4-kb 
transcript was detected in all the male-sterile cybrids, including cybrid 
13-7, whereas no transcript of this size was observed in the Brutor line, 
or in the two fertile cybrids (different from 13). Moreover, fertile 
plants possess a 1.1-kb transcript which hybridises with the probe, which 
is absent or present at a very low level in all the male-sterile cybrids 
tested. Several transcripts common to all the samples hybridise weakly 
with the probe on account of the large size of the labelled insert. It was 
confirmed that the mitochondrial transcripts can be detected in samples of 
total RNA by hybridisation of the same Northern blot with a DNA fragment 
containing the atpa gene sequence. 
The same specific 1.4 transcript was found in the Ogura mitochondrial RNA 
extracted from cauliflowers, using the 2.5 NcoI fragment as a probe. The 
exact limits of this transcript were determined using subclones of the 2.5 
NcoI fragment as a probe 
8. Study of cybrid 1 and its progeny 
Cybrid 1 was male-fertile. In its progeny, the plant 1.12 was fertile and 
the plant 1.18 sterile. The plant 1.12 gave in its progeny sterile plants 
(S.sub.3) and fertile plants (RF.sub.3). The plant 1.18 gave sterile 
plants (S.sub.2) and a fertile branch (RF.sub.2). The plants S.sub.2 and 
S.sub.3 are restored by the same nuclear gene that restores pollen 
fertility as the sterile cybrid 13. 
On hybridisation with the labelled 2.5-kb NcoI fragment, the mitochondrial 
DNA of the plants S.sub.2 and S.sub.3 does not give a signal at 2.5 kb on 
NcoI digestion, or a signal at 6.8 kb on NruI digestion. 
Similarly, hybridisation with the total RNA (Northern blotting) with a 
probe corresponding to the ORFB sequence does not give a signal at 1.4 kb 
as occurs with the sterile cybrid 13. In contrast, a probe corresponding 
to the sequence bounded by nucleotides 928 and 1569 in FIG. 1 (SEQ ID NO: 
1) gives a signal in Northern blotting at approximately 1.3 kb. This 
signal is absent from the RNA of the plants RF.sub.1, RF.sub.2, RF.sub.3 
or Brutor. Similarly, it is possible to use this sequence (928-1569) as a 
probe in dot-blotting of total RNA, and in this case, only the 
male-sterile plants, and all of them, give a signal. 
These results indicate that the S.sub.2 and S.sub.3 plants, although 
male-sterile, have not retained the nucleotide sequence (SEQ ID NO: 1) 
described in FIG. 1 in its original conformation, and demonstrate that, in 
this sequence, the portion bounded by nucleotides 928 and 1569 is that 
which carries the "Ogura sterility" specific determinant, which makes the 
plants male-sterile when this sequence is transcribed. 
This sequence has no significant homology with the sequences present in the 
data banks. 
EXAMPLE 2 
DEMONSTRATION OF UNDESIRABLE SEQUENCES IN THE OGURA MITOCHONDRIAL GENOME 
A collection of cybrids was obtained in the species B. napus by protoplast 
fusion between a colza carrying Ogura cytoplasm and a normal colza. The 
former is male-sterile and chlorophyll-deficient at low temperature, while 
the latter is normally green and fertile. The cybrids were sorted from 
among the regenerated plants and those which were male-sterile and 
normally green were adopted. 
In the same manner, a collection of cybrids was obtained in the species B. 
oleracea by protoplast fusion between a cabbage carrying Ogura cytoplasm 
and a normal cabbage. The cybrids which were male-sterile and normally 
green were adopted from among the regenerated plants. 
These cybrids were crossed with different varieties, of colza in the first 
case and cabbage in the second. The crosses were repeated at each 
generation with the same varieties so as to obtain a defined genotype 
close to that of the recurring variety. 
These different varieties, thereby converted with the cytoplasms of 
different cybrids, were subjected to agronomic tests to measure seed 
production, which depends on several factors: a sufficient production of 
nectar to effect pollination by insects, and a normal floral morphology in 
order that this pollination is effective and the fruits develop normally. 
The collection of cybrids could thus be divided into two batches: 
a batch of cybrids possessing a male sterility suited to commercial seed 
production, 
a batch of cybrids not possessing all the characteristics favourable to a 
good commercial seed production. 
The colza cybrids Nos. 27, 58 and 85 and the cabbage cybrids Nos. 9, 17, 
21, 24 and 27c, for example, belong to the first batch. 
The colza cybrids Nos. 23s, 77 and 118 and the cabbage cybrids Nos. 1, 6 
and 14, for example, belong to the second batch. 
The total DNA of these cybrids was subjected to enzymatic digestions with 
SalI, NcoI, NruI, BglI, PstI and KpnI. The Southern blots obtained were 
hybridised with various mitochondrial probes Atpa, Cob, Cox1, Atp6, 26S 
and 18S and two fragments of Ogura genome, of 2.5 kb derived from an NcoI 
digestion and of 19.4 kb derived from an NruI digestion. 
The two batches of cybrids differ in that: 
a) Nos. 23s, 77 and 11s in colza and 1, 16 and 11 in cabbage do possess the 
region of the Ogura genome which surrounds the Cox1 gene, recognisable by 
10.7-kb BglI or 11-kb NruI fragments, and the region of the Ogura genome 
which surrounds one of the formylmethionine transfer RNA genes, 
recognisable by 5.1-kb SalI or 15-kb NruI fragments. 
b) Nos. 27, 58 and 85 in colza and 9, 17, 21, 24 and 27c in cabbage do not 
possess the corresponding regions, which have been replaced, as a result 
of recombinations between the genomes of the two parents which have been 
fused, by analogous regions of the mitochondrial genome of colza in Nos. 
27, 58 and 85 and of cabbage in Nos. 9, 17, 21, 24 and 27c. 
It is deduced from this that the two regions in question of the Ogura 
genome are undesirable if it is desired to have a male sterility system 
suited to commercial seed production. 
EXAMPLE 3 
This example illustrates the value of knowing the "Ogura male sterility" 
sequences and the undesirable sequences for performing an immediate 
sorting of the cybrids obtained without having to wait several years for 
back-crossing and agronomic tests. 
Protoplasts of a Brassica plant carrying the Ogura cytoplasm are fused with 
protoplasts of the Brassica species in question. The colonies derived from 
fusion are cultured in vitro and set up to regenerate on a medium which 
promotes bud formation (see Pelletier et al., 1983). 
From one gram of fresh material, either a callus or a fragment of the 
regenerated plantlet, it is possible by the techniques described above to 
isolate the total DNA. After SalI digestion, Southern type hybridisation 
with the probe bounded by nucleotides Nos. 389 and 1199 (see FIG. 1) 
should give a signal only for a size of 4.4 kb (should not give a signal 
at 5.1 kb). Similarly, after NruI digestion and hybridisation with a probe 
carrying the Cox1 gene, a signal should be obtained for a size different 
from 11 kb. 
These hybridisations enable it to be predicted that the plant which has 
been obtained will indeed be male-sterile and suited to commercial seed 
production. 
EXAMPLE 4 
This example is a variant of Example 3, based on the idea of carrying out 
sexual crossing between the two parents under special conditions or with 
particular genotypes instead of carrying out protoplast fusions, with the 
result that, in contrast to the known processes of fertilisation in 
plants, there is mixing of the cytoplasms of the oosphere and of the 
pollen tube or the male gamete. If such methods were described, an early 
sorting could be performed in the same manner on young plants derived from 
these artificial fertilisations, using the same probes and the same 
criteria as in Example 3. 
EXAMPLE 5 
This example illustrates the value of knowing the Ogura sterility sequence 
in a type of genetic manipulation which has already been described in 
yeast (Johnston et al., 1988). 
Starting with a normal Brassica plant, meristems or alternatively cells in 
vitro are bombarded with micro-particles coated with DNA carrying the 
Ogura sterility sequence. The plants obtained in the progeny of the 
treated meristems or the regenerated plantlets will be cytoplasmic 
male-sterile if the DNA has been able to enter the mitochondria and become 
integrated in the genome of these organelles. This procedure will avoid 
the problems that are created by the undesirable sequences when Ogura 
radish chloroplasts or the sequences so defined of the Ogura mitochondrial 
genome are involved. 
EXAMPLE 6 
This example illustrates the value of knowing the "Ogura sterility" 
sequence in the construction by genetic engineering of a nuclear, instead 
of a cytoplasmic, male sterility conforming to Mendelian inheritance. 
Starting with the mitochondrial DNA sequence bounded by nucleotides 928 and 
1569 (SEQ ID NO: 1), a chimeric gene may be constructed which will be 
transcribed, after genetic transformation of Brassica cells or cells of 
another genus, in the nucleus of the cells of the transformed plants 
obtained. If the chimeric gene contains a pre-sequence which enables its 
protein translation product to be imported into the mitochondrion, these 
transformants will be male-sterile and this character will behave as a 
dominant Mendelian character. 
REFERENCES 
Amann E., Ochs B. and Abel K.-J. (1988) Gene 69:301-315 
Bannerot H., Boulidard L., Cauderon Y. and Tempe J. (1974) 
Proc Eucarpia Meeting 
Cruciferae 25:52-54 
Bannerot H., Boulidard L. and Chupeau Y. (1977) Eucarpia Cruciferae Newsl: 
2-16 
Chetrit P., Mathieu C., Vedel F., Pelletier G. and Primard C. (1985) Theor 
Appl Genet 69:361-366 
Dellaporta S. L., Wood J. and Hicks J. B. (1983) Plant Mol Biol Rep 1:19-21 
Ducruet J. M. and Gasquez J. (1978) Chemosphere 8:691-696 
Frishauf A. M., Lehrach H., Poutska A. and Murray N. (1983) J Mol Biol 
170:827-842 
Gough J. and Murray N. (1983) J Mol Biol 166:1-19 
Hiesel R., Shobel W., Schuster W. and Brennicke A. (1987) EMBO J 6:29-34 
Johnston S. A., Anziano P. Q., Shark K., Sanford J. C. and Butow R. A. 
(1988) Science 240:1538-1541 
Logemann J., Schell J. and Willmitzer L. (1987) Analytical Biochem 
163:16-20 
Ogura H. (1968) Mem Fac Agric Kagoshima Univ 6:39-78 
Pelletier G., Primard C., Vedel F., Chetrit P., Remy R., 
Rousselle P. and Renard M. (1983) Mol Gen Genet 191:244-250 
Sambrook J., Fritsch E. F. and Maniatis T. (1989) Molecular cloning, Cold 
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 
Stern D. B. and Newton K. J. (1986) Methods Enzymol 118:488-496 
Vedel F. and Mathieu C. (1982) Anal Biochem 127:1-8 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 1 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2427 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CCATGGACAATAATCTTAGTCGGAGTCAAATTCCTTACCTTTCCACCCAAAGCTGAACAT60 
ATCCGCACAGATATTCCATTTTTTTTATTGAGGATCCATTTTCGAACTGAACTACTCATG120 
CTTAGGCAAAACAAGCAAGGGAGTTGTTAATAAGGAGCTAGCTACAGTGCTGCGGAGGGT180 
TCCGTGCTTATTAAGGAGCCGGGCAGCTACGCAACACTTCCTTGCAACTCATACCTACTA240 
ACAAACTGTTTACTCTTTTTTAAGAGTTAGCTGCATTCCTGCGGGAGGTACGTACGCAAT300 
CAAAGCAGCAGGGCACGTTCGCAACACCTGCTTCAACTTCATGCACATTAGCAACAAGAT360 
TGGGTAGTTGATTGTTGGGAGGATAGCTGCAGCTCCCTACGGGAGTGAACTACAGTTCCA420 
GGGGGAGCACAGCAAGGGCCAATACCGGCTGTGAGGCGCGTAGCGGGAAGAGATGTATGG480 
TAAGGGATAGCTGTTTAACCATTTGTAATGGAATGGGATGTTGATCCTCCTTGGAATAAT540 
ACGTATATAAGAAGATTTTCATTCCAGTTGGAAAGCAATCGAGAAAACGCCGCCCAAATA600 
CGCTTCGCCACGTGTAGCCCTGTATGGACTCGCGAAGCAGGTCTCCGGTCGGTGTCCAAG660 
ATTTGATCTAACTATTGAGTGAGGACTACTTACCGATTGATAGAATAATACGTATATAAG720 
AAGAAGCTGCTTTGTGGAGTGATCTTTCTCGAAATGAATTAAGTAAGGCGCTATGTTCAG780 
ATTCTGAACCAAAGCACTAGTTGAGGTCTGAAGCCTTATGAGCAGAAGTAATAAATACCT840 
CGGGGAAGAAGCGGGGTAGAGGAATTGGTCAACTCATCAGGCTCATGACCTGAAGATTAC900 
AGGTTCAAATCCTGTCCCCGCACCGTAGTTTCATTCTGCATCACTCTCCCTGTCGTTATC960 
GACCTCGCAAGGTTTTTGAAACGGCCGAAACGGGAAGTGACAATACCGCTTTTCTTCAGC1020 
ATATAAATGCAATGATTACCTTTTTCGAAAAATTGTCCACTTTTTGTCATAATCTCACTC1080 
CTACTGAATGTAAAGTTAGTGTAATAAGTTTCTTTCTTTTAGCTTTTTTACTAATGGCCC1140 
ATATTTGGCTAAGCTGGTTTTCTAACAACCAACATTGTTTACGAACCATGAGACATCTAG1200 
AGAAGTTAAAAATTCCATATGAATTTCAGTATGGGTGGCTAGGTGTCAAAATTACAATAA1260 
AATCAAATGTACCTAACGATGAAGTGACGAAAAAAGTCTCACCTATCATTAAAGGGGAAA1320 
TAGAGGGGAAAGAGGAAAAAAAAGAGGGGAAAGGGGAAATAGAGGGGAAAGAGGAAAAAA1380 
AAGAGGGGAAAGGGGAAATAGAGGGGAAAGAGGAAAAAAAAGAGGTGGAAAATGGACCGA1440 
GAAAATAATGCTTTGTGAACCCAATTGCTTTGACAAAAATAAAGAAAGAAGCAAAATCTC1500 
ATTCAATTTGAAATAGAAGAGATCTCTATGCCCCCTGTTCTTGGTTTTCTCCCATGCTTT1560 
TGTTGGTCAACAACCAACCACAACTTTCTATAGTTCTTCACTACTCCTAGAGGCTTGACG1620 
GAGTGAAGCTGTCTGGAGGGAATCATTTTGTTGAAATCAATTAATCTAATCATGCCTCAA1680 
CTGGATAAATTCACTTATTTTTCACAATTCTTCTGGTTATGCCTTTTCTTCTTTACTTTC1740 
TATATTTTCATATGCAATGATGGAGATGGAGTACTTGGGATCAGCAGAATTCTAAAACTA1800 
CGGAACCAACTGCTTTCACACCGGGGGAAGACCATCCAGAGCAAGGACCCCAACAGTTTG1860 
GAAGATCTCTTGAGAAAAGGTTTTAGCACTGGTGTATCCTATATGTATGCTAGTTTATTC1920 
GAAGTATCCCAATGGTGTAAGGCCGTCGACTTATTGGGAAAAAGGAGGAAAATCACTTTG1980 
ATCTCTTGTTTCGGAGAAATAAGTGGCTCACGAGGAATGGAAAGAAACATATTATATAAT2040 
ATATCGAAGTCCTCTCCTTCAAATACTGGAAGGTGGATCACTTGTAGGAATTGTAGGAAT2100 
GACATAATGCTAATCCATGTTGTACATGGCCAAGGAAGCATAAAATGATTCTTTCATTCT2160 
ATAGATACCTCTGGTAGGTAAAGCACTCTACTGTGCTTTATTGAAAGTTCCCATCGCGGG2220 
GGCGAGGATACTTGCCTTCGCGGTTCGACTTTCTTTTCAGGCTTGACTCATTATTTTCCG2280 
GTCCTCTCACACCCCTTTAGAGCTCTTTATGATGCCCACTGAGTAAGATTCGGGGGCTTC2340 
CCGGCGCAGAAGCTCATTCTGAACCGCGGGAACCTTCGTCTCTTCGACACAAACGTTTTA2400 
TGAAGAGGCTGATGGTGATGAGGATCC2427 
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