The sequence of the T.sub.L -DNA of Ri plasmids found in Agrobacterium rhizogenes strains HRI and A4 is disclosed. Sixteen open reading frames bounded by eukaryotic promoters, ribosome binding sites, and polyadenylation sites were found, five of which were observed to be transcripted in a developmentally and phenotypically regulated manner. The use of promoters and polyadenylation sites from pRi T.sub.L -DNA to control expression of heterologous foreign structural genes is taught, using as examples the structural genes for Phaseolus vulgaris storage protein (phaseolin), P. vulgaris lectin, a sweet protein (thaumatin), and Bacillus thuringiensis crystal protein. Vectors useful for manipulation of sequences of the structural genes and T-DNA are also provided.

FIELD 
The present invention is in the fields of genetic engineering and plant 
husbandry, and especially provides means for promotion of transcription in 
plant. 
BACKGROUND 
Following are publications which disclose background information related to 
the present invention. These publications are discussed in greater depth 
in the Background sections indicated. Restriction maps of Ri plasmids are 
disclosed by G. A. Huffman et al. (1984) J. Bacteriol. 157:269-276; L. 
Jouanin (1984) Plasmid 12:91-102; and M. Pomponi et al. (1983) Plasmid 
10:119-129 (see TIP Plasmid DNA). L. Herrera-EstreIla et al. (1983) Nature 
303: 209-213, provides examples of use of the nos promoter to drive 
expression in plants of heterologous foreign structural genes. N. Murai et 
al:. (1983) Science 222:476-482, reported the ocs promoter could drive 
expression of an intron-containing fusion gene having foreign coding 
sequences. (Manipulations of the TIP Plasmids). R. F. Barker et al. (1983) 
Plant Molec. Biol. 2:335-350, and R. F. Barker and J. D. Kemp, U.S. Patent 
application Ser. No. 553,786 disclose the complete sequence of the T-DNA 
from the octopine-type plasmid pti15955; homologous published sequences of 
other Ti plasmid genes are referenced therein. Barker and Kemp also taught 
use of various octopine T-DNA promoters to drive expression in plants of 
various structural genes (Genes on the TIP Plasmids). 
Shuttle Vectors 
Shuttle vectors, developed by G. B. Ruvkun and F. M. Ausubel (1981) Nature 
289:85-88, which provide means for inserting foreign genetic material into 
large DNA molecules, include copies of recipient genome DNA sequences into 
which the foreign genetic material is inserted. Shuttle vectors can be 
introduced a recipient cell, by well known methods, including the 
tri-parental mating technique (Ruvkin and Ausubel, supra), direct transfer 
of a self-mobilizable vector in a bi-parental mating, direct uptake of 
exogenous DNA by Agrobacterium cells ("transformation"), spheroplast 
fusion of Agrobacterium with another bacterial cell, uptake of 
liposome-encapsulated DNA. After a shuttle vector is introduced into a 
recipient cell, possible events include a double cross-over with one 
recombinational event on either side of the marker (homogenotization). 
Phenotypically dominant traits may be introduced by single cross-over 
events (cointegration) (A. Caplan et al. (1983) Science 222:815-821; R. B. 
Horsch et al. (1984) Science 223:496-498); one must guard against deletion 
of the resulting tandem duplication. Shuttle vectors have proved useful in 
manipulation of Agrobacterium plasmids. 
"Suicide Vectors" (e.g. R. Simon et al. (1983) Biotechnol. 1:784-791), are 
shuttle vectors having replicons not independently maintainable within the 
recipient cell. Use of suicide vectors to transfer DNA sequences into a Ti 
plasmid has been reported (e.g. E. Van Haute et al. (1983) EMBO J. 
2:411-417; L. Comai et al. (1983) Plasmid 10:21-30; P. Zambryski et al. 
(1983) EMBO J. 2:2143-2150; P. Zambryski et al. (1984) in Genetic 
Engineering, Principles, and Methods, 6, eds: A. Hollaender and J. Setlow; 
P. Zahm et al. (1984) Mol. Gen. Genet. 194:188-194; and Caplan et al., 
supra; and C. H. Shaw et al. (1983) Gene 28:315-330. 
Overview of Agrobacterium 
Included within the gram-negative genus Agrobacterium are the species A. 
tumefaciens and A. rhizogenes, respectively the causal agents of crown 
gall disease and hairy root disease of gymnosperm and dicotyledonous 
angiosperm plants. In both diseases, the inappropriately growing plant 
tisssue usually produces one or more amino acid derivatives known as 
opines which may be classified into families whose type members include 
octopine, nopaline, mannopine, and agropine. 
Virulent strains of Agrobacterium harbor large plasmids known as Ti 
(tumor-inducing) plasmids (pTi) in A. tumefaciens and Ri (root-inducing) 
plasmids in A. rhizogenes (pRi), often classified by the opine which they 
caused to be synthesized. Ti and Ri plasmids both contain DNA sequences, 
referred to as T-DNA (transferred-DNA), which in tumors are found to be 
integrated into the genome of the host plant. Several T-DNA genes are 
under control of T-DNA promoters which resembles the canonical eukaryotic 
promoter in structure. The Ti plasmid also carries genes outside the T-DNA 
region. The set of genes and DNA sequences responsible for transforming 
the plant cell are hereinafter collectively referred to as the 
transformation-inducing principle (TIP). The term TIP the therefore 
includes, but is not limited to, both Ti and Ri plasmids. 
General reviews of Agrobacterium-caused disease include those by D. J. 
Merlo (1982), Adv. Plant. Pathol. 1:139-178; L. W. Ream and M. P. Gordon 
(1982), Science 218:854-859; M. W. Bevan and M.-D. Chilton (1982), Ann. 
Rev. Genet. 16:357-384; G. Kahl and J. Schell (1982) Molecular Biology of 
Plant Tumors; K. A. Barton and M.-D. Chilton (1983) Meth. Enzymol. 
101:527-539; A. Depicker et al. (1983) in Genetic Engineering of Plants: 
an Agricultural Perspective, eds: T. Kosuge et al., pp. 143-176; A. Caplan 
et al. (1983) Science 222:815-821; T. C. Hall et al., European Patent 
application 126,546; and A. N. Binns (1984) Oxford Surveys Plant Mol. Cell 
Biol. 1:130-160. A number of more specialized reviews can be found in A. 
Puhler, ed. (1983) Molecular Genetics of the Bacteria-Plant Interaction, 
including a treatment by D. Tepfer of A. rhizogenes-mediated 
transformation (pp. 248-258). R. A. Schilperoort (1984) in Efficiency in 
Plant Breeding (Proc. 10th Congr. Eur. Assoc. Res. Plant Breeding), eds: 
W. Lange et al., pp. 251-285, discusses the Agrobacterium-based plant 
transformation in the context of the art of plant genetic engineering and 
plant improvement. 
Infection of Plant Tissues 
Plant cells can be transformed by Agrobacterium by several methods known to 
the art. For a review of recent work, see K. Syono (1984) Oxford Surveys 
Plant Mol. Cell Biol. 1:217-219. In the present invention, any method will 
suffice as long as the gene is stably transmitted through mitosis and 
meiosis. 
The infection of plant tissue by Agrobacterium is a simple technique well 
known to those skilled in the art. Typically after being wounded, a plant 
is inoculated with a suspension of tumor-inducing bacteria. Alternatively, 
tissue pieces are inoculated, e.g. leaf disks (R. B. Horsch et al. 
(1985)Science 227:1229-1231) or inverted stem segments (K. A. Barton et 
al. (1983) Cell 32:1033-1043). After induction, the tumors can be placed 
in tissue culture on media lacking phytohormones usually included for 
culture of untransformed plant tissue. Traditional inoculation and culture 
techniques may be modified for use of disarmed T-DNA vectors incapable of 
inducing hormone independent growth (e.g. see P. Zambryski et al. (1984) 
in Genetic Engineering, Principles, and Methods, 6, eds.: A. Hollaender 
and J. Setlow). 
Agrobacterium is also capable of infecting isolated cells, cells grown in 
culture, callus cells, and isolated protoplasts (e.g. R. B. Horsch and R. 
T. Fraley (1983) in Advances in Gene Technology: Molecular Genetics of 
Plants and Animals (Miami Winter Symposium 20), eds.: K. Downey et al., p. 
576; R. T. Fraley et al. (1984) Plant Mol. Biol. 3:371-378; R. T. Fraley 
and R. B. Horsch (1983) in Genetic Engineering of Plants: an Agricultural 
Perspective, eds.: T. Kosuge et al., pp. 177-194; A. Muller et al. (1983) 
Biochem. Biophys. Res. Comm. 123:458-462). The transformation frequency of 
inoculated callus pieces can be increased by addition of an opine or opine 
precursors (L. M. Cello and W. L. Olsen, U.S. Pat. No. 4,459,355). 
Plant protoplasts can be transformed by the direct uptake of TIP DNA in the 
presence of a polycation, polyethelene glycol, or both (e.g. F. A. Krens 
et al. (1982) Nature 296:72-74), though integrated Ti plasmid may include 
non-T-DNA sequences. 
An alternative method involves uptake of DNA surrounded by membranes. 
pTi-DNA may be introduced via liposomes or by fusion of plant and 
bacterial cells after removal of their respective cell walls (e.g. R. Hain 
et al. (1984) Plant Cell Rept. 3:60-64). Plant protoplasts can take up 
cell wall delimited Agrobacterium cells. T-DNA can be transmitted to 
tissue regenerated from fused protoplasts. 
The host range of crown gall pathogenesis may be influenced by 
T-DNA-encoded functions such as onc genes (A. Hoekema et el. (1984) J. 
Bacteriol. 158:383-385; A. Hoekema et al. (1984) EMBO J. 3:3043-3047; W. 
C. Buchholz and M. F. Thomasshow (1984) 160:327-332). R. L. Ausich, 
European Patent Application 108,580, reports transfer of T-DNA from A. 
tumefaciens to green algal cells, and expression therein of octopine 
synthase and Tn5 kanamycin resistance genes. G. M. S. Hooykaasvan 
Slogteren et al. (1984) Nature 311:783-764, and J.-P. Hernalsteens et al. 
(1984) EMBO J. 3:3039-3041, have demonstrated transformation of monocot 
cells by Agrobacterium without the customary tumorigenesis. 
Regeneration of Plants 
Differentiated plant tissues with normal morphology have been obtained from 
crown gall tumors. For example, L. Otten et al. (1981) Molec Gert. Genet. 
183:209-213, used tms (shoot-inducing, root-suppressing) Ti plasmid 
routants to create tumors which proliferated shoots that formed 
self-fertile flowers. The resultant seeds germinated into plants which 
contained T-DNA and made opines. The tms phenotype can be partly overcome 
by washing of the rooting area and can be bypassed by grafting onto a 
normal stock (A. Wostemeyer et al. (1984) Mol. Gen. Genet. 194:500-507). 
Similar experiments with tmr (root-inducing, shoot-suppressing) mutant 
showed that full-length T-DNA could be transmitted through meiosis to 
progeny and that in those progeny nopaline genes could be expressed, 
though at variable levels (K. A. Barton et al. (1983) Cell 32:1033-1043). 
Genes involved in opine anabolism were capable of passing through meiosis, 
though the plants were male sterile if the T-DNA was not disarmed. 
Seemingly unaltered T-DNA and functional foreign genes can be inherited in 
a dominant, closely linked, Mendelian fashion. Genetically, T-DNA genes 
are closely linked in regenerated plants (A. Wostemeyer et al. (1984) Mol. 
Gert. Genet. 194:500-507; R. B. Horsch et al. (1984) Science 223:496-498; 
D. Tepfer (1984) Cell 37:959-967). 
The epigenetic state of the plant cells initially transformed can affect 
regeneration potential (G. M. S. van Slogteren et al. (1983) Plant Mol. 
Biol. 2:321-333). 
Roots resulting from transformation from A. rhizogenes have proven 
relatively easy to regenerate directly into plantlets (M.-D. Chilton et 
al. (1982) Nature 295:432-434; D. Tepfer (1984) Cell 37:959-967; Tepfer 
(1983) in Puhler, supra), and are easily cloned. Regenerability from 
transformed roots may be dependent on T-DNA copy-number (C. David et al. 
(1984) Biotechnol. 2:73-76). Hairy root regenerants have a rhizogenic 
potential and isozyme pattern not found in untransformed plants (P. 
Costantino et al. (! 984) J. Mol. Appl. Genet. 2:465-470). The phenotype 
of these plants is generally altered, although not necessarily 
deleteriously. 
Genes on the TIP Plasmids 
The complete sequence of the T-DNA of an octopine-type plasmid found in 
ATCC 15955, pti15955, has been reported (R. F. Barker et al. (1983) Plant 
Molec. Biol. 2:335-350), as has that of the T.sub.L region of pTiAch5 (J. 
Gielen et al. (1984) EMBO J. 3:835-846). Published T-DNA genes do not 
contain introns and do have sequences that resemble canonical eukaryotic 
promoter elements and polyadenylation sites. 
Ti plasmids having mutations in the genes tms, tmr, tinl, and ocs 
respectively incite tumorous calli of Nicotiana tabacum which generate 
shoots, proliferate roots, are larger than normal, and do not synthesize 
octopine; all but ocs are onc (oncogenicity) genes. In other hosts, 
routants of these genes can induce different phenotypes (see M. W. Beyan 
and M.-D. Chilton (1982) Ann. Rev. Genet. 16:357-384). Mutations in T-DNA 
genes do not seem to affect the insertion of T-DNA into the plant genome 
(J. Leemans et al. (1982) EMBO J. 1:147-152; L. W. Ream et al. (1983) 
Proc. Natl. Acad. Sci. USA 80:1660-1664). 
Octopine Ti plasmids carry an ocs gene which encodes octopine synthase 
(lysopine dehydrogenase). All upstream signals necessary for expression of 
the ocs gene are found within 295 bp of the ocs transcriptional start site 
(C. Koncz et al. (1983) EMBO J. 2:1597-1603). P. Dhaese et al. (1983) EMBO 
J. 2:419-426, reported the utilization of various polyadenylation sites by 
"transcript 7" (ORF3 of Barker et al., supra) and ocs. The presence of the 
enzyme octopine synthase within a tissue can protect that tissue from the 
toxic effect of various amino acid analogs (G. A. Dahl and J. Tempe (1983) 
Theor. Appl. Genet. 66:233-239; M. G. Koziel et al. (1984) J. Mol. Appl. 
Genet. 2:549-562). 
Nopaline Ti plasmids encode the nopaline synthase gene (nos) (sequenced by 
A. Depicker et al. (1982) J. Mol. Appl. Genet. 1:561-573). The "CAAT" box, 
but not upstream sequences therefrom, is required for wild-type levels of 
nos expression; a partial or complete "TATA" box supports very low level 
nos activity (C. H. Shaw et al. (1984) Nucl. Acids Res. 12:7831-7846). 
Genes equivalent to tms and tmr have been identified on a nopaline-type 
plasmid and a number of transcripts have been mapped (L. Willmitzer et al. 
(1983) Cell 32:1045-1056). 
Transcription from hairy root T-DNA has also been detected (L. Willmitzer 
et al. (1982) Mol. Gen. Genet. 186:16-22). Ri plasmids and tms.sup.- Ti 
plasmids can complement each other when inoculated onto plants, resulting 
in calli capable of hormone-independent growth (G. M. S. van Slogteren 
(1983) Ph.D. thesis, Rijksuniversiteit te Leiden, Netherlands). 
TIP plasmid genes outside of the T-DNA region include the vir genes, which 
when mutated result in an avirulent Ti plasmid. Several vir genes have 
been accurately mapped and have been found to be located in regions 
conserved among various Ti plasmids (V. N. Iyer et al. (1982) Mol. Gen. 
Genet. 188:418-424). The vir genes function in trans, being capable of 
causing the transformation of plant cells with T-DNA of a different 
plasmid type and physically located on another plasmid (e.g. A. J. de 
Framond et al. (1983) Biotechnol. 1:262-269; A. Hoekema et al. (1983) 
Nature 303:179-180; J. Hille et al. (1984) J. Bacteriol. 158:754-756; A. 
Hoekema et al. (1984) J. Bacteriol. 1.58:383-385); such arrangements are 
known as binary systems. Chilton et al. (18 Jan. 1983) 15th Miami Winter 
Symp., described a "micro-Ti" plasmid made by resectioning the "mini-Ti" 
of de Framond et al., supra (see European Patent application 126,546 for a 
description). G. A. Dahl et al., U.S. patent application Ser. No. 532,280, 
and A. Hoekema (1985) Ph.D. Thesis, Rijksuniversiteit te Leiden, The 
Netherlands, disclose micro-Ti plasmids carrying ocs genes constructed 
from pti15955. M. Bevan (1984) Nucl. Acids Res. 12:8711-8721, discloses a 
kanamycin-resistant micro-Ti. T-DNA need not be on a plasmid to transform 
a plant cell; chromosomally located T-DNA is functional (A. Hoekema et al. 
(1984) EMBO J. 3:2485-2490). Ti plasmid-determined characteristics have 
been reviewed by Merlo, Supra (see especially Table II therein), and Ream 
and Gordon, supra. 
TIP Plasmid DNA 
Ri plasmids have been shown to have extensive homology among themselves (P. 
Costantino et al. (1981) Plasmid 5:170-182), and to both octopine (F. F. 
White and E. W. Nester (1980) J. Bacteriol. 144:710-720) and nopaline (G. 
Risuleo et al. (1982) Plasmid 7.:45-51) Ti plasmids, primarily in regions 
encoding vir genes, replication functions, and opine metabolism functions 
(L. Jouanin (1984) Plasmid 12:91-102; K. Lahners et al. (1984) Plasmid 
1I:130-140; E. E. Hood et al. (1984) Biotechnol. 2:702-709; F. Leach 
(1983) Ph.D. Thesis, Universire de Paris-Sud, Centre d'Orsay, France); 
none of the homologies are in pRi T.sub.L -DNA. pRi T-DNA contains 
extensive though weak homologies to T-DNA from both types of Ti plasmid 
(L. Willmitzer et al. (1982) Mol. Gen. Genet. 186:16-22). DNA from several 
plant species contains sequences, referred to as cT-DNA (cellular T-DNA), 
having homology with the Ri plasmid (F. F. White et al. (1983) Nature 
301:348-350, L. Spano et al. (1982 ) Plant Molec. Biol. 1:291-300; D. 
Tepfer (1982) in 2e Colloque sur les Recherches Fruitieres Bordeaux, pp. 
47-59). G. A. Huffman et al. (1984) J. Bacteriol. 157:269-276 and Jouanin, 
supra, and Leach, supra, have shown that, in the region of 
cross-hybridization, the Ri plasmid pRiA4.sub.b is more closely related to 
a pTiA6 (octopine-type) than pTiT37 (nopaline-type) and that this Ri 
plasmid appears to carry sequence homologous to tms but not tmr. Their 
results also suggested that Ri T-DNA may be discontinuous, analogous to 
the case with octopine T-DNA (see below). The restriction maps of 
pRiA4.sub.b, pri1855, and pRiHRI were respectively disclosed by Huffman et 
al., supra, M. Pomponi et al. (1983) Plasmid 10:119-129, and L. Jouanin 
supra. Ri plasmids are often characterizable as being agropine-type or 
mannopine-type (A. Petit et al. (1983) Mol. Gen. Genet. 190:204-214). 
A portion of the Ti or Ri plasmid is found in the DNA of tumorous plant 
cells, T-DNA may be integrated (i,e. inserted) into host DNA at multiple 
sites in the nucleus, Flanking plant DNA may be either repeated or low 
copy number sequences, Integrated T-DNA can be found in either direct or 
inverted tandem arrays and can be separated by spacers, Much non-T-DNA Ti 
plasmid DNA appears to be transferred into the plant cell prior to T-DNA 
integration (H, Joos et al, (1983) EMBO J. 2:2151-2160). T-DNA has direct 
repeats of about 25 base pairs associated with the borders, i.e. with the 
T-DNA/plant DNA junctions, which may be involved in either transfer from 
Agrobacterium or integration into the host genome. 
Ri plasmids integrate two separate T-DNAs, T.sub.L -DNA and T.sub.R -DNA, 
left and right T-DNAs, respectively. T.sub.L (about 15-20 kbp) and T.sub.R 
(about 8-10 kbp) are separated by about 15-20 kbp (Huffman et al., supra, 
Jouanin, supra). The region of agropine-type pRi T.sub.L and T.sub.R 
integrated can vary between individual plants or species inoculated (F. F. 
White et al. (1983) Nature 301:348-350; D. A. Tapfar (1984) Cell 
37:959-967). Though T-DNA is occasionally deleted after integration in the 
plant genome, it is generally stable. Tumors containing a mixture of cells 
which differ in T-DNA organization or copy number are the result of 
multiple transformation events. 
The exact location relative to the border repeats of T-DNA/flanking plant 
DNA junctions varies and need not be within a border repeat. Virulence is 
not always eliminated after deletion of one of either of the usual 
nopaline T-DNA border sequences (compare H. Joos et al. (1983) Cell 
32:1057-1067 with K. Wang et al. (1984) Cell 38:455-462 and C. H Shaw et 
al. (1984) Nucl. Acids Res. 12:6031-6041, concerning the right border). 
The orientation of the right nopaline-border can be reversed without total 
loss of functionality, and a single border sequence is capable of 
transforming closely-linked sequences (M. De Block et al. (1984) EMBO J. 
3:1681-1689). A synthetic 25 bp nopaline right border repeat is functional 
(Wang et al., supra). Circular intermediates associated with T-DNA 
transfer appear to be spliced precisely within the 25 bp direct repeats 
(Z. Koukolikova-Nicola et al. (1985) Nature 313:191-196). 
Manipulations of the TIP Plasmids 
Altered DNA sequences, including deletions, may be inserted into TIP 
plasmids (see Shuttle Vectors). Some pTi derivatives can be transferred to 
E. coli and mutagenized therein (J. Hille et al. (1983) J. Bacteriol. 
154:693-701). P. Zambryski et al. (1983) EMBO J. 2:2143-2150, report use 
of a vector, deleted for most T-DNA genes to transform tobacco and 
regenerate morphologically normal plants. 
The nopaline synthase promoter can drive expression of drug resistance 
structural genes useful for selection of transformed plant cells. M. W. 
Bevan et el. (1983) Nature 3.04:184-187; R. T. Fraley et el. (1983) Proc. 
Natl. Acad. Sci. USA 80:4803-4807; and L. Herrera-Estrella et el. (1983) 
EMBO J. 2:987-995, have inserted the bacterial kanamycin resistance 
structural gene (neomycin phosphotransferase II, NPT2), or kan, from Tn5 
downstream from (i.e. behind or under control of) the nopaline synthase 
promoter. The constructions were used to transform plant cells which in 
culture were resistant to kanamycin and its analogs such as neomycin and 
G418. Promoters for octopine T.sub.L genes ORF24 and ORF25 can also drive 
kan structural gene expression (J. Velten et el. (1984) EMBO J. 
3:2723-2730). Herrera-Estrella et el., supra, reported a similar 
construction, in which a methotrexate resistance gene (dihydrofolate 
reductase, DHFR) from Tn7 was placed behind the nos promoter; transformed 
plant cells were resistant to methotrexate. Furthermore, L. 
Herrera-Estrella et el. (1983) Nature 303:209-213, have obtained 
expression in plant cells of enzymatic activity of octopine synthase and 
chloramphenicol acetyltransferase by placing their structural genes under 
control of nos promoters. G. Helmer et el. (1984) Biotechnol. 2:520-527, 
have created a fusion gene useful as a screenable marker having the 
promoter and 5'-end of the nos structural gene fused to E. coli 
.beta.-galactosidase (lacZ) sequences. 
N. Murai et el. (1983) Science 222:476-482, reported fusion of the promoter 
and the 5'-end of the octopine synthase structural gene to a phaseolin 
structural gene. The encoded fusion protein was produced under control of 
the T-DNA promoter. Phaseolin-derived introns underwent proper 
post-transcriptional processing. 
SUMMARY OF THE INVENTION 
One object of this invention is to provide means for promoting the 
expression of structural genes within plant cells wherein said genes are 
foreign to said cells. In pursuance of this goal, other objects are to 
provide pRi T-DNA promoters and transcript terminators, and especially pRi 
T.sub.L -DNA-derived promoters and pRi T.sub.L -DNA-derived 
polyadenylation sites, which are DNA sequences capable of controlling 
structural gene transcription and translation within plant cells, and to 
provide developmental and phenotypic regulation of said foreign structural 
genes. Another object is to provide specialized plant tissues and plants 
having within them proteins encoded by foreign structural genes and, in 
cases where the protein is an enzyme, having or lacking metabolites or 
chemicals which respectively are not or are otherwise found in the cells 
in which the genes is inserted. Other objects and advantages will become 
evident from the following description. 
The invention disclosed herein provides a plant comprising a genetically 
modified plant cell having a foreign structural gene introduced and 
expressed therein under control of pRi T.sub.L -DNA-derived plant 
expressible transcription controlling sequences (TxCS). Further, the 
invention provides plant tissue comprising a plant cell whose genome 
includes T-DNA comprising a foreign structural gene inserted in such 
orientation and spacing with respect to pRi T.sub.L -DNA-derived 
plant-expressible TxCS as to be expressible in the plant cell under 
control of those sequences. Also provided are novel strains of bacteria 
containing and replicating T-DNA, the T-DNA being modified to contain an 
inserted foreign structural gene in such orientation and spacing with 
respect to a T-DNA-derived, plant-expressible TxCS as to be expressible in 
a plant cell under control of said TxCS. Additionally, the invention 
provides novel vectors having the ability to replicate in E. coli and 
comprising T-DNA, and further comprising a foreign structural gene 
inserted within T-DNA contained within the vector, in such manner as to be 
expressible in a plant cell under control of a pRi T.sub.L -DNA TxCS. 
Furthermore, strains of bacteria harboring said vectors are disclosed. 
Much is known about the location, size, and function of many transcripts 
activated when A. tumefaciens T-DNA regions are transferred into the 
genome of plants (see Background). Most pTi T-DNA T.sub.L -DNA open 
reading frames (ORFs) correlate with known gene products. However, until 
the disclosure of the present invention, the art knew little about the 
number, size, and function of genes activated when the T.sub.L -DNA 
regions from A. rhizogenes plasmids, such as pRiA4, are transferred into a 
plant genome. Agropine synthase, tms-1 and tms-2 genes have been 
identified by homology with pTi T-DNA in Ri plasmids, but these loci are 
located in pRi T.sub.R -DNA (G. A. Huffman et al. (1984) J. Bacteriol. 
157:269-276; , L. Jouanin (1984) Plasmid 12:91-102). The experimental work 
presented herein is believed to be the first disclosure of a pRi T.sub.L 
-DNA sequence or of any sequence homologous thereto. The availability of 
this sequence will enable and otherwise facilitate work in the art of 
plant transformation to express foreign structual genes and to engage in 
other manipulations of pRi T.sub.L -DNA and pRi T.sub.L -DNA-derived 
sequences. Without the newly disclosed pRi T.sub.L -DNA sequence, those of 
ordinary skill in the art would be unable to use promoters and 
polyadenylation sites contained therein to promote transcription and 
translation in plant cells of foreign structural genes. The disclosed 
sequence reveals the existence of previously unknown T-DNA ORFs and 
associated transcription controlling sequences, and makes possible 
construction of recombinant DNA molecules using promoters and 
polyadenylation sites from pRi T.sub.L -DNA genes whose sequences were 
hitherto unknown and unavailable to the public. The work presented herein 
is also believed to be the first disclosure of developmental and 
phenotypic regulation of T-DNA genes. Results newly disclosed herein will 
allow those of ordinary skill in the art to use T-DNA transcription 
controlling sequences which are so regulated to express heterologous 
foreign structural genes in transformed plants. T-DNA genes known to the 
art before the present disclosure are not known to be so regulated. 
Furthermore, knowledge of pRi T.sub.L -DNA sequence enables one to bring 
to utility promoters and polyadenylation sites that are presently 
unrecognized; in the future, should a new pRi T.sub.L -DNA transcript be 
discovered and mapped, the sequence disclosed herein will permit 
associated TxCSs to be combined with heterologous foreign structural 
genes. 
The present invention comprises foreign structural genes under control of 
pRi T.sub.L -DNA promoters expressible in plant cells, the promoter/gene 
combination being inserted into a plant cell by any means known to the 
art. More specifically, in its preferred embodiment the invention 
disclosed herein comprises expression in plant cells of foreign structural 
genes under control of certain pRi T.sub.L -DNA-derived plant expressible 
TxCSs, after introduction via T-DNA, that is to say, by inserting the 
foreign structural gene into T-DNA under control of a pRi T.sub.L -DNA 
promoter and/or ahead of a pRi T.sub.L -DNA polyadenylation site and 
introducing the T-DNA containing the TxCS/structural gene combination into 
a plant cell using known means. Once plant cells transformed to contain a 
foreign structural gene expressible under control of a pRi T.sub.L -DNA 
TxCS are obtained, plant tissues and whole plants can be regenerated 
therefrom using methods and techniques well known in the art. The 
regenerated plants are then reproduced by conventional means and the 
introduced genes can be transferred to other strains and cultivars by 
conventional plant breeding techniques. The invention in principle applies 
to any introduction of a foreign structural gene combined with a pRi 
T.sub.L -DNA promoter or polyadenylation site into any plant species into 
which foreign DNA (in the preferred embodiment pTi T-DNA) can be 
introduced and maintained by any means. In other words, the invention 
provides a means for expressing a structural gene in a plant cell and is 
not restricted to any particular means for introducing foreign DNA into a 
plant cell and maintaining the DNA therein. Such means include, but are 
not limited to, T-DNA-based vectors (including pTi-based vectors), viral 
vectors, minichromosomes, non-T-DNA integrating vectors, and the like. 
The invention is useful for genetically modifying plant cells, plant 
tissues, and whole plants by inserting useful structural genes from other 
species, organisms, or strains that change phenotypes of plants or plant 
cells when expressed therein. Such useful structural genes include, but 
are not limited to, genes conveying phenotypes such as improved tolerance 
to extremes of heat or cold; improved tolerance to drought or osmotic 
stress; improved resistance or tolerance to insect (e.g. insecticidal 
toxins), arachnid, nematode, or epiphyte pests and fungal, bacterial, or 
viral diseases, or the like; the production of enzymes or secondary 
metabolites not normally found in said tissues or plants; improved 
nutritional (e.g. storage proteins or lectins), flavor (e.g. sweet 
proteins), or processing properties when used for fiber or human or animal 
food; changed morphological traits or developmental patterns (e.g. leaf 
hairs which protect the plant from insects, aesthetically pleasing 
coloring or form, changed plant growth habits, dwarf plants, reduced time 
needed for the plants to reach maturity, expression of a gene in a tissue 
or at a time that gene is not usually expressed, and the like); male 
sterility; improved photosynthetic efficiency (including lowered 
photorespiration); improved nitrogen fixation; improved uptake of 
nutrients; improved tolerance to herbicides; increased crop yield; 
improved competition with other plants; and improved germplasm 
identification by the presence of one or more characteristic nucleic acid 
sequences, proteins, or gene products, or phenotypes however identified 
(to distinguish a genetically modified plant of the present invention from 
plants which are not so modified, to facilitate transfer of a linked 
artificially introduced phenotype by other (e.g. sexual) means to other 
genotypes or to facilitate identification of plants protected by patents 
or by plant variety protection certificates); selectable markers (i.e. 
genes conveying resistance in cell or tissue culture to selective agents); 
screenable markers; and the like. 
The invention is exemplified by introduction and expression of a structural 
gene for phaseolin, the major seed storage protein of the bean Phaseolus 
vulgaris L., into plant cells. The introduction and expression of the 
structural gene for phaseolin, for example, can be used to enhance the 
protein content and nutritional value of forage or other crops. The 
invention is also exemplified by the introduction and expression of a 
lectin structural gene, in this case also obtained from P. vulgaris, into 
plant cells. The introduction and expression of a novel lectin may be used 
to change the nutritional or symbiotic properties of a plant tissue. The 
invention is exemplified in yet other embodiments by the introduction and 
expression of DNA sequences encoding thaumatin, and its precursors 
prothaumatin, prethaumatin, and preprothaumatin. Mature thaumatin is a 
heat-labile, sweet-tasting protein found naturally in katemfe 
(Thaumatococcus daniellii) which can be used to enhance the flavor of 
vegetables which are eaten uncooked without significantly increasing the 
caloric content of the vegetables. The invention is further exemplified by 
introduction and expression of a structural gene for a crystal protein 
from B. thuringensis var. kurstaki HD-73 into plant cells. The 
introduction and expression of the structural gene for an insecticidal 
protein can be used to protect a crop from infestation with insect larvae 
of species which include, but are not limited to, hornworm (Manduca sp.), 
pink bollworm (Pectionophora gossypiella), European corn borer (Ostrinia 
nubilalis), tobacco budworm (Heliothis virescens), and cabbage looper 
(Trichoplusia ni). Applications of insecticidal protein prepared from 
sporulating B. thuringliensis does not control insects such as the pink 
bollworm in the field because of their particular life cycles and feeding 
habits. A plant containing in its tissues insecticidal protein will 
control this recalcitrant type of insect, thus providing advantage over 
prior insecticidal uses of B. thuringiensis. By incorporation of the 
insecticidal protein into the tissues of a plant, the present invention 
additionally provides advantage over such prior uses by eliminating 
instances of nonuniform application and the costs of buying and applying 
insecticidal preparations to a field. Also, the present invention 
eliminates the need for careful timing of application of such preparations 
since small larvae are most sensitive to insecticidal protein and the 
protein is always present, minimizing crop damage that would otherwise 
result from preapplication larval foraging. Other uses of the invention, 
exploiting the properties of other structural genes introduced into 
various plant species, will be readily apparent to those skilled in the 
art.

DETAILED DESCRIPTION OF THE INVENTION 
The following terms are defined in order to remove ambiguities to the 
intent or scope of their usage in the Specification and Claims. 
TxCS 
Transcription controlling sequences refers to a promoter/transcript 
terminator combination flanking a particular structural gene or open 
reading frame (ORF). The promoter and transcript terminator DNA sequences 
flanking a particular inserted foreign structural gene need not be derived 
from the same source genes (e.g. pairing two different pRi T.sub.L -DNA) 
genes or the same taxonomic source (e.g. pairing sequences from pRi 
T.sub.L -DNA with sequences from non-pRi-T.sub.L -DNA sources such as 
other types of T-DNA, plants, animals, fungi, yeasts, and eukaryotic 
viruses). Therefore the term TxCS refers to either combination of a 
claimed promoter with an unclaimed transcript terminator, or combination 
of a unclaimed promoter with a claimed polyadenylation site, or 
combination of a promoter and a polyadenylation site which are both 
claimed. Examples of non-pRi-T.sub.L -DNA plant-expressible promoters 
which can be used in conjunction with a pRi T.sub.L -DNA polyadenylation 
site include, but are not limited to, those from genes for nos, ocs, 
phaseolin, RuBP-Case small subunit and the 19S and 35S transcripts of 
cauliflower mosaic virus (CaMV). 
Promoter 
Refers to sequences at the 5'-end of a structural gene involved in 
initiation of translation or transcription. Expression under control of a 
pRi T-DNA promoter may take the form of direct expression in which the 
structural gene normally controlled by the promoter is removed in part or 
in whole and replaced by the inserted foreign structural gene, a start 
codon being provided either as a remnant of the pRi T-DNA structural gene 
or as part of the inserted structural gene, or by fusion protein 
expression in which part or all of the structural gene is inserted in 
correct reading frame phase within the existing pRi T-DNA structural gene. 
In the latter case, the expression product is referred to as a fusion 
protein. The promoter segment may itself be a composite of segments 
derived from a plurality of sources, naturally occurring or synthetic. 
Eukaryotic promoters are commonly recognized by the presence of DNA 
sequences homologous to the canonical form 5' . . . TATAA . . . 3' about 
10-30 bp 5' to the location of the 5'-end of the mRNA (cap site). About 30 
bp 5' to the TATAA another promoter sequence is often found which is 
recognized by the presence of DNA sequences homologous to the canonical 
form 5' . . . CCAAT . . . 3'. Translational initiation often begins at the 
first 5' . . . AUG . . . 3' 3'-from the cap site (see Example 1.5). 
Transcript terminator 
Refers to any nucleic acid sequence capable of determining the 3'-end of a 
eukaryotic messenger RNA (mRNA). The transcript terminator DNA segment may 
itself be a composite of segments derived from a plurality of sources, 
naturally occurring or synthetic, and may be from a genomic DNA or an 
RNA-derived cDNA. Some eukaryotic RNAs, e.g. histone mRNA (P. A. Krieg and 
D. A. Melton (1984) Nature 308:203-206), ribosomal RNA, and transfer RNA, 
are not 3'-terminated by polyadenylic acid or by polyadenylation sites; it 
is intended that the term transcript terminator include, but not be 
limited to, both nucleic acid sequences determining the 3'-ends of such 
transcripts and polyadenylation site sequences (see below). 
Polyadenylation site 
Refers to any nucleic acid sequence capable of determining the 3'-end of a 
eukaryotic polyadenylated mRNA. After transcriptional termination 
polyadenylic acid "tails" are added to the 3'-end of most mRNA precursors. 
The polyadenylation site DNA segment may itself be a composite of segments 
derived from a plurality of sources, naturally occurring or synthetic, and 
may be from a genomic DNA or an mRNA-derived cDNA. Polyadenylation sites 
are commonly recognized by the presence of homology to the canonical form 
5' . . . AATAAA . . . 3', although variation of distance, partial 
"read-thru", and multiple tandem canonical sequences are not uncommon. It 
should be recognized that a canonical "polyadenylation site" may in fact 
not actually cause polyadenylation per se (N. Proudfoot (1984) Nature 
307:412-413) and that sequences 3' to the "AATAAA" and the 3'-end of the 
transcript may be needed (A. Gil and N. J. Proudfoot (1984 ) Nature 
312:473-474). 
Foreign structural gene 
As used herein includes that portion of a gene comprising a DNA segment 
coding for a foreign RNA, protein, polypeptide or portion thereof, 
possibly including a translational start codon, but lacking at least one 
other functional element of a TxCS that regulates initiation or 
termination of transcription and inititation of translation, commonly 
referred to as the promoter region and transcript terminator. As used 
herein, the term foreign structural gene does not include pRi T.sub.L -DNA 
structural genes unless the structural gene and pRi T.sub.L -DNA 
transcription controlling sequences combined with the structural gene are 
derived from different pRi T.sub.L -DNA genes; i.e. unless the structural 
gene and either a pRi promoter or a pRi polyadenylation site combined with 
the structural gene are heterologous. (Note that such foreign functional 
elements may be present after combination of the foreign structural gene 
with a pRi T.sub.L -DNA TxCS, though, in embodiments of the present 
invention, such elements may not be functional in plant cells). A foreign 
structural gene may encode a protein not normally found in the plant cell 
in which the gene is introduced. Additionally, the term refers to copies 
of a structural gene naturally found within the cell but artificially 
introduced. A foreign structural gene may be derived in whole or in part 
from sources including but not limited to eukaryotic DNA, prokaryotic DNA, 
episomal DNA, plasmid DNA, plastid DNA, genomic DNA, cDNA, viral DNA, 
viral cDNA, or chemically synthesized DNA. It is further contemplated that 
a foreign structural gene may contain one or more modifications in either 
the coding segments or untranslated regions which could affect the 
biological activity or chemical structure of the expression product, the 
rate of expression or the manner of expression control. Such modifications 
include, but are not limited to, mutations, insertions, deletions, and 
substitutions of one or more nucleotides, and "silent" modifications that 
do not alter the chemical structure of the expression product but which 
affect intercellular localization, transport, excretion or stability of 
the expression product. The structural gene may constitute an 
uninterrupted coding sequence or it may include one or more introns, 
bounded by the appropriate plant functional splice junctions, which may be 
obtained from synthetic or a naturally occurring source. The structural 
gene may be a composite of segments derived from a plurality of sources, 
naturally occurring or synthetic, coding for a composite protein, the 
composite protein being foreign to the cell into which the gene is 
introduced and expressed or being derived in part from a foreign protein. 
The foreign structural gene may be a fusion protein,.: and in particular, 
may be fused to all or part of a structural gene derived from the same ORF 
as was the TxCS. 
Plant tissue 
Includes differentiated and undifferentiated tissues of plants including, 
but not limited to roots, shoots, pollen, seeds, tumor tissue, such as 
crown galls, and various forms of aggregations of plant cells in culture, 
such as embryos and calluses. The plant tissue may be in planta or in 
organ, tissue, or cell culture. 
Plant cell 
As used herein includes plant cells in planta and plant cells and 
protoplasts in culture. 
Production of a genetically modified plant, plant seed, plant tissue, or 
plant cell expressing a foreign structual gene under control of a pRi 
T-DNA TxCS, and especially a pRi T.sub.L -DNA-derived TxCS, combines the 
specific teachings of the present disclosure with a variety of techniques 
and expedients known in the art. In most instances, alternative expedients 
exist for each stage of the overall process. The choice of expedients 
depends on variables such as the choice of the basic vector system for the 
introduction and stable maintenance of the pRi T.sub.L -DNA 
TxCS/structural gene combination, the plant species to be modified and the 
desired regeneration strategy, and the particular foreign structural gene 
to be used, all of which present alternative process steps which those of 
ordinary skill are able to select and use to achieve a desired result. For 
instance, although the starting point for obtaining pRi T.sub.L -DNA TxCSs 
is exemplified in the present application by pRi T.sub.L -DNA isolated 
from pRiA4 and pRiHFI, DNA sequences of other homologous agropine-type Ri 
Ti plasmids might be substituted as long as appropriate modifications are 
made to the TxCS isolation and manipulation procedures. Additionally, 
T-DNA genes from other types of pRi T.sub.L -DNA homologous to the 
agropine-type pRi T.sub.L -DNA genes having TxCSs disclosed herein may be 
substituted, again with appropriate modifications of procedural details. 
Homologous genes may be identified by those of ordinary skill in the art 
by the ability of their nucleic acids to cross-hybridize under conditions 
of stringency appropriate to detect 70% homology; such conditions are well 
understood in the art. It will be understood that there may be minor 
sequence variations within gene sequences utilized or disclosed in the 
present application. These variations may be determined by standard 
techniques to enable those of ordinary skill in the art to manipulate and 
bring into utility the T-DNA promoters and transcript terminators of such 
homologous genes. (Homologs of foreign structural genes may be identified, 
isolated, sequenced, and manipulated as is in a similar manner as homologs 
of the pRi genes of the present invention.) As novel means are developed 
for the stable insertion of foreign genes in plant cells, those of 
ordinary skill in the art will be able to select among those alternate 
process steps to achieve a desired result. The fundamental aspects of the 
invention are the nature and structure of pRi T-DNA genes and their use as 
a means for expression of a foreign structural gene in a plant genome. The 
remaining steps of the preferred embodiment for obtaining a genetically 
modified plant include inserting the pRi T.sub.L -DNA TxCS/structural gene 
combination into T-DNA, transferring the modified T-DNA to a plant cell 
wherein the modified T-DNA becomes stably integrated as part of the plant 
cell genome, techniques for in vitro culture and eventual regeneration 
into whole plants, which may include steps for selecting and detecting 
transformed plant cells and steps of transferring the introduced gene from 
the originally transformed strain into commercially acceptable cultivars. 
An advantage, which will be readily understood by those skilled in the art, 
of use of transcription controlling sequences disclosed herein for 
controlling structural gene expression over previously published T-DNA 
TxCSs is that transcription of many pRi T-DNA ORFs is phenotypically and 
developmentally regulated (see Example 1.9). pTi T-DNA genes are not known 
to be so regulated. Transcripts of ORFs 8, 11, 13, and 15 (Sequence ID 
Nos. 8, 11, 13 and 15, respectively) are more prevalent in roots than 
leaves, with the case of ORF 15 (SEQ ID NO. 15) being particularly 
striking, while ORF 12 (SEQ ID NO. 12) expression is specific to leaves 
and to a particular phenotype (T', see Example 1.9). Therefore, choice of 
a particular pRi T.sub.L -DNA TxCS allows modulation of expression of a 
structural gene with which the TxCS is combined. For example, should one 
want expression of a structural gene to be much higher in roots than 
leaves; ORF15 (SEQ ID NO. 15) provides the TxCS of choice. 
A principal feature of the present invention in its preferred embodiment is 
the construction of T-DNA having an inserted foreign structural gene under 
control of a pRi T.sub.L -DNA TxCS, i.e., between a promoter and a 
polyadenylation site, as these terms have been defined, supra, at least 
one of which is derived from pRi T.sub.L -DNA. The structural gene must be 
inserted in correct position and orientation with respect to the desired 
pRi T.sub.L -DNA promoter. Position has two aspects. The first relates to 
which side of the promoter the structural gene is inserted. It is known 
that the majority of promoters control initiation of transcription and 
translation in one direction only along the DNA. The region of DNA lying 
under promoter control is said to lie "downstream" or alternatively 
"behind" or "3' to" the promoter. Therefore, to be controlled by the 
promoter, the correct position of foreign structural gene insertion must 
by "downstream" from the promoter. The second aspect of position refers to 
the distance, in base pairs, between known functional elements of the 
promoter, for example the transcription initiation site, and the 
translational start site of the structural gene. Substantial variation 
appears to exist with regard to this distance, from promoter to promoter. 
Therefore, the structural requirements in this regard are best described 
in functional terms. As a first approximation, reasonable operability can 
be obtained when the distance between the promoter and the inserted 
foreign structural gene is similar to the distance between the promoter 
and the T-DNA gene it normally controls. Orientation refers to the 
directionality of the structural gene. That portion of a structural gene 
which ultimately codes for the amino terminus of the foreign protein is 
termed the 5'-end of the structural gene, while that end which codes for 
amino acids near the carboxyl end of the protein is termed the 3'-end of 
the structural gene. Correct orientation of the foreign structural gene is 
with the 5'-end thereof proximal to the promoter. An additional 
requirement in the case of constructions leading to fusion protein 
expression is that the insertion of the foreign structural gene into the 
pRi T.sub.L -DNA promoter-donated structural gene sequence must be such 
that the coding sequences of the two genes are in the same reading frame 
phase, a structural requirement which is well understood in the art. An 
exception to this requirement exists in the case where an intron separates 
coding sequences derived from a foreign structural gene from the coding 
sequences of the pRi T.sub.L -DNA structural gene. In that case, both 
structural genes must be provided with compatible splice sites, and the 
intron splice sites must be so positioned that the correct reading frame 
for the pRi T.sub.L -DNA promoter-donated structural gene and the foreign 
structural gene are restored in phase after the intron is removed by 
post-transcriptional processing. Differences in rates of expression or 
developmental control may be observed when a given foreign structural gene 
is inserted under control of different pRi T.sub.L -DNA TxCSs. Rates of 
expression may also be greatly influenced by the details of the resultant 
mRNA's secondary structure, especially stem-loop structures. Stability, 
ability to be excreted, intercellular localization, intracellular 
localization, solubility, target specificity, and other functional 
properties of the expressed protein itself may be observed in the case of 
fusion proteins depending upon the insertion site, the length and 
properties of the segment of pRi T.sub.L -DNA protein included within the 
fusion protein and mutual interactions between the components of the 
fusion protein that effect folded configuration thereof, all of which 
present numerous opportunities to manipulate and control the functional 
properties of the foreign protein product, depending upon the desired 
physiological properties within the plant cell, plant tissue, and whole 
plant. Similarly to the promoter, the polyadenylation site must be located 
in correct position and orientation relative to the 3'-end of the coding 
sequence. Fusion proteins are also possible between the 3'-end of the 
foreign structural gene protein and a polypeptide encoded by the DNA which 
serves as a source of the polyadenylation site. 
A TxCS is comprised by two major functionalities: a promoter, which is 
absolutely necessary for gene expression, and a transcript terminator, 
being in the preferred embodiment a polyadenylation site, positioned 
respectively 5' and 3' to the structural gene. Although as exemplified 
herein these two portions of the TxCS are obtained from the same gone, 
this is not a requirement of the present invention. These 5' and 3' 
sequences may be obtained from diverse pRi T-DNA genes, especially pRi 
T.sub.L -DNA genes, or one of these sequences may even be obtained from a 
non-pRi T-DNA gene. For instance, a promoter may be taken from a pRi 
T.sub.L -DNA gone while the polyadenylation site may come from a plant 
gene. 
In the Examples, a foreign structural gene is nested within a pRi T.sub.L 
-DNA TxCS, suturing the structural gene into the TxCS at NdeI sites and 
placing the entire TxCS/structural gone combination between a pair of 
BamHI sites. As will be apparent to those of ordinary skill in the art, 
the TxCS/gene combination may be placed between any restriction sites 
convenient for removing the combination from the plasmid it is carried on 
and convenient for insertion into the plant transformation or shuttle 
vector of choice. Alternatives to the use of paired NdeI sites (5' . . . 
CATATG . . . 3') at the ATG translational start include, but are not 
limited to, use of ClaI (5' . . . (not G)ATCGAT(G) . . . 3') or NcoI (5' . 
. . CCATGG . . . 3') sites. As will be understood by persons skilled in 
the art, other sites may be used for the promoter/structural gene suture 
as long as the sequence at the junction remains compatible with 
translational and transcriptional functions. An alternative to the suture 
of the promoter to the foreign structural gene at the ATG translational 
start is suturing at the transcriptional start or cap site. An advantage, 
especially for eukaryotic structural genes, of the use of this location is 
the secondary (stem-loop) structure of the foreign structural gene mRNA 
will not be disrupted thereby leading to an mRNA having translational 
activity more nearly resembling the activity observed in the organism 
which was the source of the gone. The restriction sites at the 5'- and 
3'-ends of the structural gone need not be compatible. Use of cut sites 
cut by two different restriction enzymes at the two TxCS/structural gene 
junctions will automatically correctly orient the structural gene when it 
is inserted between the TxCS elements, though use of an extra restriction 
enzyme may necessitate removal of an additional set of inconvenient 
restriction sites within the TxCS and the structural gene. The use of a 
single restriction enzyme to link both a promoter and a polyadenylation 
site to a particular structural gene is not required. Convenient sites 
within the pRi T.sub.L -DNA structural gene and 3' to the translational 
stop of the foreign structural gene may be used. When these sites have 
incompatible ends, they may be converted to blunt-ends by methods well 
known in the art and blunt-end ligated together. 
Location of the TxCS/foreign structural gene combination insertion site 
within T-DNA or a T-DNA-derived vector is not critical as long as the 
transfer function of the T-DNA borders and any other necessary vector 
elements (e.g. a selectable or screenable marker) are not disrupted. The 
T-DNA into which the TxCS/structural gene combination is inserted may be 
obtained from any of the TIP plasmids, including both Ti and Ri plasmids. 
The TxCS/structural gene combination is inserted by standard techniques 
well known to those skilled in the art. The orientation of the inserted 
plant gene, with respect to the direction of transcription and translation 
of endogenous T-DNA or vector genes is not critical, either of the two 
possible orientations is functional. Differences in rates of expression 
might be observed when a given gene is inserted at different locations 
within T-DNA. 
A convenient means for inserting a TxCS/foreign structural gene combination 
into T-DNA involves the use of a shuttle vector, as described in the 
Background. An Agrobacterium strain transformed by a shuttle vector is 
preferably grown under conditions which permit selection of a 
double-homologous recombination event which results in replacement of a 
preexisting segment of a Ti or Ri plasmid with a segment of T-DNA of the 
shuttle vector.. However, it should be noted that the present invention is 
not limited to the introduction of the TxCS/structural gene combination 
into T-DNA by a double homologous recombination mechanism; a homologous 
recombination event with a shuttle vector (perhaps have only a single 
continuous region of homology with the T-DNA) at a single site will also 
prove an effective means for inserting that combination into T-DNA as will 
insertion of a combination-carrying bacterial transposon. 
An alternative to the shuttle vector strategy involves the use of plasmids 
comprising T-DNA or modified T-DNA, into which an TxCS/foreign structural 
gene is inserted, said plasmids lacking vir genes and being capable of 
independent replication in an Agrobacterium strain. As reviewed in the 
Background, the T-DNA of such plasmids can be transferred from an 
Agrobacterium strain (e.g. A. rhizogenes, A. tumefaciens, or derivatives 
thereof) to a plant cell provided the Agrobacterium strain contains 
certain trans-acting vir genes whose function is to promote the transfer 
of T-DNA to a plant cell. Plasmids that contain T-DNA and are able to 
replicate independently in an Agrobacterium strain are herein termed 
"sub-TIP" plasmids. A spectrum of variations is possible in which the 
sub-TIP plasmids, which may be derived from Ri or Ti plasmids, differ in 
the amount of T-DNA contained. A "mini-TiP" plasmid retains all of the 
T-DNA from a TIP. "Micro-TIP" plasmids are deleted for all T-DNA but that 
surrounding the T-DNA borders, the remaining portions being the minimum 
necessary for the sub-TIP plasmid to be transferrable and integratable in 
the host cell. Sub-TIP plasmids are advantageous in that they are 
relatively small and relatively easy to manipulate directly, eliminating 
the need to transfer the gene to T-DNA from a shuttle vector by homologous 
recombination. After the desired structural gene has been inserted, they 
can easily be introduced directly into a Agrobacterium cell containing the 
trans-acting genes that promote T-DNA transfer. Introduction into an 
Agrobacterium strain is conveniently accomplished either by transformation 
of the Agrobacterium strain or by conjugal transfer from a donor bacterial 
cell, the techniques for which are well known to those of ordinary skill. 
pRi T-DNA TxCS/structural gene combinations may be combined with 
pTi-derived Ti plasmids or sub-TIP vectors. 
Modified T-DNA carrying a pRi T.sub.L -DNA TxCS/structural gene combination 
can be transferred to plant cells by any technique known in the art (see 
Background). The resultant transformed cells must be selected or screened 
to distinguish them from untransformed cells. Selection is most readily 
accomplished by providing a selectable marker known to the art 
incorporated into the T-DNA in addition to the TxCS/foreign structural 
gene combination. Indeed, a pRi T.sub.L -DNA TxCS can be a component of 
such a marker. In addition, the T-DNA provides endogenous markers such as 
the gene or genes controlling hormone-independent growth of Ti-induced 
tumors in culture, the gene or genes controlling abnormal morphology of 
Ri-induced tumor roots, and genes that control resistance to toxic 
compounds such as amino acid analogs, such resistance being provided by an 
opine synthase (e.g. ocs). Screening methods well known to those skilled 
in the art include assays for opine production, specific hybridization to 
characteristic RNA or T-DNA sequences, or immunological assays. 
Additionally the phenotype of expressed foreign gene can be used to 
identify transformed plant tissue (e.g. insecticidal properties of the 
crystal protein). 
Although the preferred embodiment of this invention uses a T-DNA-based 
Agrobacterium-mediated system for incorporation of the TxCS/foreign 
structural gene combination into the genome of the plant which is to be 
transformed, other means for transferring and incorporating the gene are 
also included within the scope of this invention. Other means for the 
stable incorporation of the combination into a plant genome additionally 
include, but are not limited to, use of vectors based upon viral genomes 
(e.g. see N. Brisson et al. (1984) Nature 310:511-514), minichromosomes, 
transposons, and homologous or nonhomologous recombination into plant 
chromosomes. Alternate forms of delivery of these vectors into a plant 
cell additionally include, but are not limited to, direct uptake of 
nucleic acid (e.g. see J. Paszkowski et al. (1984) EMBO J. 3:2717-2722), 
fusion with vector-containing liposomes or bacterial spheroplasts, 
microinjection, and encapsidation in viral coat protein followed by an 
infection-like process. After introduction into a plant cell of a pRi 
T.sub.L -DNA TxCS/structural gene combination, the combination will be 
contained by a plant cell. Furthermore, the combination will be flanked by 
plant DNA, unless utilizing a nonintegrating vector, e.g. a virus or 
minichromosome. 
Regeneration of transformed cells and tissues is accomplished by resort to 
known techniques. An object of the regeneration step is to obtain a whole 
plant that grows and reproduces normally but which retains integrated 
T-DNA. The techniques of regeneration vary somewhat according to 
principles known in the art, depending upon the origin of the T-DNA, the 
nature of any modifications thereto and the species of the transformed 
plant. In many plant species, cells transformed by pRi-type T-DNA are 
readily regenerated, using techniques well known to those of ordinary 
skill, without undue experimentation. Plant cells transformed by pTi-type 
T-DNA can be regenerated, in some instances, by the proper manipulation of 
hormone levels in culture. Preferably, however, the Ti-transformed tissue 
is most easily regenerated if the T-DNA has been mutated in one or both of 
the tmr and tms genes. It is important to note that if the mutations in 
tmr and tms are introduced into. T-DNA by double homologous recombination 
with a shuttle vector, the incorporation of the mutation must be selected 
in a different manner than the incorporation of the TxCS/structural gene 
combination; e.g. one might select for tmr and tms inactivation by 
chloramphenicol resistance while one might select for TxCS/foreign gene 
integration by kanamycin resistance. The inactivation of the tms and tmr 
loci may be accomplished by an insertion, deletion, or substitution of one 
or more nucleotides within the coding regions or promoters of these genes, 
the mutation being designed to inactivate the promoter or disrupt the 
structure of the encoded proteins (e.g. the T-DNA of NRRL B-15821, or the 
pTi of A3004, L. W. Ream et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 
80:1660-1664). Resultant transformed cells are able to regenerate plants 
which carry integrated T-DNA and express T-DNA genes, such as an opine 
synthase, and also express an inserted pRi T.sub.L -DNA TxCS/structural 
gene combination. These serve as parental plant material for normal 
progeny plants carrying and expressing the pRi T.sub.L -DNA 
TxCS/heterologous foreign structural gene combination, and for seeds 
containing the combination, in the preferred embodiments the combination 
being integrated into a plant chromosome and flanked by plant DNA. 
The genotype of the plant tissue transformed is often chosen for the ease 
with which its cells can be grown and regenerated in in vitro culture and 
for susceptibility to the selective agent to be used. Should a cultivar of 
agronomic interest be unsuitable for these manipulations, a more amenable 
variety is first transformed. After regeneration, the newly introduced 
TxCS/foreign structural gene combination is readily transferred to the 
desired agronomic cultivar by techniques well known to those skilled in 
the arts of plant breeding and plant genetics. Sexual crosses of 
transformed plants with the agronomic cultivars yielded initial hybrid. 
These hybrids can then be back-crossed with plants of the desired genetic 
background. Progeny are continuously screened and selected for the 
continued presence of integrated T-DNA or for the new phenotype resulting 
from expression of the inserted foreign gene. In this manner, after a 
number of rounds of back-crossing and selection, plants can be produced 
having a genotype essentially identical to the agronomically desired 
parents with the addition of a inserted pRi T-DNA promoter/foreign 
structural gene combination or of a foreign structural 
gene/polyadenylation site combination. 
EXAMPLES 
The following Examples are presented for the purpose of illustrating 
specific embodiments within the scope of the present invention without 
limiting the scope; the scope being defined by the Claims. Numerous 
variations will be readily apparent to those of ordinary skill in the art. 
These Examples utilize many techniques well known and accessible to those 
skilled in the arts of molecular biology and manipulation of TIPs and 
Agrobacterium; such methods are fully described in one or more of the 
cited references if not described in detail herein. Enzymes are obtained 
from commercial sources and are used according to the vendor's 
recommendations or other variations known to the art. Reagents, buffers 
and culture conditions are also known to those in the art. Reference works 
containing such standard techniques include the following: R. Wu, ed.. (! 
979) Herb. Enzymol. 68, R. Wu et al., eds. (1983) Meth. Enzymol. 100 and 
101, L. Grossman and K. Moldave, eds. (1980) Meth. Enzymol. 65, J. H. 
Miller (1972) Experiments in Molecular Genetics, R. Davis et al. (1980) 
Advanced Bacterial Genetics, R. F. Schleif and P. C. Wensink (1982) 
Practical Methods in Molecular Biology, and T. Maniatis et al. (1982) 
Molecular Cloning. Additionally, R. F. Lathe et al. (1983) Genet. Engin. 
4:1-56, make useful comments on DNA manipulations. 
Textual use of the name of a restriction endonuclease in isolation, e.g. 
"BclI" refers to use of that enzyme in an enzymatic digestion, except in a 
diagram where it can refer to the site of a sequence susceptible to action 
of that enzyme, e.g. a restriction site. In the text, restriction sites 
are indicated by the additional use of the word "site", e.g. "BclI site". 
The additional use of the word "fragment", e.g. "BclI fragment", indicates 
a linear double-stranded DNA molecule having ends generated by action of 
the named enzyme (e.g. a restriction fragment). A phrase such as 
"BclI/SmaI fragment" indicates that the restriction fragment was generated 
by the action of two different enzymes, here BclI and SmaI, the two ends 
resulting from the action of different enzymes. Note that the ends will 
have the characteristics of being "sticky" (i.e. having a single-stranded 
protrusion capable of base-pairing with a complementary single-stranded 
oligonucleotide) or "blunt" and that the sequence of a sticky-end will be 
determined by the specificity of the enzyme which produces it. 
In the Examples and Tables, the underlining of a particular nucleotide in a 
primer or other sequence indicates the nucleotide which differs from the 
naturally found sequence, being an insertion or substitution of one or 
more nucleotides. The use of lower case for two adjacent nucleotides 
brackets one or more nucleotides that have been deleted from the native 
sequence. Unless otherwise noted, all oligonucleotide primers are 
phosphorylated at their 5'-ends are represented 5'-to-3', and are 
synthesized and used as referenced in Example 5. 
Plasmids are usually prefaced with a "p", e.g., pRiA4 or p8.8, and strain 
parenthetically indicate a plasmid harbored within, e.g., A. rhizogenes 
(pRiA4) or E. coli HB101 (p8.8). Self-replicating DNA molecules derived 
from the bacteriophage M13 are prefaced by an "m", e.g. mWB2341, and may 
be in either single-stranded or double-strand form. A. tumefaciens 
(pTi15955) is on deposit in ATCC 15955, E. coli C600 (pRK-203-Kan-103-Lec) 
as NRRL B-15821, E. coli HB101 (pLJ40) as NRRL B-15957, and E. coli HB101 
(EcoRI e36) as NRRL B-15958 (as deposited EcoRI e36 was designated EcoRI 
3a); other deposited strains are listed in column 3 of Table 7. 
The DNA constructions described in these Examples have been designed to 
enable any one of the eukaryotic TxCSs of pRi T.sub.L -DNA to be combined 
with any of four foreign structural genes, Towards that end, the 
structural genes, the TxCSs, and the TxCS/structural gene combinations 
have been placed on DNA "cassettes", having the properties that, after 
initial modifications have been made, any structural gene may be readily 
inserted into any TxCS without further modification, and any 
TxCS/structural gene combination may be isolated by a simple procedure 
applicable to all such combinations. All combinations are thereby 
equivalent when being inserted into the plant transformation vector of 
choice. The initial modifications of the TxCSs are all analogous to each 
other and the initial modifications of the structural genes are also all 
analogous to each other. These Examples often involve the use of a common 
strategy for multiple constructions that differ only in items such as 
choice of restriction enzymes, DNA fragment size, ORFs encoded, plasmids 
generated or used as starting material, specific numbers and sequences of 
oligonucleotides used for mutagenesis, sources of plasmids, and enzyme 
reactions utilized. For the sake of brevity, the DNA manipulations and 
constructions are generally described once, the differing items being 
detailed by reference to a particular column in a particular Table, a 
particular series of manipulations used in a particular construction 
occupying horizontal lines within that Table. One combination, the ORF 11 
(SEQ ID NO. 11) TxCS with the crystal protein structural gene, is also 
detailed in the text. 
The following is an outline, diagrammed schematically in FIG. 3, of a 
preferred strategy used to make the exemplified DNA constructions detailed 
in Examples 3 through 6. Endogenous NdeI sites are removed from the 
M13-based vector mWB2341, resulting in a vector designated mWB2341(Nde) 
(Example 3.1). Large fragments of T-DNA are introduced into mWB2341(Nde) 
in a manner that also eliminates the vector's BamMI Site (Example 3.2). 
Endogenous T-DNA NdeI and BamHI sites are then removed (Example 3.3) and 
novel sites are introduced. NdeI sites are introduced at and near the 
translational start and stop sites, respectively, so that a foreign 
structural gene on a NdeI fragment may replace the endogenous ORF 
structural gene. BamHI sites are introduced approximately 0.3 kbp 5'0 to 
and 3' from the transcriptional start and stop signals, respectively, so 
that the TxCS/structural gene combination eventually constructed may be 
removed on a BamHI fragment (Example 3.4). The structural genes, which 
fortuitously have no internal NdeI or BamHI sites, are introduced into 
mWB2341 (Nde) (Example 4.1 ) and NdeI sites are introduced at and after 
the translational start and stop sites (Examples 4.2 and 4.3). The 
structural genes are removed from their vectors on "DNA cassettes" by 
digestion with NdeI and are inserted into any desired TxCS which has had 
its endogenous structural gene removed by NdeI digestion (Example 6.1). 
The TxCS/foreign structural gene combinations are then removed from their 
vector by digestion with BamHI and inserted into the plant transformation 
vectors of choice (Example 6.2). It is recognized that construction 
strategies utilizing fortuitously located restriction sites might be 
designed by persons of ordinary skill which might be simpler for some 
particular TxCS/structural gene combination than the generalized DNA 
cassette strategy utilized herein; however, DNA cassettes are a better 
approach when one is trying to achieve flexibility in the choice and 
matching of many diverse TxCSs and structural genes. 
Example 1 
This Example provides disclosure, analysis, and discussion of the pRi 
T.sub.L -DNA sequencing results. 
1.1 Summary of Results 
pRi T.sub.L -DNA was sequenced (SEQ ID NO. 19) and eighteen open reading 
frames (ORFs) (SEQ ID NOS: 1-18), two of which (7 and 18) (SEQ ID NOS: 7 
and 18, respectively) were clearly prokaryotic in nature, were found. 
Eleven ORFs had canonical eukaryotic promoter and polyadenylation elements 
(ORFs 1, 2, 3, 6, 8, 11, 12, 13, 14, 15 and 16) (Sequence ID Nos. 1, 2, 3, 
6, 8, 11, 12, 13, 14, 15 and 16, respectively). These ORFs were 
distributed within an about 19.4 kilobase pair (kbp) segment of pRi 
T.sub.L -DNA (SEQ ID NO. 19) integrated into the genome of C. arvensis 
clone 7. DNA encoding ORFs 8, 11, 12, 13, and 15 (Sequence ID Nos. 8, 11, 
12, 13 and 15, respectively) was observed to be transcribed in tobacco. 
1.2 Sequence of pRi T.sub.L -DNA 
A physical map of the pRi T.sub.L -DNA region is shown in FIG. 1 along with 
pRi subclones and the nucleotide sequencing strategy used. Nine-tenths of 
the sequence obtained was determined from both DNA strands, the remaining 
tenth being sequenced more than once from the same DNA strand. A 
nucleotide sequence of 21,126 base pairs (bp) was obtained, which included 
a 19.4 kbp pRi T.sub.L -DNA region identified in the genome of C. arvensis 
clone 7, and is presented in FIG. 2, 5'-to-3' corresponding to 
left-to-right as mapped in FIG. 1. DNA was sequenced from the 5'-end of 
BamHI fragment 32 to about 2216 bp into EcoRI fragment 3b (3'-end) (see 
FIG. 1). The cleavage sites for over seventy restriction enzymes were 
determined; cleavage positions for enzymes with less than nineteen sites 
are listed in Table 1. 
1.3 T.sub.L -DNA Border Repeats 
Genomic hybridization and DNA sequence analyses of the T.sub.L -DNA region 
integrated into the genome of C. arvensis clone 7 showed the exact 
location of a left plant/T-DNA junction and an approximate position for a 
right pRi T.sub.L -DNA/plant junction (F. Leach (1983) Ph.D. Thesis, 
Universite de Paris-Sud, Centre d'Orsay, France). The left plant DNA/T-NA 
junction was between position 570 and 571, as defined in FIG. 2. The left 
25 bp T-DNA border repeat sequence was located between positions 520 and 
544. The right boundary of T.sub.L -DNA of RiA4-transformed C. arvensis 
could vary over a 8 kbp region. The complete 21,126 bp of pRi T.sub.L -DNA 
region was scanned for the presence of a 25 bp consensus sequence derived 
by comparison with published sequences, 
##STR1## 
Twenty-seven nucleotide sequences matching this consensus at 15 or more 
bases were identified. Included among these sequences were the 25 bp 
nucleotide sequences starting (5') at positions 520 (matching at 23 of 25 
bases) and 19,966 (17 of 25) (see FIG. 2). These two positions were near 
the T-DNA/plant junctions of a transformed Nicotiana glauca tissue (F. F. 
White et al. (1983) Nature 301:348-350) and C. arvensis clone 7, as 
determined by comparison of genomic restriction maps of transformed plant 
DNA and pRiA4 DNA. Other matches were found at positions 154, 576, 725, 
3244, 6316, 6365, 7209, 7379, 8697, 10339, 10436, 11079, 11232, 12313, 
13832, 14235, 14510, 15145, 16285, 17071, 17483, 18121, 18273, 18368, and 
18797. The eleven previously published 25 bp border repeat sequences were 
as little as 64% homologous to each other, thus indicating that many of 
these pRi border sequences could be functional. Genomic hybridization 
analysis of the pRi T.sub.L -DNA region in tobacco (D. Tepfer (1984) Cell 
37:959-967) showed a much smaller T.sub.L -DNA with the left junction 
probably involving a border sequence at either position 6316 or 6365. 
1.4 Identification of Open Reading Frames 
Analysis of the nucleotide sequence presented in FIG. 2 revealed the 
presence of sixteen ORFs starting with an ATG initiation codon and 
extending over 300 nucleotides. The locations, sizes, and molecular 
weights of the putative translational polypeptides of these ORFs are 
listed in Table 2. Two additional ORFs (9 and 10) (Sequence ID Nos. 9 and 
10, respectively) were shorter than 300 nucleotides but were included in 
Table 2 because they satisfied other criteria (see below). The size of the 
ORFs ranged from 255 nucleotides (ORF 9) (SEQ ID NO. 9) up to 2280 
nucleotides (ORF 8) (SEQ ID NO. 8), encoding polypeptides ranging in size 
from 9600 to 85,000 daltons, respectively. However, the actual size of an 
RNA transcript encoding an ORF could be considerably larger than that 
listed in Table 2 because 5' and 3' noncoding regions and 3'-polyadenylic 
acid tails were not included. 
Though to date no introns have been found in any of the fourteen sequenced 
pTi T-DNA genes, (R. F. Barker et el. (1983) Plant Mol. Biol. 2:335-350), 
J. Gielen et el. (1984) EMBO J. 3:835-846), introns are present in some 
plant nuclear genes; pRi T.sub.L -DNA genes could have introns. Transcript 
mapping (Example 1.9) did not generally indicate spliced mRNA. However, 
analysis of mRNA encoded between positions 6500 and 9000 detected two 
transcripts, a 2300 base transcript as predicted for ORF 8 and an 
unpredicted 650 base transcript. The nucleotide sequence of the only other 
ORF in this region, ORF 9 (SEQ ID NO. 9) , suggested a transcript of about 
450 bases, about half the size as found. The coding region of ORF 8 (SEQ 
ID NO. 8) was scanned for sequences which matched consensus donor 
##STR2## 
the "*" indicating the splice site) and acceptor 
##STR3## 
intron splice sequences and conformed to the G-T/A-G rule (R. Breathnach 
et al. (1978) Proc. Natl. Aced. Sci. USA 75:4853-4857) and a plant 
consensus sequence (J. L. Slightore et al. (1983) Proc. Natl. Aced. Sci. 
USA 80:1897-1901). Splicing between an acceptor at position 8943 and a 
donor at positions 7283, 7327, 7374, 7701, or 7894 would result in a 
second transcript having a translation initiation codon-polyadenylation 
site distance of 724, 758,943, 1270, or 1325 bp, respectively, which is in 
the size range observed. Proper processing of an intron-containing genes 
in T. DNA has been observed (e.g. N. Murai et. al. (1983) science 
222:476-482). 
No homology greater than random was found to exist in coding or noncoding 
sequences between pRi T.sub.L -DNA and octopine pTi T-DNA (Barker et al., 
supra), consistent with the lack of cross-hybridization between pRi 
T.sub.L -DNA and octopine pTi T-DNA observed by G. A. Huffman et el. 
(1984) J. Bacteriol. 157:269-276, and L. Jouanin (1984) Plasmid 12:91-102 
1.5 Translational Initiation Codons 
Eukaryotic translation is preferentially initiated at the first AUG of an 
mRNA; and A or G at position -3 and G at position +4 may facilitate 
recognition of functional AUG codons. This 
##STR4## 
concensus is referred to as the ribosome binding site (M. Kozak (1981) 
Nucl. Acids Res. 9:5233-5252; M. Kozak (1983) Cell 34:971-978). The number 
of amino acids and calculated molecular weights for the putative pRi 
T.sub.L -DNA protein products (Table 3) were derived by assigning the 
first in-phase AUG codon as the initiator codon. The art has not ruled out 
use of secondary AUG codons as translation initiation codons (M. Kozak 
(1983) Microbiol. Rev. 47:1-45). 
Initiator codon DNA sequences are listed in Table 2 below the consensus 
eukaryotic ribosome binding site. Eight of the eighteen ORFs had first AUG 
codons which conform with this consensus sequence (ORFs 1, 7, 8, 10, 11, 
12, 14, and 18) (Sequence ID Nos. 1, 7, 8, 10, 11, 12, 14, and 18, 
respectively). Of the ten remaining ORFs, four had downstream, in-phase 
AUG codons which conformed with the consensus sequence: ORF 2(SEQ ID NO. 
2), 287 bp downstream; ORF 3 (SEQ ID NO. 3), 160 bp; ORF 6 (SEQ ID NO. 6), 
344 bp; ORF 13 (SEQ ID NO. 13), 203 bp; and ORF 17 (SEQ ID NO. 17), 105 bp 
(see FIG. 2). The remaining six ORFs (2, 4, 5, 9, 15, and 16) (Sequence ID 
No. 2, 4, 5, 9, 15 and 16, respectively) did not have any AUG codons which 
conform to the consensus sequence followed by 300 bp in-phase ORFs. The 
presence of of consensus ribosome binding AUG codon is not necessary for 
translation initiation of T-DNA mRNAs; four abundantly transcribed 
octopine pTi T.sub.L -DNA genes are initiated at AUG codons which do not 
conform to the consensus sequences. 
Several pTi T-DNA ORFs are actively transcribed in E. coli minicells (G. 
Schr/e,uml/o/ der et al. (1983) EMBO J. 2:403-409). Translational 
initiation in E. coli and most prokaryotes generally start at an AUG codon 
that is proceeded by a G-rich ribosome binding site (J. Shine and L. 
Dalgarno (1974) Proc. Natl. Acad. Sci. USA 71:1342-1346). Sequences which 
may function as prokaryotic ribosome binding sites were observed ahead of 
the pRi T.sub.L -DNA ORF 4, 5, 7, 9, and 18 (Sequence ID Nos. 4, 5, 7, 9, 
and 18, respectively) initiation codons. 
1.6 Codon Usage 
Most pRi T.sub.L -DNA ORFs were observed to fit pTi T.sub.L -DNA codon 
preference patterns, thereby indicating that they are functional after 
integration into a plant genome, notable exceptions being ORFs 7 and 18 
(Sequence ID Nos. 7 and 18, respectively). 
1.7 Locations of Transcription Controlling Sequences 
Comparisons of nucleotide sequences from the 5'-flanking regions of many 
eukaryotic genes have revealed consensus locations and sequences of 
several DNA elements which may be important in regulating RNA polymerase 
II-mediated transcript:ion (S. L. McKnight and R. Kingsbury (1982) Science 
217:316-324). These characteristic eukaryotic promoter elements are the 
"TATA-element", located 25-30 bp upstream (5') from the start of 
transcription, and the "CCAAT-element", located 40-50 nucleotides upstream 
from the TATA-element (C, Benoist et al., (1980) Nucl. Acids. Res. 
8:127-142; A. Efstratiades et al. (1980) Cell 21:653-668). Similar 
promoter elements have been found in the 5'-flanking regions of many plant 
and pTi-T-DNA genes; pTi15955 T-DNA (Barker et al., supra) an dpTiAch5 
T.sub.L -DNA GieTen et al., supra) have sequences resembling these TATA 
and CCAAT promoter elements located in the 5'-flanking regions of eight 
T.sub.L -DNA and six T.sub.R -DNA ORFs (i.e. have "eukaryotic-looking" 
promoters). All eight eukaryotic-looking pTt. T.sub.L -DNA ORFs are 
transcribed and at least five of six eukaryotic-looking pTi TR-DNA ORFs 
are known to be transcribed, 
The presence of TATA and CCAAT promoter elements in 5'-flanking regions of 
pRi T.sub.L -DNA ORFs indicated that a particular ORF was part of a 
functional gone, Most pRi T.sub.L -DNA ORFs (16 of 18) were flanked by 
sequences (Table 3) that closely resembled these eukaryotic promoter 
elements. The amount of sequence identity between the promoter elements 
and the consensus sequences was very high; ORFs 2 and 12 (Sequence ID Nos. 
2 and 12), respectively) had promoter elements which matched the consensus 
sequences while the promoter elements from the other thirteen ORFs did not 
vary by more than three mismatches. These results were consistent with the 
degree of homology found for promoter elements from pTi T-DNA ORFs (Barker 
et al., supra; Gielen et al., supra), 
pRi T.sub.L -DNA open reading frames 1, 4, 8, 10, 13, 14, and 17 (Sequence 
ID Nos. 1, 4, 8, 10, 13, 14 and 17), respectively, were flanked by 
multiple promoter elements, ORFs 7 and I8 were not flanked by sequences 
resembling eukaryotic promoter elements and were not expected to be 
transcribed in plant tissues, ORFs 4, 5, 7, and 9 overlapped ORFs 5, 6, 
and 8 (Sequence ID Nos. 5, 6 and 8, respectively) on the opposite strand 
(FIG. 1, Table 2); the larger ORFs (5, 6, and 8) (Sequence ID Nos. 5, 6 
and 8, respectively) were more likely to be transcribed because DNA 
encoding overlapping, antiparallel ORFs in pTi T-DNA was found to be 
transcribed from either one strand or the other (Gielen et al., supra). 
Comparison of polyadenylation sites present in the 3'-noncoding regions of 
plant genes indicates a preference for the hexanucleotide, AATAAA (J. 
Messing et al. (1983) in Genetic Engineering of Plants, ed.: A. 
Hollaender, pp. 211-227), however, variations have been observed for plant 
genes, e.g. AATAAG and GATAAA. Many pTi T-DNA ORFs are also followed by 
AATAAA sequences. The remaining pTi T-DNA ORFs are followed by 
polyadenylation sites which vary only slightly, e.g. AATAAT, TATAAA, or 
AATGAA; AATAAT is known to function for the ocs gene (H. DeGreve et al. 
(1982) J. Mol. Appl. Genet. 499-511). 
Presumptive pRi T.sub.L -DNA polyadenylation sites and their locations are 
listed in Table 3. Ten ORFs (2, 4, 6, 8, 9, 11, 12, 13, 14, and 15) 
(sequence ID Nos. 2, 4, 6, 8, 9, 11, 12 13, 14 and 15, respectively) had 
the consensus hexanucleotide, AATAAA, near their 3'-ends, whereas only two 
(ORFs 7 and 18) (Sequence ID Nos. 7 and 18, respectively) did not contain 
any related sequence (Table 3, FIG. 2). The remaining ORFs (1, 3, 10, and 
16) (Sequence ID Nos. 1, 3, 10 and 16), respectively) had polyadenylation 
sites closely related to those described above. ORFs 8, 10, 12, 13, and 14 
(Sequence ID Nos. 8, 10, 12, 13 and 14, respectively) were followed by 
multiple polyadenylation signals. Multiple polyadenylation sites have also 
been observed in several pTi T-DNA genes (P. Dhaese et al. (1983) EMBO J. 
2:419-426; Gielen et al., supra). 
1.8 ORF Locations with Respect to Base Composition 
The G+C content of the large Agrobacterium plasmids is about (S. 
Sheikholeslam et al. (1979) Phytopathol. 69:54-58). In contrast, pRi 
T.sub.L -DNA had very A+T-rich regions flanking the eukaryotic ORFs while 
coding regions had G+C contents in the range of 50%. Plant genes can also 
have A+T-rich flanking sequences. 
1.9 Detection of Transcripts 
The T.sub.L -DNA-left junction with plant DNA found in an A. rhizogenes 
transformed tobacco tissue, clone 9, was between the position 6361 HindIII 
site and the position 7585 EcoRI site, while the right border was to the 
right of the position 19,918 KpnI site (see Example 1.3). Hybridization of 
nick-translated pRi T.sub.L -DNA probes to membrane filter-bound replicas 
of the gels ("Northern blots") clearly showed transcripts carrying ORFs 8 
and 13 (SEQ ID NOS. 8 and 13, respectively). An observed transcript of 
about 950 nucleotides which hybridized with pRi T.sub.L -DNA between EcoRI 
sites at positions 9077 and 13,445 was assigned to ORF 11 (SEQ ID NO. 11). 
An observed transcript of about 1400 nucleotides which hybridized with 
sequences spanning the position 17,059 EcoRI site was assigned to ORF 15 
(SEQ ID NO. 15). An observed transcript of about 800 nucleotides which 
hybridized with pRi T.sub.L -DNA between the positions 9077 and 13,445 
EcoRI sites was assigned to ORF 12 (SEQ ID NO. 12). 
The relative abundances of pRi T.sub.L -DNA transcripts in clone 9-derived 
plants were observed to be a function of organ (leaves vs. roots) and 
phenotype (T vs. T'; see Tepfer (1984) supra). With the exception of the 
transcript corresponding to ORF 12 (SEQ ID NO. 12), pRi T.sub.L -DNA 
transcripts were more prevalent in roots than in leaves, with a 
particularly striking case being the mRNA assigned to ORF 15. Expression 
of the transcript assigned to ORF 12 (SEQ ID NO. 12) was leaf-specific and 
was correlated with the T' phenotype. 
RNA from C. arvensis tissue transformed by pRi T.sub.L -DNA which included 
sequences encoding ORFs 1-6 (SEQ ID NO. 8-6, respectively) also hybridized 
with pRi T.sub.L -DNA. 
1.10 Conclusions 
The data discussed above (Examples 1.2, 1.4-1.8) indicated that of the ORFs 
flanked by eukalyotic transcription controlling sequences (ORFs 1, 2, 3, 
4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) (Sequence ID Nos. 1-6 and 
8-17), respectively) ORFs 1, 2, 3, 6, 8, 11, 12, 13, 14, 15, and 16 
(Sequence ID Nos. 1-3, 6, 8, 11-16, respectively) were most likely to be 
transcribed. In tobacco tissue transformed by DNA encoding ORFs 8-18 (SEQ 
ID NOS. 8-18, respectively), transcription of DNA region encoding ORFs 8, 
11, 12, 13, and 15 (Sequence ID Nos. 8, 11, 12, 13 and 15, respectively) 
has been detected (Example 1.9). 
Example 2 
This Example discloses materials and methods used to obtain the results 
disclosed in Example 1. 
2.1 Materials 
Restriction endonucleases AvaI, BamHI, BglII , EcoRI, HindIII, KPnI, PstI, 
PuvII, SalI, StuI, Xbal, and XhOI were obtained from Promega-Biotec. 
Enzymes AccI, ClaI, DraI, MstI, MstII, NarI, NcoI, XmnI, and XorII were 
obtained from New England Biolabs. Polynucleotide kinase was from P-L 
Biochemicals and bovine alkaline phosphatase was from Boehringer-Mannheim. 
[.gamma.-.sup.32 P] ATP (2000-3000 Ci/mmole) was obtained from New England 
Nuclear. Chemicals used for DNA sequencing were obtained from the vendors 
recommended by A. M. Maxam and W. Gilbert (1980) Meth. Enzymol. 65:499- 
560. X-ray film on rolls (20 cm.times.25 m) XAR-351 was obtained from 
Kodak. DuPont Quanta II intensifying screens (35 cm.times.1 m) were cut in 
half to fit sequencing gels (17.5 cm.times.1 m). DNA sequencing gel 
stands, designed for gels measuring 20 cm.times.104 cm, and safety 
cabinets were from Fotodyne Inc., New Berlin, Wis. Water jacket 
thermostating plates were constructed using 1/4 inch thick plate glass 
glued together by 100% silicone rubber. 
2.2 DNA Isolation 
Procedures for the isolation and mapping of plasmid and cosmid subclones of 
the closely-related Ri plasmids pRiA4 and pRiHRI have been published: A4 
subclones: EcoRI e36 (EcoRI 3a), BamHI 8a, e16 (contains Ri EcoRI 
fragments 15, 36, and 37a) by F. Leach (1983) Ph.D, Thesis, Universite de 
Paris-Sud, Centre d'Orsay; and pRiHRI subclones: pLJ40 (i.e. cosmid 40) 
and EcoRI 3b by L. Jouanin (1984) Plasmid 12:81-102. Plasmid DNAs were 
prepared as described by H. C. Birnboim and J. Doly (1979) Nucl, Acids 
Res. 7.:1513-1523, followed by two CsCl, ethidium bromide gradient 
bandings. 
2.3 DNA Sequencing 
DNA sequences were determined using the chemical method, essentially as 
described by Maxam and Gilbert, Supra. Generally, 10-20 .mu.g of plasmid 
2.3 DNA sequencing 
DNA sequences were determined using the chemical method, essentially DNA 
was digested with the appropriate restriction enzyme, followed by removal 
of the 5' terminal phosphate with 2-3 units of calf intestinal alkaline 
phosphatase Reactions were done in 100 mM Tris pH 8.4, 55.degree. C. for 
30 min, Both restriction enzyme and phosphatase were removed by two phenol 
and one chloroform extractions. DNA samples were then precipitated with 
ethanol, desalted with 70% ethanol, dried, and then resuspended in 15 
.mu.l denaturation buffer (50 mM Tris-HCl (pH 9.5), 5 mM spermidine, and 
0.5 mM EDTA) and 15 .mu.l H.sub.2 O. End-labeling with [.gamma.-.sup.32 P] 
ATP and isolation of end-labeled fragments were as described by Maxam and 
Gilbert, supra. Care was taken to avoid sequencing errors resulting from 
the presence of hydrazine-unreactive 5-methycytosine bases, found after 
growth in E. coli at the second cytosine base of EcoRII or BstNI 
restriction enzyme sites (J. L. Slightom et al. (1980) Cell 21:627-638 ). 
Conditions for chemical reactions, at 20..degree. C. were as follows: 1 
.mu.l dimethyl sulfate for G, 30 sec.; 30 .mu.l of formic acid 95% for A, 
2.5 min.; 30 .mu.l of hydrazine 95% for C+T and C, 2.5 min. DNA samples 
were electrophoresed 14 hours, at 2500 V at constant voltage on gels 20 cm 
wide, 104 cm long and 0.2 mm thick. Constant gel temperatures (50.degree. 
C.) were maintained using a water-jacketed plate on one side of the gel 
sandwich. The opposite plate of the sandwich was treated with 
.gamma.-methacryloxypropyltrimethoxy silane (Sigma 6514) as described by 
H. Garoff and W. Ansorge (1980) Analyt. Biochem. 115:450-457, to bind the 
acrylamide chemically to the glass. Gel pouring, loading, and 
autoradiography have been described by R. F. Barker et al. (1983) Plant 
Mol. Biol. 2:335-350, and J. L. Slightom et al. (1983) Proc. Natl. Acad. 
Sci. USA 80:1897-1901. 
Computer programs for DNA sequence analysis were supplied by the. 
University of Wisconsin Genetics Computer Group. 
Example 3 
This Example teaches the manipulation of pRi T.sub.L -DNA TxCSs preparatory 
to insertion of a foreign structural gene. 
3.1 Removal of NdeI Sites From an M13-based Vector 
These Examples extensively use oligonucleotide-directed, site-specific 
mutageneiss of DNA (see Example 5.2). Although individuals skilled in the 
art may choose to use double-stranded DNA methods for such mutagenesis, as 
exemplified herein single-stranded methods are used. In general, 
single-stranded methods utilize M13-based vectors having inserted E. coli 
lac gene sequences. Wild-type M13 contains three NdeI sites while the lac 
sequences contain no NdeI site; BamHI sites are absent from both M13 and 
lac. Removal of these NdeI sites, described below, by site-specific 
mutagenesis may prove essential when replacing a T-DNA structural gene 
with a heteroIogous foreign structural gene (Example 6.1). M13-based 
vectors include mWB2341 and related vectors (W. M. Barnes et. al. (1983) 
Meth. Enzymol. 101:98-122; W. M. Barnes and M. Bevan (1983) Nucl. Acids 
Res. 11:349-368), and the M13mp-series of vectors (e.g. see J. Norrander 
et al. (1983) Gene 26:101-106, J. Messing and J. Vieira (1982) Gene 
19:269-276). mWB2341 and related vectors are linearized by digestion with 
EcoRI and HindIII and the resultant sticky-ends are converted to 
blunt-ends by incubation with the Klenow fragment of E. coli DNA 
polymerase I. Most of the M13mp-series vectors can be linearized by at 
least one blunt-end-forming restriction endonuclease (e.g. SmaI or 
HincII). In the alternative, particular single-stranded DNA vectors may be 
preferred for some operations; other vectors may be substituted for those 
referred to above with minor modification of procedures described herein, 
as will be understood by those of ordinary skill in the art. Also in the 
alternative, double-stranded DNA vectors might be substituted (see 
references cited in Example 5.2). 
Single-stranded DNA (ssDNA) of the viral form of an M13-based vector is 
isolated and .subjected to oligonucleotide-directed site-specific 
mutagenesis, described in detail in Examples 3.3 and 5, after 
hybridization to 5 'CAATAGAAAATTCATAGGGTTTACC3', 
5'CCTGTTTAGTATCATAGCGTTATAC3', and 5'CATGTCAATCATTTGTACCCCGGTTG3, thereby 
removing three NdeI sites which will later prove to be inconvenient 
without changing the translational properties of the encoded proteins. A 
mutated M13-based vector lacking three NdeI sites is identified and 
designated m13(Nde). 
3.2 Subcloning pRi T.sub.L -DNA Into an M13-based Vector 
DNA of a plasmid listed in Table 4, column 1 (e.g. pLJ40 for manipulations 
of the ORFs 11, 12, and 13 (SEQ ID NOS. 11, 12 and 13) promoters and 
polyadenylation sites) (see Example 2.2 for the sources of these plasmids) 
is isolated and digested to completion with the restriction enzyme(s) 
listed in Table 4, column 2 (e.g. SmI and MstII for ORFs 11, 12, and 13) 
(SEQ ID NOS. 11, 12 and 13). DNAs of e36 and pLJ40 are respectively 
harbored by the deposited strains NRRL B-15958 and NRRL B-15957. 
(Alternatively, pRiA4 DNA or pRiHRI DNA may be isolated and digested with 
the enzyme(s) listed in Table 4, column 2.) 5' or 3'- protruding-ends are 
then converted to blunt-ends by incubation with the Klenow fragment of E. 
coli DNA polymerase I or T4 DNA polymerase, respectively, and all four 
deoxynucleotide triphosphates. The resulting mixture of DNA fragments 
separated by agarose gel electrophoresis and a fragment whose size is 
listed in Table 4, column 3 (e.g. 5.2 kbp for ORFs 11, 12, and 13 ) (SEQ 
ID NOS. 11, 12, and 13) is eluted from the gel. 
Covalently-closed-circular DNA (cccDNA) of the replicative form (RF) of the 
M13-based vector m13(Nde) is isolated, converted to a linear, blunt-ended 
DNA, and has its 5'-phosphates removed by incubation with phosphatase. The 
resulting linearized vector is purified by gel electrophoresis is and is 
mixed with and ligated to the T-DNA fragment isolated above. After 
transformation of the resulting mixture into E. coli, viral DNAs and RFs 
are isolated from transformants and screened by restriction and 
hybridization analysis for the presence of inserts which when in 
single-stranded viral form, are complementary to the sequence as presented 
in FIG. 1 and which carry the complete DNA sequence of ORFs listed in 
Table 4, column 4. The virus which infects the selected colony is 
designated as listed in Table 4, column 5 (e.g. mR4 for ORFs 11, 12, and 
13) (SEQ ID NOS. 11, 12 and 13). 
3.3 Removal of Endogenous NdeI and BamHI Sites from pRi T.sub.L -DNA 
A vector designated as listed in Table 5, column 1 (e.g. mR4' for 
manipulations of the ORFs 11, 12, and 13 (SEQ ID NOS. 11, 12 and 13) 
promoters and polyadenylation sites) is prepared from the vector listed in 
the corresponding line of Table 5, column 2 (e.g. mR4 for ORFs 11, 12, and 
13) (SEQ ID NOS. 11, 12 and 13) by primer extension after hybridization to 
the oligonucleotides listed in Table 5, column 3 (e.g. 
5'GATTAGATAGTCAGATGAGCATGTGC3', 5'GCAAATCGGAGCCCCTCGAATAGG3', 
5'GCAATTTGGGAGCCATTGTGATGTGAG3' and 5'CGGTTACGCGGAGCCTATGCGGAGCGCC3' for 
ORFs 11, 12, and 13) (SEQ ID NOS. 11, 12 and 13). This operation removes 
indigenous BamHI sites and NdeI sites, the sites designated in Table 5, 
column 4 being at pRi T.sub.L -DNA positions listed in column 5 (e.g. for 
ORFs 11, 12, and 13, (SEQ ID NOS. 11, 12 and 13) an NdeI site at position 
10,305 and BamHI sites at positions 11,198, 11,278, and 12,816), which may 
be present which may prove inconvenient in later manipulations. (Note that 
there are no BamHI or NdeI sites in mR5. ) The sites may be removed one at 
a time by hybridization of a particular oligonucleotide to the ssDNA viral 
form of the vector listed in Table 5, column 2 (e.g. mR4 for ORFs 11, 12, 
and 13) (SEQ ID NOS. 11, 12 and 13), incubation of the primer/viral DNA 
complex with the Klenow fragment of E. coli DNA polymerase I, all four 
deoxynucleotide triphosphates, and DNA ligase, enrichment of resulting 
cccDNA molecules, transformation into E. coli selection of transformants, 
and isolation of RF followed by restriction enzyme analysis to identify a 
clone missing the undesired restriction sites. These steps are repeated 
for each site which is to be removed. Alternatively, the vector listed in 
Table 5, column 2 (e.g. mR4 for ORFs 11, 12, and 13) (SEQ ID NOS. 11, 12 
and 13) may be simultaneously hybridized to all of the oligonucleotides 
listed in Table 5, column 3 and then carried through the mutagenesis 
procedure thereby attempting, the procedure not being 100% efficient, to 
eliminate all of the sites in a single operation. 
3.4. Placement of Novel NdeI and BamHI Sites in pRi T.sub.L -DNA 
A vector designated as listed in Table 6, column 1 (e.g. mORF 11 for 
manipulations of the ORF 11 promoter and polyadenylation site) is prepared 
from the vector listed in the corresponding line of Table 5, column 2 
(e.g. mR4' for ORF 11) by primer extention after hybridization to the 
oligonucleotides listed in Table 6, column 3 (e.g. 
5'GCTGCGAAGGGATCCCTTTGTCGCC3', 5'CGCAAGCTACAACATCATATGGGGCGG3', 
5'GGGATCCATATGTGATGTGAGTTGG3', 5'GCCTAAGAAGGAATGGTGGATCCATGTACGTGC3' for 
ORF 11) as described above and in Example 5. This has the effect of 
introducing NdeI sites (5' . . . CATATG . . . 3') at the translational 
start site (ATG) and near the translational stop site (TAA, TGA, or TAG), 
and of introducing BamHI sites (5' . . . GGATCC . . . 3') in the sequences 
flanking the T-DNA gene, usually approximately 0.3 kbp from the 
transcriptional start and polyadenylation sites. The first and fourth 
oligonucleotide of each quartet listed in Table 6, column 3 introduces 
BamHI sites while the second and thirds introduce NdeI sites. These sites 
are located in the corresponding pRi T.sub.L -DNA at the approximate 
position listed in Table 6, column 4. For example, for manipulation of ORF 
11(SEQ ID NO. 11), 5'GCTGCGAAGGGATCCCTTTGTCGCC3' and 
5'GCCTAAGAAGGAATGGTGGATCCATGTACGTGC3' introduce BamHI sites and position 
9,974 and 12,001, respectively, while 5'CGCAAGCTACAACATCATATGGGGCGG3' and 
5'GGGATCCATATGTGATGTGAGTTGG3' introduce NdeI sites at positions 10,679 and 
11,286, respectively. The size and locations of the TxCS-carrying DNA 
segments used in these Examples may be calculated from the positions 
listed in Table 6, column 4 and the orientations defined in Table 2 and 
FIG. 1. Positions listed in Table 6, column 4, of pairs of NdeI and BamHI 
sites define promoter-bearing (P) and polyadenylation site-bearing (A) DNA 
segments as indicated by "P"s and "A"s, respectively, in column 5, the 
segments having approximate sizes as indicated in column 6. For example, 
the ORF 11 promoter is on an approximately 715 bp DNA segment located 
between artificial NdeI and BamHI sites at approximate positions 11,286 
and 12,001, respectively, while the ORF 11 polyadenylation sites is on an 
approximately 705 bp DNA segment located between artificial BamHI and NdeI 
sites at approximate positions 9,974 and 10,679, respectively. Note that 
mORF12-13 and mORF16-17 provide examples of combinations of a promoter and 
a polyadenylation site from two different T-DNA genes. 
Example 4 
This Example teaches the manipulation of four exemplary foreign structural 
genes preparatory for insertion into a pRi T.sub.L -DNA TxCS. The genes 
are for the proteins phaseolin (a nutritionally important seed storage 
protein from Phaseolus vulgaris), P. vulgaris lectin (a nutritionally 
important protein found in seeds and other plant tissues which may be 
involved in symbiotic nitrogen fixation and making seeds unpalitable to 
herbivores), thaumatin (a protein which tastes sweet to primates, 
naturally found in Thaumatococcus daniellii), and crystal protein (a 
protein produced by Bacillus thuringiensis which is used commercially to 
control larval pests of a large number of lepidopteran insect species). 
The crystal protein structural gene used here, though lacking its 3' end, 
encodes a protein toxic to insect larvae. Phaseolin, lectin, and thaumatin 
are eukaryotic genes; crystal protein is prokaryotic. Phaseolin contains 
introns; lectin and crystal protein do not. The lectin gene itself 
contains no introns and could be obtained on a 5.7 kbp HindlII fragment 
from a genomic clone (L. M. Hoffman (1984) J. Mol. Appl. Genet. 2:447-453) 
which is part of a plasmid harbored by the deposited strain NRRL B-15821 
(see also Example 6.4). However, in this Example the lectin structural 
gene is obtained from a cDNA clone (L. M. Hoffman et al. (1982) Nucl. 
Acids Res. 10:7819-7828), as is the thaumatin gene. 
4.1 Subcloning Structural Genes into M13 
The genes listed in Table 7, column 1 are carried by the plasmids listed in 
Table 7, column 2, which may be isolated from the deposited stains listed 
in Table 7, column 3 (e.g. the crystal protein structural gene is carried 
by p123/58-10 which is harbored within NRRL B-15612). DNA of a plasmid 
listed in Table 7, column 2 is digested to completion with the restriction 
enzyme(s) listed in the corresponding row of Table 6, column 4 and 
protruding ends are removed by incubation with the enzyme listed in Table 
6, column 5 (e.g. for manipulation of the crystal protein structural gene, 
p123/58-10 DNA is digested with HindlII and the resulting sticky-ends are 
removed by incubation with the Klenow fragment of E. coli DNA polymerase 
I). A DNA fragment whose size is listed in Table 7, column 6 (e.g. 6.6 kbp 
for the crystal protein) is isolated by elution from an agarose gel after 
electrophoretic separation, The resulting fragment is mixed with and 
ligated to dephosphorylated, blunt-ended, linearized m13(Nde), prepared as 
described in Example 3.1, and is transformed into E. coli. Viral DNAs and 
RFs are isolated from transformants and screened by restriction and 
hybridization analyses for the presence of inserts which are complementary 
to the sequence when in single-stranded viral form as present in the mRNA. 
The vector which infects the selected colony is designated as listed in 
Table 7, column 7 (e.g. mBtCP for the crystal protein). 
4.2 Placement of NdeI Sites Flanking Three Structural Genes 
DNA of a vector listed in Table 8, column 1 is used to prepare a vector 
designated as listed in Table 8, column 2 by primer extension after 
hybridization to the oligonucleotides listed in Table 8, column 3 (e.g. 
for crystal protein, mBtCP is used to make mBtCP' by extending the primers 
5'GGAGGTAACATATGGATAACAATCCG3' and 5'GCGGCAGATTAACGTGTTCATATGCATTCGAG3') 
as described in Examples 3.3 and 5. This has the effect of introducing 
NdeI sites at the translational start site and near the translational stop 
site; there are no BamHI or NdeI sites present within the structural gene 
which might otherwise be removed. In the case of the B. thuringiensis 
crystal protein gene, a translational stop codon (TAA) is additionally 
introduced. The structural genes listed in Table 7, column 1 may be 
isolated as a DNA fragment whose size is listed in Table 8, column 4 after 
digesting DNA of a vector listed in the corresponding line of Table 8, 
column 2 to completion with NdeI (e.g, the crystal protein structural gene 
is isolated from mBtCP' on a 2.8 kbp NdeI fragment). 
4.3 Mutagenesis of Thaumatin 
Thaumatin cDNA-containing vectors have been disclosed by C. T. Verrips et 
al., Eur. Pat. applications 54,330 and 54,331, and L. Edens et al. (1982) 
Gene 18:1-12. Thaumatin is originally synthesized as preprothaumatin, the 
prefix "pre" representing the presence of a "signal peptide" having the 
function of causing the export of thaumatin from the cytoplasm into the 
endoplasmic reticulum of the cell in which it is being synthesized, and 
the prefix "pro" representing that the protein is not in mature form. A 
thaumatin cDNA structural gone is present as the complement to thaumatin 
mRNA in M13-101-B (Eur. Pat. application 54,331). The viral form of this 
vector is used as a source of a thaumatin structural gone after 
site-specific mutagenesis directed by two of the following 
oligonucleotides: (a) 5'GGCATCATACATCATATGGCCGCCACC3', (b) 
5'CCTCACGCTCTCCCGCGCATATGGCCACCTTCGAGATCGTCAACCGC3', (c) 
5'CGAGTAAGAGGATGAAGACGGACATATGAGGATACGC3', or (d) 
5'GGGTCACTTTCTGCCCTACTGCCTAACATATCAAGACGACTAAGAGG3'. When mutated by 
oligonucleotides (a) and (.cent.), which bind to the 5'- and 3'-ends of 
the structural gene, respectively, a preprothaumatin sequence is extracted 
from the resultant vector by NdeI digestion. When mutated by 
oligonucleotides (b) and (d), which bind to the 5'- and 3'-ends, 
respectively, a mature thaumatin sequence is similarly extracted. Use of 
the combinations of (a) with (d) and (b) with (c) yields fragments 
encoding what might be termed prethaumatin and prothaumatin, respectively. 
All of these sequences are obtained on fragments having a size of 
approximately 0.7 kbp having no internal NdeI or BamHI sites which may be 
isolated as usual by gel electrophoresis. 
4.4 Other Possible Manipulations 
Phaseolin and lectin, as initially translated have signal peptides at their 
amino-termini, as is the case with thaumatin. If desired, these signal 
peptides may be eliminated by placing the 5'-NdeI site between the codons 
forming the junction between the signal peptide and the mature protein. 
When under control of a T-DNA in a plant cell nucleus, such a structural 
gene will cause the synthesis of a phaseolin or lectin protein which is 
not exported from the cell's cytoplasm. Sequences useful for designing 
oligonucleotides for manipulating for phaseolin and lectin structural 
genes are respectively reported by J. L. Slightom et al. (1983) Proc. 
Natl. Acad. Sci. USA 80:1897-1901, and Hoffman et al. (1982) supra. 
Example 5 
This Example describes techniques for the synthesis and use of synthetic 
oligonucleotides. Other useful references can be found in the list of 
works cited in the section introductory to these Examples. 
5.1 Oligonucleotide Synthesis 
Techniques for chemical synthesis of DNA utilize a number of techniques 
well known to those skilled in the art of DNA synthesis. Modification of 
nucleosides is described by H. Schaller et al. (1963) J. Amer. Chem. Soc. 
85:3821-3827, and H. Buchi and H. G. Khorana (1972) J. Mol. Biol. 
72:251-288. Preparation of deoxynucleoside phosphoramidites is described 
by S. L. Beaucage and M. H. Caruthers (1981) Tetrahedron Lett. 
22:1859-1862. Preparation of solid phase resin is described by S. P. Adams 
et al. (1983) J. Amer. Chem. Soc. 105:661-663. Hybridization procedures 
useful during the formation of double-stranded molecules are described by 
J. J. Rossi et al. (1982) J. Biol. Chem. 257:9226-9229. 
5.2 Oligonucleotide-directed Site-specific Mutagenesis 
General methods of directed mutagenesis have been reviewed by D. Shortle et 
al. (1981) Ann. Rev. Genet. 15:265-294. Of special utility in manipulation 
of genes is oligonucleotide-directed site-specific-mutagenesis, reviewed 
recently by C. S. Craik (1985) Biotechniques 3:12-19; M. J. Zoller and M. 
Smith (1983) Meth. Enzymol. 100:468-500; M. Smith and S. Gillam (1981) in 
Genetic Engineering Principals and Methods, Vol. 3, eds.: J. K. Setlow and 
A. Hollaender; and M. Smith (1982) Trends in Biochem. 7:440-442. This 
technique permits the change of one or more base pairs in a DNA sequence 
or the introduction of small insertions or deletions. Recent examples of 
oligonucleotide-directed mutagenesis include W. Kramer et al. (1984) Nucl. 
Acids Res. 12:9441-9456; Zoller and Smith (1983) supra; M. J. Zoller and 
M. Smith (1982) Nucleic Acids Res. 10:6487-6500; G. Dalbadie-McFarland et 
al. (1982) Proc. Natl. Acad. Sci. USA 79:6409-6413; G. F. M. Simons et al. 
(1982) Nucleic Acids Res. 10:821-832; and C. A. Hutchison III et al. 
(1978) J. Biol. Chem. 253:6551-6560. Oligonucleotide-directed mutation 
using double-stranded DNA vectors is also possible (R. B. Wallace et al. 
(1980) Science 209:1396-1400; G. P. Vlasuk et al. (1983) J. Biol. Chem. 
258:7141-7148; E. D. Lewis et al. (1983) Proc. Natl. Acad. Sci. USA 
80:7065-7069; Y. Morinaga et al. (1984) Biotechnol. 2:636-639). See 
Example 3.1 for useful M13-based vectors. 
Example 6 
This Example teaches use of the pRi T.sub.L -DNA TxCSs and the foreign 
structural genes manipulated in Example 3 and 4, respectively. Specific 
Examples of plant transformation vectors, plant transformation, and plant 
regeneration are given below in Examples 6.4-6.7. 
6.1 Assembly of TxCS/structural Gene Combinations 
A plasmid listed in Table 6, column 1 (e.g. mORF 11) is digested with NdeI 
and dephosphorylated with phosphatase, and the opened vector may be 
separated from the T-DNA structural gene found nested within the TxCS. A 
plasmid listed in Table 8, column 2 is digested with NdeI and the 
corresponding structural gene listed in Table 7, column 1 is isolated as a 
fragment whose size is listed in Table 8, column 4 by agarose gel 
electrophoresis followed by elution from the gel (e.g. crystal protein 
structural gene is isolated from mBtCP' on a 2.8 kbp NdeI fragment). 
Additionally, a thaumatin-encoding fragment may be isolated as described 
in Example 4.3. Any desired combination of an opened TxCS vector and an 
isolated foreign structural gene may now be mixed with each other and 
ligated together. For example, crystal protein structural gene may be 
placed between an ORF 11 promoter and an ORF 11 polyadenylation site, 
thereby replacing the structural gene of ORF 11 with that of the crystal 
protein, by ligating the 2.8 kbp NdeI fragment of mBtCP' into 
NdeI-digested mORF 11 DNA. The ligation mixtures are individually 
transformed into E. coli and RFs are isolated from the resultant 
transformants and characterized by restriction analysis. A colony is 
chosen for each transformation which lacks the endogenous pRi T.sub.L -DNA 
structural gene and has a single copy of the heterologous foreign 
structural gene inserted within the TxCS, the structural gene and the TxCS 
being in such orientation with respect to each other that the gene is 
expressible under control of the TxCS when within a plant cell. 
6.2 Assembly of Plant Transformation Vectors 
A TxCS/heterologous foreign structural gene combination may be removed from 
the M13-based vector constructed in Example 6.1 by digestion with BamHI 
followed by agarose gel electrophoresis and elution. The size of the 
BamHI-fragment bearing the promoter/structural gen/polyadenylation site 
may be calculated by adding the size of the structural gene-bearing 
fragment, as listed in Table 8, column 4, to the sizes of the promoter and 
polyadenylation site-bearing segments, as listed in Table 6, column 6. For 
example, an ORF 11 TxCS/crystal protein structural gene combination, as 
exemplified herein, may be obtained on a 4.2 kbp BamHI fragment (2.8 
kbp+715 bp+705 bp). A TxCS/gene combination may be inserted directly into 
a 5'GATC . . . 3' sticky-ended site, which may be generated by BamHI, 
BclI, BglII, MboI, or SaU3AI. Alternatively, the combination may be 
inserted into any desired restriction site by conversion of sticky-ends 
into blunt-ends followed by blunt-end ligation or by use of appropriate 
oligonucleotide linkers. 
An alternative to assembly of a pRi T.sub.L -DNA TxCS/structural gone 
combination followed by insertion of that combination into a plant 
transformation vector is the insertion of a pRi TxCS into a plant 
transformation vector followed by insertion of the structural gene into 
the TxCS/transformation vector combination. It is advantageous that the 
plant transformation vector not contain NdeI sites if the particular 
manipulation strategy exemplified herein is to be used. Otherwise 
TxCS/vector combination may be linearized by partial NdeI digestion, as 
will be understood in the art. 
6.3 Vector Choice, Transformation and Plant Regeneration 
The plant transformation vector into which the TxCS/gene combination is to 
be inserted may be a TIP-based system such as a TIP plasmid, a shuttle 
vector for introduction of novel DNAs into TIP plasmids, or a sub-TIP 
plasmid, e.g. mini-Ti or micro-Ti. Alternatively, a vector based upon a 
DNA virus, minichromosome, transposon, and homologous or nonhomologous 
recombination into plant chromosomes may be utilized. Any mode of delivery 
into the plant cell which is to be initially transformed may be used which 
is appropriate to the particular plant transformation vector into which 
the TxCS/structural gone combination is inserted. These forms of delivery 
include transfer from a Agrobacterium cell, fusion with vector-containing 
liposomes or bacterial spheroplasts, direct uptake of nucleic acid, 
encapsidation in viral coat protein followed by an infection-like process, 
or microinjection. 
The initially transformed plant cell s are propagated and used to produce 
plant tissue and whole plants by any means known to the art which is 
appropriate for the plant transformation vector and delivery mode being 
used. Methods appropriate for TIP-based transformation systems include 
those described by M.-D. Chilton et al. (1982) Nature 295:432-434, for 
carrots, K. A. Barton et al. (1983) Cell 32:1033-1043, for tobacco. 
Selection of transformed cells may be done with the drugs and selectable 
markers as described in the Background. The exact drug, concentration, 
plant tissue, plant species and cultivar must be carefully matched and 
chosen for ability to regenerate and efficient selection. Screening of 
transformed tissues for tissues expressing the foreign structural gene may 
be done using immunoassays known to the art. Southern, northern, and dot 
blots, all methods well known to those skilled in the art of molecular 
biology, may be used to detect incorporated or expressed nucleic acids. 
Screening for opine production is also often useful. 
6.4 Preparation of a disarmed T-DNA vector 
E. coli C600 (pRK-203-Kan-103-Lec), which is on deposit as NRRL B-15821, is 
a pRK290 derivative containing T-DNA sequences of pTi15955 from between 
EcoRI sites at positions 4,494 and 12,823, as defined by R. F. Barker et 
al. (1983) Plant Mol. Biol. 2:335-350, except for a deletion of sequences 
between position 5,512 HindIII site and position 9,062 BamHI site. 
Inserted into the deletion, i.e. substituting for the deleted T-DNA, is a 
Tn5-derived kanamycin resistance (kan) gene and a Phaseolus vulgaris seed 
lectin gene (see Example 4, Hoffman (1984) supra.). The lectin gene may be 
deleted from pRK-203-Kan-103-Lec by digestion with HindIII followed by 
religation; the resultant vector is designated pRK-203-Kan-103- 
BamHI-digested, dephosphorylated pRK-203-Kan-103 is mixed with and ligated 
to a BamHI fragment bearing the pRi T.sub.L -DNA TxCS/heterologous foreign 
structural gene combination assembled in Example 6.2; the resultant vector 
is designated pRK-203-Ri-Kan-103. pRK-203-Ri-Kan-103 is introduced in A. 
tumefaciens ATCC15955 using methods well known in the art, and a 
double-homologous recombinant, designated RS-Ri-Kan, is identified. 
RS-Ri-Kan does not harbor pRK-203-Ri-Kan-103, but contains a mutated 
pti15955 having a T-DNA substitution between the positions 5,512 HindIII 
site and 9,062 and BamHI site of a TxCS/structural gene combination and a 
kan gene for pTi T-DNA. This substitution deletes some tmr and tms 
sequences, thereby disarming the T-DNA. RS-Ri-Kan T-DNA transforms 
inoculated plant tissue without conferring the phenotype of 
hormone-independent growth. Tobacco tissues transformed by RS-Ri-Kan may 
be regenerated into normal plants using protocols well known in the art 
for regeneration of untransformed tissue. 
6.5 Construction of a Micro-Ti Plasmid 
p102, a pBR322 clone of the pti15955 T-DNA fragment between HindIII sites 
at positions 602 and 3,390 (as defined by R. F. Barker et al., supra 
carries the left border of T.sub.L and promoter sequences associated with 
ORF 1. p233 is a pBR322 clone of the pti15955 T-DNA BamHI/EcoRI fragment 
spanning positions 9,062 and 16,202. The T-DNA of p233 includes a 
SmaI/BclI fragment spanning positions 11,207 and 14,711, having ocs, a 
3'-deleted tml, and the right border of T.sub.L. p233 was linearized with 
SmaI, mixed with and ligated to a commercially available blunt-end BglII 
linker, trimmed with BglII, religated to itself, and transformed into E. 
coli GM33 (a dam.sup.- host that does not methylate DNA in a manner 
incompatible with the action of BclI, M. G. Marinus and N. R. Morris 
(1974) J. Mol. Biol. 85:309-322). A colony was identified which harbored a 
plasmid, designated p233G, having a BglII site in the location formerly 
occupied by the position 11,207 SmaI site. p233G DNA was digested with 
BglII and BclI and a 3.5 kbp fragment was isolated by agarose gel 
electrophoresis followed by elution. The 3.5 kbp BglII/BclI fragment was 
mixed with and ligated to BglII-digested, phosphatase-treated p102 DNA. 
The ligation mixture was transformed into E. coli K802 (W. B. Wood (1966) 
J. Mol. Biol. 16:118). Plasmid DNAs from ampicillin-resistant 
transformants were characterized by restriction analysis and a colony was 
identified, designated pAK-4, having the BglII/BClI fragment of p233G 
inserted into the BglII site of p102 and oriented so that the ocs gene was 
located between the left and right T.sub.L borders. One BglII site, also 
between the borders, was regenerated, and a BglII/BclI suture, not 
susceptable to the action of either enzyme, was generate to the right of 
the right border. pAK-4 may be represented as follows: 
EQU . . . pBR322 . . . HindIII . . . left border . . . BglII . . . ocs . . . 
right border . . . (BglII/BclI) . . . HindIII . . . pBR322 . . . 
The T-DNA of pAK-4 may be removed on a 6 kbp HindIII fragment. 
HindIII-digested pAK-4 DNA was mixed with and ligated to 
HindIII-linearized, phosphatase-treated pSUP106 DNA. pSUP106, a 10 kbp 
wide host-range plasmid capable of maintenance in both E. coli and 
Agrobacterium (R. Simon et el. (1983) in Molecular Genetics of the 
Bacteria-Plant Interaction, ed.: A. Puhler, pp. 98-106), is harbored by E. 
coli CSH52 (pSUP106) which is on deposit as NRRL B-15486. The reaction 
mixture was transformed into K802 and plasmid DNAs from 
chloramphenicol-resistant transformants were characterized by restriction 
analysis. A colony was identified harboring a plasmid, designated pAN6, 
having the Agrobacterium DNA of pAK-4 inserted into the HindIII site of 
pSUP106 oriented so that BglII/BclI suture was proximal to the pSUP106 
EcoRI site. pAN6 is a micro-Ti plasmid having within its two T-DNA borders 
a functional ocs gene and a BglII site that is unique to the plasmid. The 
BglII site is flanked by an incomplete tml gene and the pTi ORF 1 
promoter, both of which are transcribed towards the BglII site. 
BamHI-digested, dephosphorylated pAN6 is mixed with and ligated to a BamHI 
fragment bearing the pRi T.sub.L -DNA TxCS/heterologous foreign structural 
gene combination assembled in Example 6.2; the resultant vector is 
designated pAN6-Ri. pAN6-Ri may be introduced into an Agrobacterium strain 
having a helper plasmid, e.g. LBA4404 (G. Ooms et el. (1981) Gene 
14:33-50), using methods well known in the art. 
6.6 Inoculation of Tobacco Stems 
Stems of sterile Nicotiana tabacura var. Xanthi are cut into segments 
approximately 1 cm long. These segments are placed basal end up in Petri 
dishes containing Murashige and Skoog medium (MS medium: 1.65 g/l NH.sub.4 
NO.sub.3, 1:9 g/l KNO.sub.3, 440 mg/l CaCl.sub.2.2H.sub.2 O, 370 mg/l 
MgSO.sub.4.7H.sub.2 O, 170 mg/l KH.sub.2 PO.sub.4, 0.83 mg/l KI, 6.2 mg/l 
H.sub.3 BO.sub.3, 22.3 mg/l MnSO.sub.4.4H.sub.2 O, 8.6 mg/l 
ZnSO.sub.4.7H.sub.2 O, 0.25 mg/l Na.sub.2 MoO.sub.4.2H.sub.2 O, 0.025 mg/l 
CuSO.sub.4.5H.sub.2 O, 0.025 mg/l COCl.sub.2.6H.sub.2 O, 37.23 mg/l 
Na.sub.2 EDTA, 27.85 mg/l FeSO.sub.4.7H.sub.2 O, 1 g/l inositol, 50 mg/l 
nicotinic acid, 50 mg/l pyroxidine. HCl.50 mg/l thiamine.HCl, 30 g/l 
sucrose, and 8 g/l agar, pH 5.8) without hormonal supplement, a medium 
well known in the art. The basal (upper) ends are then inoculated with 
Agrobacterium cells by puncturing the cut surface of the stem with a 
syringe needle. After two weeks of incubation at 28.degree. C. with 16 hr 
light and 8 hr dark, calli develop at the upper surface of all stem 
segments. The callus regions are then transferred to MS medium containing 
2.0 mg/l NAA (1-naphthalene acetic acid), 0.3 mg/l kinetin and 0.5 mg/ml 
carbinicillin. After two weeks on this medium, the tissues are free of 
bacteria and can be assayed for the presence of opines, a methodology well 
known in the art. 
Once free of inciting bacteria, the transformed plant tissues are grown on 
MS medium with hormones at 25.degree. C. with 16 hr light and 8 hr dark. 
These tissues are cloned using a suspension method described by A. N. 
Binns and F. Meins (1979) Planta 145:365-369. Briefly, tissues are placed 
in liquid MS medium supplemented with 2.0 mg/l NAA and 0.1 mg/l kinetin, 
and shaken at 135 rpm at 28.degree. C. for 10-14 days. The resultant 
suspensions are filtered successively through 0.543 and 0.213 mm mesh 
sieves, concentrated, and plated at a final density of 8.times.10.sup.3 
cells/ml in MS medium supplemented with 2.0 mg/l NAA and 0.3 mg/l kinetin. 
After these grow to approximately 100 mg, colonies are split into two 
pieces. One piece is placed on complete MS medium and the other is 
screened for the presence of opines. Approximately 0-50% of the colonies 
are found to be opine-positive, depending on the particular parental 
uncloned callus piece from which the colonies were descended. Uncloned 
pieces having higher concentrations of opine tended to yield a higher 
percentage of opine-positive clones. 
6.7 Regeneration of Recombinant Plants 
Tissues from various opine-positive clones are transferred onto MS medium 
supplemented with 0.3 mg/l kinetin and cultured at 28.degree. C. with 16 
hr light and 8 hr dark. Shoots initiated are subsequently rooted by 
placing them in MS medium without hormones. Rooted plantlets are 
transferred to soil and placed at high humidity in a greenhouse. After 
7-10 days, the plants are then grown with normal greenhouse conditions. 
Regenerated plants derived from opine-positive clones contain opines. The 
presence of opines indicates thereby that these normal looking plants are 
transformed by T-DNA. 
TABLE 1 
______________________________________ 
Restriction Enzyme Sites in pRi T.sub.L -DNA Region 
En- No. 
zyme Sites Locations 
______________________________________ 
Bst 1 3 993 
E II 
Sna I 1 6 459 
Apa I 2 3 390 17 851 
Mst II 
2 4 806 15 021 
Sma I 2 3 075 9 863 
Xba I 2 676 4 999 
Kpn I 3 3 364 14 133 
19 918 
Mlu I 3 17 606 20 793 
20 856 
Nco I 3 2 262 10 133 
21 021 
Sst II 
3 3 431 14 691 
17 037 
Xho I 3 9 242 11 003 
20 700 
Bam 4 1 343 11 198 
11 278 
12 816 
HI 
Hpa I 4 8 375 12 459 
13 700 
18 818 
Nde I 4 3 519 3 861 4 822 10 308 
Nru I 4 5 281 10 968 
11 617 
18 901 
Sal I 4 4 515 6 047 12 655 
15 821 
Ava 5 13 684 14 382 
15 480 
16 415 
18 262 
III 
BssH 5 5 727 6 847 19 761 
20 260 
20 660 
II 
BstX I 
5 2 269 4 226 9 912 16 016 
18 309 
Cla I 5 35 753 11 421 
12 598 
21 110 
Nar I 5 465 4 114 11 356 
16 441 
20 385 
Nsi I 5 13 688 14 386 
15 484 
16 419 
18 266 
Sca I 5 1 794 4 546 10 166 
11 500 
13 858 
Tth 5 3 413 3 816 8 217 8 769 11 369 
III I 
Xma 5 5 814 7 970 8 502 10 613 
20 347 
III 
Aat II 
6 974 5 615 6 054 7 521 9 272 19 089 
Asu II 
6 4 792 10 026 
12 954 
16 897 
19 418 
19 436 
Hind 6 5 602 6 361 9 814 11 587 
15 827 
17 404 
III 
Mst I 6 4 004 8 091 11 427 
16 088 
19 690 
20 408 
Pst I 6 2 244 4 892 7 003 10 486 
10 533 
17 780 
Xor II 
6 230 2 659 4 480 5 694 8 509 16 962 
Bcl I 7 992 1 364 6 710 10 564 
18 673 
19 403 
19 827 
Bgl II 
7 4 197 5 525 7 879 11 239 
13 097 
15 517 
15 760 
EcoR I 
7 7 585 9 077 13 445 
15 358 
17 059 
18 766 
18 911 
Acc I 8 333 4 516 6 048 6 460 9 514 12 656 
15 822 19 089 
Bal I 8 497 3 568 5 488 9 233 9 339 9 916 
12 001 17 544 
Sph I 8 582 11 476 
15 013 
15 057 
15 486 
17 175 
19 027 20 404 
Xmm I 8 1 759 2 725 4 498 4 546 10 103 
12 206 
17 338 17 917 
EcoR 9 5 134 6 738 7 775 10 098 
10 626 
13 173 
14 048 16 080 
17 491 
Sst I 9 1 967 4 152 10 879 
11 068 
12 395 
14 105 
17 016 19 214 
19 866 
Stu I 9 5 590 6 696 7 512 11 442 
12 066 
15 967 
16 656 20 186 
20 467 
Bgl I 10 1 571 3 125 5 872 5 956 6 832 9775 
10 912 14 290 
16 606 
21 065 
Ava I 11 3 073 3 765 5 268 7 012 9 242 9 861 
10 573 10 629 
11 003 
14 402 
20 700 
Aha 12 2 486 11 334 
12 233 
13 427 
14 580 
13 666 
III 
15 577 15 599 
16 168 
18 135 
18 573 
20 070 
Nae I 13 316 446 1 664 3 931 3 962 5 733 
7 616 9 771 15 000 
16 622 
18 474 
20 380 
20 652 
Pvu II 
13 250 1 235 1 859 1 395 2 752 7 888 
8 451 12 042 
13 715 
15 590 
15 620 
16 056 
18 688 
______________________________________ 
Ban II 19 Hph I 37 Hpa II 72 
HgiA I 19 Rsa I 38 Cfo I 80 
Ban I 20 HinF I 41 Hinp I 80 
Hinc II 21 Hga I 42 Ala I 87 
Xho II 22 Fok I 48 Sau 3a 87 
Hae II 23 Dde I 55 Hae III 
99 
Nci I 23 Mbo II 63 Taq I 113 
Aha II 24 Sau 96 66 Fnu 4A 132 
Ava II 26 Fnu II 68 Mnl I 171 
BstN I 35 Bbv I 69 
______________________________________ 
TABLE 2 
__________________________________________________________________________ 
Open-Reading Frames in pRi-T.sub.L -DNA 
Sequence location 
Ribosome binding Calculated molecular 
Sequence 
After first 
Before 
binding sites 
Coding sequence 
Amino 
weight (daltons) of 
ORF 
ID No. 
ATG in-frame 
terminator 
.sub.G.sup.A XX ATG G 
base pairs 
acids 
ORF-encoded 
__________________________________________________________________________ 
protein 
1. 1 2262 937 GCC ATG G 
1326 442 47,400 
2. 2 3458 2649 GAT ATG T 
810 270 29,400 
3. 3 3726 4799 ATC ATG C 
1074 358 38,200 
4. 4 4400 4041 GGG ATG C 
360 120 13,200 
5. 5 4918 4607 GGG ATG C 
312 104 12,000 
6. 6 5143 6216 CGT ATG C 
1074 358 40,300 
7. 7 5643 5071 GGC ATG G 
573 191 21,700 
8. 8 6609 8888 GTG ATG G 
2280 760 85,000 
9. 9 6830 6576 GCC ATG A 
255 85 9,600 
10. 
10 9748 10044 AGA ATG G 
297 99 11,400 
11 11282 10509 ACA ATG G 
774 258 29,500 
12 12466 13002 AAC ATG G 
537 179 20,100 
13 13723 14319 TGA ATG G 
597 199 22,100 
14 15659 16210 AGC ATG G 
552 184 20,300 
15 17545 16517 CAG ATG G 
1029 343 37,400 
16 18189 17737 AAA ATG T 
453 151 17,400 
17 18743 18177 GAG ATG A 
567 189 21,700 
18 19390 19031 AAC ATG G 
360 120 13,400 
__________________________________________________________________________ 
Coordinates represent the A of the AUG initiation codon or the last 
nucleotide before the termination codon. 
TABLE 3 
__________________________________________________________________________ 
Eukaryotic Transcription Controlling Sequences 
Sequence and position of Sequence and position 
promoter elements, of polyadenylation sites, 
Distance (bp) from 
Sequence 
positions from first ATG positions from terminator 
first Met to best 
ORF 
ID No. 
(CCAAT) (TATAA) (AATAAA) polyadenylation 
__________________________________________________________________________ 
site 
1. 1 -211 -100 -143 -92 -65 +38 +100 1364 
CAAT; CAATA TATA; ATAA; TAATAA 
AATAAT; AATATA 
2. 2 -81 -60 +116 926 
CCAAT ATAT AATAAA 
3. 3 -102 -80 +137 1211 
CAACT TATA AATGAA 
4. 4 -107 -82 -46 +380 740 
CCAAA ATAAA; AATA AATAAA 
5. 5 -131 -68 +119 431 
CCAAAT ATAA GATAAA 
6. 6 -146 -98 +87 +260 +294 1368 
CAAAAT ATAATA AATAAT; AGTAAA; AATAAA 
7. 7 -- -- -- 573 
8. 8 -133 -129 -92 -72 +96 +236 2376 
CCTACA; CAAAGT 
TAATAA; TATAA 
AATAAA; AATAAA 
9. 9 -76 -59 +187 442 
CAATT TATAA AATAAA 
10. 
10 -221 -91 -64 -144 -25 +75 +114 414 
CATAT; CAATA; CAATT 
TATATA; TAATA 
AATAAG; AATATA 
11 -116 -54 +350 1124 
CCAAA TATT AATAAA 
12 -81 -56 +83 +141 620 
CCAAT TATAAA AATAAA; AATAAA 
13 -155 -87 -51 +111 +262 708 
CAAAT ATAAT; TAAATA 
AATAAA; AATAAA 
14 -174 -116 -95 -140 -72 -50 +60 +128 +231 612 
CCAAT; CAAAA; AATA; TAAATA; 
AATAAA; AATAAA; AATAAA 
CAAAG AATA 
15 -91 -65 +149 1178 
CCAAAA TATAAA AATAAA 
16 -193 -126 +87 +120 545 
CAAAA TATA AATTAA; TATAAA 
17 -69 -50 -60 -37 +92 +164 670 
CAATC; CAAAT ATAT; ATAAT TATAAA; AATGAA 
18 -- -- -- 360 
__________________________________________________________________________ 
Element positions are negative or positive when respectively 5' or 3' to 
an ORF. 
TABLE 4 
__________________________________________________________________________ 
Construction of pRi T.sub.L -DNA ORF-carrying vectors based on M13 
1.sup.a 2.sup.b 3.sup.b 
4.sup.b 
5.sup.c 
__________________________________________________________________________ 
to use ORF 1: 
e36 
##STR5## 
3.8 kbp 
ORF 1 mR1 
to use ORFs 2-9: 
##STR6## 
##STR7## 
7.9 kbp 
ORFs 2-9 
mR2 
to use ORF 10: 
##STR8## 
##STR9## 
3.3 kbp 
ORF 10 mR3 
to use ORFs 11-13: 
pLJ40 or e16 
##STR10## 
5.2 kbp 
ORFs 11-13 
mR4 
to use ORFs 14-17: 
pLJ40 
##STR11## 
5.8 kbp 
ORFs 14-17 
mR5 
__________________________________________________________________________ 
.sup.a Plasmids listed in column 1 are listed as sources of T.sub.L -DNA 
sequences. 
.sup.b After restriction enzymes listed in column 2 are used to cut the 
plasmids listed in column 1, DNA fragments having sizes listed in column 
are isolated which carry the ORF(s) listed in column 4. 
.sup.c The resultant M13based vectors designated in column 5 carry the 
ORFs designated in column 4. 
TABLE 5 
__________________________________________________________________________ 
##STR12## 
1.sup.a 
2.sup.a 
3.sup.b 4.sup.b 
5.sup.b 
__________________________________________________________________________ 
to use ORF 1: 
mR1' 
mR1 
##STR13## 
##STR14## 
1,343 
to use ORFs 2-9: 
mR2' 
mR2 
##STR15## 
##STR16## 
3,518 
##STR17## 
##STR18## 
3,860 
##STR19## 
##STR20## 
4,821 
to use ORF 10: 
mR3' 
mR3 
##STR21## 
##STR22## 
10,305 
to use ORFs 11-13: 
mR4' 
mR4 
##STR23## 
##STR24## 
10,305 
##STR25## 
##STR26## 
11,198 
##STR27## 
##STR28## 
11,278 
##STR29## 
##STR30## 
12,816 
__________________________________________________________________________ 
.sup.a Modified vectors having designations listed in column 1 are made 
from the vectors listed in column 2. 
.sup.b Oligonucleotides listed in column 3 are used as primers in 
sitespecific mutagensis of vectors listed in column 2, thereby removing 
restriction sites specific to the enzymes listed in column 4 which are 
found in the pRi T.sub.L -DNA sequence near the positions listed in colum 
5. Underlined letters in column 3 indicate positions of introduced 
mutations. 
3 TABLE 6 
##STR31## 
1.sup.a 2.sup.a 3.sup.b 
4.sup.b 5.sup.c 6.sup.c to use ORF 1: 
mORF1 mR1' 
##STR32## 
692 
967 } A 275 bp 
##STR33## 
2,265 2,611 } P 346 bp 
to use ORF 2: mORF2 mR2' 
##STR34## 
2,324 
2,579 } A 255 bp 
##STR35## 
3,474 3,885 } P 411 bp 
to use ORF 3: mORF3 mR2' 
##STR36## 
3,281 
3,723 } P 442 bp 
##STR37## 
4,821 5,106 } A 285 bp 
to use ORF 4: mORF4 mR2' 
##STR38## 
3,531 
4,025 } A 494 bp 
##STR39## 
4,405 4,757 } P 352 bp 
to use ORF 5: mORF5 mR2' 
##STR40## 
4,277 
4,689 } A 412 bp 
##STR41## 
4,923 5,302 } P 379 bp 
to use ORF 6: mORF6 mR2' 
##STR42## 
4,675 
5,138 } P 463 bp 
##STR43## 
6,221 6,613 } A 392 bp 
to use ORF 8: mORF8 mR2' 
##STR44## 
6,156 
6,604 } P 448 bp 
##STR45## 
8,917 9,233 } A 316 bp 
to use ORF 9: mORF9 mR2' 
##STR46## 
6,156 
6,621 } A 465 bp 
##STR47## 
6,833 7,239 } P 406 bp 
to use ORF 10: mORF10 mR3' 
##STR48## 
9,332 
9,743 } P 411 bp 
##STR49## 
10,05410,349 } A 295 bp 
to use ORF 11: mORF11 mR4' 
##STR50## 
9,97410,679 } A 705 bp 
##STR51## 
11,28612,001 } P 715 bp 
to use ORF 12: mORF12 mR4' 
##STR52## 
12,00112,463 } P 462 bp 
##STR53## 
12,93013,351 } A 421 bp 
to use ORF 13: mORF13 mR4' 
##STR54## 
13,35113,718 } P 367 bp 
##STR55## 
14,29014,687 } A 397 bp 
to use ORF 14: mORF14 mR5 
##STR56## 
15,24215,656 } P 414 bp 
##STR57## 
16,17016,491 } A 321 bp 
to use ORF 15: mORF15 mR5 
##STR58## 
16,25216,590 } A 338 bp 
##STR59## 
17,54917,888 } P 339 bp 
to use ORF 16: mORF16 mR5 
##STR60## 
17,41617,741 } A 325 bp 
##STR61## 
18,74819,002 } P 254 bp 
to use ORF 17: mORF17 mR5 
##STR62## 
17,88718,201 } A 314 bp 
##STR63## 
18,74819,172 } P 424 bp to use the ORF 12promoter with the mORF12-13 
mR4' 
##STR64## 
12,00112,463 } P 462 bp ORF 13 polyadenyla-tion site: 
##STR65## 
14,29014,687 } A 397 bp to use the ORF 16promoter with the mORF16-17 
mR5 
##STR66## 
17,41617,741 } A 325 bp ORF 17 polyadenyla-tion site: 
##STR67## 
18,74819,172 } P 424 bp 
.sup.a Modified vectors having designations listed in column 1 are made 
from the vectors listed in column 2. 
##STR68## 
.sup.c Pairs of oligonucleotides marked in column 5 with "P"s define 
promoterbearing segments as exemplified herein, while paris marked with 
"A" define polyadenylation sitebearing segments, the segments having 
approximate sizes indicated in column 6. 
TABLE 7 
__________________________________________________________________________ 
Construction of vectors carrying structural genes 
1.sup.a 2.sup.a 
3.sup.a 4.sup.b 
5.sup.b 6.sup.c 
7.sup.c 
__________________________________________________________________________ 
to use phaseolin: 
p8.8 NRRL B-15393 
##STR69## 
Klenow fragment of E. coli DNA polymerase 
I 3.8 kbp 
mPhas 
to use lectin: 
pPVL134 
ATCC 39181 
##STR70## 
bacteriophage T4 DNA polymerase 
0.95 kbp 
mLec 
to use crystal protein: 
p123/58-10 
NRRL B-15612 
##STR71## 
Klenow fragment of E. coli DNA polymerase 
I 6.6 kbp 
mBtCP 
__________________________________________________________________________ 
.sup.a Structural genes encoding the proteins listed in column 1 are 
carried by plasmids listed in column 2 which are harbored by the deposite 
strains listed in column 3. 
.sup.b DNAs of plasmids listed in column 2 are digested with the 
restriction endonuclease(s) listed in column 4 and incubated with the 
enzymes listed in column 5 to convert stickyends to bluntends. 
.sup.c DNA fragments of the sizes listed in column 6 are isolated and 
combined with an M13based vector described in Example 3.1 to form the 
vectors listed in column 7. 
TABLE 8 
__________________________________________________________________________ 
##STR72## 
1.sup.a 
2.sup.a 
3.sup.b 4.sup.c 
__________________________________________________________________________ 
to use phaseolin: 
mPhas 
mPhas' 
##STR73## 2.1 kbp 
##STR74## 
to use lectin: 
mLec 
mLec' 
##STR75## 0.8 kbp 
##STR76## 
to use crystal protein: 
mBtCP 
mBtCP' 
##STR77## 2.8 kbp 
##STR78## 
__________________________________________________________________________ 
.sup.a The vectors listed in column 1 are used to make the modified 
vectors listed in column 2. 
##STR79## 
##STR80## 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 19 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1326 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
( A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (1..1326) 
(D) OTHER INFORMATION: /label=ORF1 
/note="Sequence ORF1 corresponds to bases 937 
through 2262 of Seq. ID No. 19. It is read 5'to 
3'from the complementary strand." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TGGTTGGCTCAAATTTTGGCTCTGGTGCTCGATGACGTCGAGATGAGGACAGTAGTGATC60 
AACTTGGCGGTCGATACCTTGGTTACGCCACTCCCAGAGTGCCATGTCGTCCTCCGAGCG120 
GTCTGAGATAAC CCAGTCGGCAATTGCTGCTGCATTGCCGGGCGTTCCCCAACCACGACG180 
AATATGCTTTCGTTCATCTAACTCGCGTCGCACTGCCCTCCCAGTCATGAAGTCAAAGCC240 
AAATTCTACCCTCTCTCCATTTCCCAGCTCAGTCGAGAAATCGTAACACCTCGTGGCA GC300 
TGACAGTTTCAGAAAGGGGCGTATCCCTCGAACTCCAGGGTCCTCTTTCACATAGTTAGC360 
AAGGCGTACTGCTGCATAATCTGCGTTGAAGGCTCTGATGACTACAGGATCCTCGGACAA420 
GCCCAATTGATCAGGGCGAACCCTCGCGCTCATAA TATGAATTGCGACGACCCTTGCTTC480 
CTGTCGGAGCATCGAATCAATCCAAGCCTTCCCTGCGGCATAGAGGTCATCGACTGCGAT540 
GTCATCAAGATCGAGTAGCTTTGCCAACCTAGGAAGTTCTTGAGGAAAAATCACCGGCAT600 
GACAGCAACCGT CTCTCGCCAGTCAGTTGCCGGACTGGCTTCCCTAACGCCATCCACGAA660 
TGCCTCACCGCTTGCGTATTTGAATGTGTAAAAGAGAAGGACCACTCTTTGGCGGTACTT720 
CGGACGCCGGCTTAGCCACGCGGCAATAATGTGGGCCTCAAACTCACGACCATCCAAA AA780 
TATAGTCGCGCCTGGATTGACCTCGCTGGCCTTGTCGAGAAGAGGTTCCAAAAAGGGAAC840 
GGTGTCTTTCGTAATAGTACTTAAATCTGTGAGTTCGCCATGCGAAACCTCTCGAACGAT900 
TATCGGCGTATCCCTGACATCAGCTGAATGAAATT CTCGGACGAGTTTGTCGGGCAAAGT960 
GGAGACCCGCCACGTGTTGAAGTCGTGGGAAACGATGGGCACATCGTCGCCGGTGAGTGC1020 
GGCATCGAGCTCAGAGAGGTTCCGCCTGCCAACCTCACCGAGAGCAGCTAACAACGAAGT1080 
TTCGGTGCATTC CTGTATCCCTTTACCCAGATTATACATGCCCCGGTGTTCGATAACTTG1140 
AAGAGGCAGTGGCTCCTCAAGATGTTCAAGGAGGTGGGGTACAGAGTGCCGGGCGAGGAC1200 
CTCATCCACCGTGACACCAACCGGGAGATCCCATTCGAGTTTCCACTGGGGCCAGCAT GT1260 
GCCCGCGACGGCGAAAGGTTTGCGCTGGCAAAGAACCCGGCTGCTGCAGGTGGACCTATC1320 
CTTACC1326 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 810 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (1..810) 
(D) OTHER INFORMATION: /label=ORF2 
/note="Sequence ORF2 corresponds to bases 2649 
through 3458 of Seq. ID No. 19. It is read 5'to 
3'from the complementary strand." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CTGACTGCGATCGGGAAGAAGCTCGCCAAGTTCACCGAGAATAGCAGAGAGCGCATCCTC 60 
ATCGGGTACTACGAACACATTCGTCCCAGAGGGCTTTGTTTCAGCTGCGCCAACCCAGAA120 
AGCAAGGCCATTTTCCAAGTTGCCGATGGCGGTCAGCATGTTTTGATTGTTGCTGCCGTT180 
TCCACAAGCGATGTGAAGGCCGATCCCGTGAGAGAGGCC CTTGACGAAGGTGAAATAGCC240 
TTTGGATTTTCCAACTGTTTCAACGGGCACTAGATATTGACCCTCTGGCGCGGCAACCAC300 
CTTGAATTTGCGAGATGACTGGTTGCCGATGAGCGAAGAAAGCATTTCTCCGGCTTCTTT360 
GTAAGATTTGTGAGAT TCCCACATTTGACAGCCGTAGAAATGCCCCATCGGAATGTTGCG420 
GATTCCCGGGATGCCACCAAATTTGTTCTCCATAGCCGCGTGAACGGCTTGCCAGTTGGG480 
CAGGGAGAAAGAATCGAAGCGATCATCTTTGTAGATCGTGACCATTCCATCATTTCCCTG 540 
GAATCCGATATTTTCAATGGCGCTGAAAACTGACCTTGCGATTTCTTCGCATTCCCGTGC600 
GGATGTGAGCAATTGATAATGGCCCTTGCAGGCGATCCTGGTCAAATTGGCGATGATGTT660 
GATGGCAGGATTAATATCCCAACACTGGTGATTTCGATC TTGCTTAAAGGTGGTACCATC720 
GCCGTCGAAGGCGAGCAGGGCCCGGAGAGATGAATCGGCAAGACTGCGTCGGACCCGCTC780 
CGCGGCGTCGGGAATGAGGCTGATAAGAGA810 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1074 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(i x) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 1..1074 
(D) OTHER INFORMATION: /label=ORF3 
/note="Sequence ORF3 corresponds to bases 3726 
through 4799 of Sequence ID No. 19." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CCCGCCAACGACGCGACATGCGCTGCCGCGATTGCCTTCCCCGAGGGCAACTGGAAGGAA60 
GAACTTGATGCGCTCCGCACCTTGTGTGACCCCGTCGAGGTGGTTAAGGTCGCAGTCGGC120 
AGAGGTCTTAGCGGCATATGTAATGTTGTTGCAGCAATGAATCCCACAAAGGTGAGGGGC180 
CTCGGCGATGTCATCGGGCAGATGCCGGCTCTTAATCACCGTATT GCTGCCGCCGCCGGC240 
GAAACTCCGGTGCGAGACCTTGGAATAGGTTACCAGTGCGCAATCTGCCACCCCGACATA300 
GCCAGTGCGATGTTAGCCACTTCTGAGGGGATCAGCCACGTTCTCCGTGAAAGGATTGAG360 
AAAGAAGTTGACCGGGACATTG GAGAAGGCGCCACCGTCTGCATTTTCGTTCAGCCGAGA420 
ATGAGCTCCAAGGGCTCTCCAGTTTCTGTCCATTTCACCCTCCAGTTTGCGAGATCTGGA480 
ACTCTTGTCGATGCCAGAATGATGGAGAGTTACAATTTCATGAAAGGCAATGGCACAGTG540 
ACCGCACCGGATTTGAAAAGTCATTGGAAGAAGCACGGTATTGACAGGCCAGGCCCACGT600 
CCGCCCACGTCCAAGTTTGAACTCCTCTTCGCCGCTGTCCCCGACAACAGTAAACTTGCC660 
GCCACCGATTTTACCCATCTCGGCCCTGTCGAGCGTGATAAGGAA CTACTCGGCAGCACG720 
GTATTCGGGATTGCCGCTAAGAAACCTGGTACGATCGTTTATCCGTGCGAAAAGGTTCTC780 
TGTTTGGAGGTCGACGTACACGCGCATCGCGCCCTAGAAGTACTTCACCGCCTTGGGGAA840 
CAGGCTTATAGCAATGGCCGTG GCACTAGCTTCGGTCTTCACACCGGTCCGTCCTCTTGC900 
CTTAATCTTTCCGCCGCCGCGCTCGCTACATTTTTCAAACGCTCGGATCTCTGTTCCCTT960 
CCATTGAGTGATGCTTTTGTCCTTTTCTGCGACCCGCCACCGCCTACAGCGCCAAGAAAG1020 
ATGGCCTTCCGATCACTGCCTTCTCCCCCACGAGCACCAATCAGTTCGAACTCG1074 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 360 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (1..360) 
(D) OTHER INFORMATION: /label=ORF4 
/note="Sequence ORF4 corresponds to bases 4041 
through 4400 of Seq. ID No. 19. It is read 5'to 
3'from the complementary strand." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GCCACTTCTGAGGGGATCAGCCACGTTCTCCGTGAAAGGATTGAGAAAGAAGTTGACCGG60 
GACATTGGAGAAGGCGCCACCGTCTGCATTTTCGTTCAGCCGAGAATGAGCTCCAAGGGC120 
TCT CCAGTTTCTGTCCATTTCACCCTCCAGTTTGCGAGATCTGGAACTCTTGTCGATGCC180 
AGAATGATGGAGAGTTACAATTTCATGAAAGGCAATGGCACAGTGACCGCACCGGATTTG240 
AAAAGTCATTGGAAGAAGCACGGTATTGACAGGCCAGGCCCACGTCCGC CCACGTCCAAG300 
TTTGAACTCCTCTTCGCCGCTGTCCCCGACAACAGTAAACTTGCCGCCACCGATTTTACC360 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 312 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii ) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (1..312) 
(D) OTHER INFORMATION: /label=ORF5 
/note="Sequence ORF5 corresponds to bases 4607 
through 4918 of Seq. ID No. 19. It is read 5'to 
3'from the complementary strand." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CACCGGTCCGTCCTCTTGCCTTAATCTTTCCGCCGCCGCGCTCGCTACATTTTTCAAACG60 
CTCGGATCTCTGTTCCCTTCCATTGAGTGATGCTTTTGTCCTTTTCTGCGAC CCGCCACC120 
GCCTACAGCGCCAAGAAAGATGGCCTTCCGATCACTGCCTTCTCCCCCACGAGCACCAAT180 
CAGTTCGAACTCGTAGAGCCTCAGGTCGTCAAGGCATATGTTCTCGGACTTTTCGACGCG240 
CCGACGATGGTTACGCCCCGCGACAAAACG CGAGCCAGCTTCTGCAGCCAATATGTACGT300 
TTCCGTGAACCG312 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1074 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 1..1074 
(D) OTHER INFORMATION: /label=ORF6 
/note="Sequence ORF6 corresponds to bases 5143 
through 6216 of Seq. ID No. 19." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
CGTACAGGTATCACATTTAACGTTGCTGCGGCGGACCGAGCCCGCTTGGAAGCGATTGTT60 
GCAGCTCCAACTTCTGCTCAGAAGCACGTGTGGCGAGCGAAGATCATCTTGATGAGCAG T120 
GATGGCTCGGGAACGGTCGCGATCATGGAGGCAACCGGTAAATCCAAAACCTGTGTCTGG180 
CGCTGGCAGGAGCGCTTCATGACTGAGGGCGTCGATGGCCTTTTGCACGACAAGAGCAGA240 
CCGCCCGGCATTGCGCCGCTTGATGGCGAACTCGTT GAGCGTGTCGTCGCACTGACGCTT300 
GAGACGCCTCAACAGGAAGCAACGCACTGGACTGTTCGTGCGATGGCCAAGGCCGTTGGG360 
ATTGCAGCCTCTTCGGTTGTGAAGATCTGGCACGAGCATGGTCTTGCGCCGCATCGCTGG420 
CGCTCTTTCAAAC TGTCGAACGACAAGGCCTTTGCCGAGAAGCTTCACGACGTCGTTGGC480 
CTCTACGTCTCGCCACCGGCCCATGCCATTGTCCTGTCCGTCGATGAGAAGAGCCAGATC540 
CAGGCACTCGATCGGACGCAACCGGGACTCCCCTTGAAGAAAGGGCGCGCCGGCACAAT G600 
ACCCACGATTACAAGCGCCACGGCACCACCACCCTATTTGCCGCCCTCAACATCCTCGAC660 
GGCTCGGTGATCGGCCGAAACATGCAGCGTCACCGGCATCAGGAGTTCATCCGTTTTCTC720 
AACGCCATCGAGGCGGAACTGCCAAAGGACAAGGCC GTCCACGTCATTCTCGACAATTAC780 
GCGACCCATAAGCAGCCGAAGGTCCGCGCCTGGCTGGCAAGGCATCCGCGCTGGACCTTC840 
CACTTCGTCCCAACATCATGTTCATGGCTGAACGCCGTCGAGGGATTCTTCGCTAAATTG900 
ACACGTCGACGTC TGAAGCACGGTGTCTTTCATTCCGTCGTTGACCTCCAGGCCACCATC960 
AACCGCTTCGTCAGAGAGCATAATCAGGAACCAAAGCCGTTCATCTGGAGAGCAGATCCA1020 
GACGAGATCATTGCAGCCGTCAAACGTGGGCACCAAGCGTTGGAATCAATCCAC 1074 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 573 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (1..573) 
(D) OTHER INFORMATION: /label=ORF7 
/note="Sequence ORF7 corresponds to bases 5071 
through 5643 of Seq. ID No. 19. It is read 5'to 
3'from the complementary strand." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GTGGATTGATTGAAACA AAGGAGTCCGAGTTGGGATTCCCTTTCGGTCTTCGTCGTGCAA60 
CGATATCGTATGCGTACAGGTATCACATTTAACGTTGCTGCGGCGGACCGAGCCCGCTTG120 
GAAGCGATTGTTGCAGCTCCAACTTCTGCTCAGAAGCACGTGTGGCGAGCGAAGATCATC 180 
TTGATGAGCAGTGATGGCTCGGGAACGGTCGCGATCATGGAGGCAACCGGTAAATCCAAA240 
ACCTGTGTCTGGCGCTGGCAGGAGCGCTTCATGACTGAGGGCGTCGATGGCCTTTTGCAC300 
GACAAGAGCAGACCGCCCGGCATTGCGCCGCTTGATGGCG AACTCGTTGAGCGTGTCGTC360 
GCACTGACGCTTGAGACGCCTCAACAGGAAGCAACGCACTGGACTGTTCGTGCGATGGCC420 
AAGGCCGTTGGGATTGCAGCCTCTTCGGTTGTGAAGATCTGGCACGAGCATGGTCTTGCG480 
CCGCATCGCTGGCGCTC TTTCAAACTGTCGAACGACAAGGCCTTTGCCGAGAAGCTTCAC540 
GACGTCGTTGGCCTCTACGTCTCGCCACCGGCC573 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2279 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 1..2279 
(D ) OTHER INFORMATION: /label=ORF8 
/note="Sequence ORF8 corresponds to bases 6609 
through 8888 of Seq. ID No. 19. " 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
GATCTTCCATATCAGCGCCCACGTTTCACCCCGTTTGCCGTCACCCATCCACGTAGTGGA60 
GTCAACCTGAACCGTGCAATTTCTCAGGCCTTTGTCTGCTATGAT CAGTTCTGCGAACGG120 
CTCTTGCGATATCAGCAAAGCTGGACGGATTGGGTGTTCGACCACGGATTTGCAGAAGCC180 
ATTGAAGACGTGGCGCTGGTGTTCCAGGTTGCACCTTGCCTTCATGGCCCCCGAATAGGC240 
GCGCTCGAAGTGTTGATACCTC GTCGCACCCAGGTCTTCATTTATATGTCGAACAACCAA300 
TTGCAGCGCTTTGTTGCACACCAGTGCATTGCTCAACTTGGCGACGCCGTGCTTGCTTGC360 
ATGATCCCGCCCTACGCGAGTGACCTCTCGCTGCAGGAAATGGCTCGGGCGCACAACAGA420 
TTTTGCCCAGGCAGTTACACGAGGTCCGCAGACGTACAGTGCTTTATCGCCATCCAACTC480 
AGCAGCCGATTCGTTGAGGAGGGCACATGTAACGTGCACGGGCGAAATGGCTTAAAAAGA540 
ACCTGCCGCTTCTTTCGTCGCCCTGCTGAGTTCTTCAGCCGTTAT GACATCGTTGCCATT600 
GGGCCGGTGCTCTTCCATGATGAACTGGATTGCCCAGCAAACTGCAATGAGCCTCTTTCC660 
TGCTTTGACCTGCGGTACGACTATCAGGTTTTCCTCCAGGAGTGCGATGCCCATGATGGT720 
GTGGGGCATTATCCGGAAGGCG CACCACTACCTAGTGTTGCCATCGTAGGAGGCGGGCTG780 
TCTGGCCTTGTTGCTGCCACAGAACTACTTGGCGCTGGCGTCAAGGAAATCACTCTTTTC840 
GATACCGTTGATGAGATCCGTAGTTTTGGGGCATCGCCGATGCCAAACGGCGACGCTCAC900 
CAGGCCTTGACGTCGTTCGGTGTCATGCCTTTCTCCGCCAACCAACTTTGCCTGTCATAC960 
TATCTGGATAAGTTTAGAATTCCGTCCAGCCTTCGTTTTCCTTGTGCCGGCAACGACCAC1020 
ACAGCACTATATTTCCGCCAGAAACGCTACGCATGGCACGCGGGG CAAGCTCCGCCGGGG1080 
ATATTTCAGCGGGTACATGTCGGATGGAAGACACTACTCTACCAAGGGTGTGAACGGAAT1140 
GGCAGGAGACTGATGGCTCCGATGGATATCTCTTTCATGTTGAAAGAGCGTCGTCGTGAT1200 
GAAGCCTCAGAAGCACGGCAGC TTTGGCTCCGAGAGTTCGGAAAATTCACTTTCCATGCC1260 
GTTTTGGTCGAGATCTTCAGCTGTGGTAATTCGAGTCCTGGTGGCAAGGCATGGCAAACA1320 
CCCCATGATTTCGAGGCTTTCGGGATACTGAGGTTGGGATACGGCCGAGTTTCGTCCTAT1380 
TACAACGTGTTGTTTTCAACGATCCTGGACTGGATTATCAATGGCTACGAGGAGGACCAG1440 
CATCTTTCTATTGGTGGGGTTCAACTTTTGCAGGCTCTGATGCGCATTGAAATATTCCAG1500 
AAAAGCCATGCGAAAGCACGACTCTGTTTTGATCCCGTGCGTGGA ATAGCCAAGGAGGGC1560 
GGGAGATTGAAGGTATGCTTGAAACACGGTCATTCGCGTGTTTTTGACCAGGTCATCATT1620 
GGCGGCAGTGCTGAGGCCGCTACAGTTGATAACAGACTGGCCGGGGATGAGACTTCCTTC1680 
AGCTACAATATCGAACCCGCCG TCGGAAACTCGTCTGCCGCTGTCAATTCAGCACTCTTC1740 
ATGGTCACGAAGCAAAAGTTTTGGGTTAACTCCGGCATCCCAGCAGTGATATGGACCGAT1800 
GGGCTTGTCCGTGAGCTGTGTTGCATTGACATCGAATCGCCAGCTGGAGAGGGCCTTGTC1860 
GTTTTTCACTATGCTTTGGATGACTATCTATCCCGGCCGATCGAGCATCATGACAAGAAG1920 
GGACGGTGCTTGGAATTGGTCAGGGAGCTTGCTGCTGCCTTTCCTGAACTGGCTTGTCAC1980 
CTGGTCCCAGTCAACGAAGACTACGAACGATATGTCTTCGACGAC CACCTAACGGATGGT2040 
TTTAAGGGAGCTTTGTGGAGGGAAAATTCTCTGGAAAAAGGTCAGTATATCCAGGATCTG2100 
CCTGGGAATAATTTTCCTATTGGGGATCACGGGGGAGCCTATCTGATTGACCGTGACGAC2160 
TGCGTCACCGGAGCCTCGTTCG AGGAGCAGGTGAAGGCGGGCATCAAAGCGGCCTGCGCC2220 
GTCATCCGCAGCACCGGCGGGACGCTCTCTTCACTCCAACCGGTGGACTGGAATAAAAA2279 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 255 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (1..255) 
(D) OTHER INFORMATION: /label=ORF9 
/note="Sequence ORF9 corresponds with bases 6576 
through 6830 of Seq. ID No. 19. It is read 5'to 
3'from the complementary strand." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
TTCTCCCAAGCAATCCTGAGTAGCTGCGTGATGGATCTTCCATATCAGCGCCCACGTTTC60 
ACCCCGTTTGCCGTCACCCATCCACG TAGTGGAGTCAACCTGAACCGTGCAATTTCTCAG120 
GCCTTTGTCTGCTATGATCAGTTCTGCGAACGGCTCTTGCGATATCAGCAAAGCTGGACG180 
GATTGGGTGTTCGACCACGGATTTGCAGAAGCCATTGAAGACGTGGCGCTGGTGTTCCAG240 
GTT GCACCTTGCCTT255 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 297 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 1..297 
(D) OTHER INFORMATION: /label=ORF10 
/note="This sequence, designated ORF10, 
corresponds to bases 9748 through 10044 of 
Sequence ID No. 19. " 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GAATTAGCCGGACTAAACGTCGCCGGCATGGCCCAGACCTTCGGAGTATTATCGCTCGTC60 
TGTTCTAAGCTTGTTAGGCGTGCAAAGGCCAAGAGGAAGGCCAAACGGGTATCCCCGGGC120 
GAACGCGACCATCT TGCTGAGCCAGCCAATCTGAGCACCACTCCTTTGGCCATGACTTCC180 
CAAGCCCGACCGGGACGTTCAACGACCCGCGAGTTGCTGCGAAGGGACCCTTTGTCGCCG240 
GACGTGAAAATTCAGACCTACGGGATTAATACGCATTTCGAAACAAACCTACGGGAT 297 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 774 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (1..774) 
(D) OTHER INFORMATION: /label=ORF11 
/note="Sequence ORF11 corresponds to bases 10509 
through 11282 of Seq. ID No. 19. It is read 5'to 
3'on the complementary strand." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GGCTTCTTTCTTCAGGTT TACTGCAGCAGGCTTCATGACGCCCTCCTCGCCTTCCTGATC60 
AGGCCCCGAGAGTCGCAGGGTTAGGTCTGGCTCCGGTGAGGAGGCGGCCGGACGTGATAT120 
CCCGAGGGCATTTTTGGTGAATTGTGTGGTGCCGCAAGCTACAACATCATAGGGGCGGTT 180 
TTCAGTCCCTCGCCGCAGAAAGAAGGTGCAAGCTACCTCTCTCCCGTAAACGTTGGTCAC240 
TTTTAACTCCAGCAAGTGAATGAACAAGGAACTTGCGAAAATGGCGATGAAGCATTCTAA300 
ATCAGGTTCCTCCGTGCGGCTGTGCGGCCAAGCAAGGTTG TGAACACGGAGCATCTCCTG360 
GAGGGCGAGCTCGCTCCGATATGGTTGAATCGTTGTCGCCAGCACGGCCTCCATTCCAAA420 
TGTAATGGATTGTTCCTTCAGCACTTTCTGCATCTTCTCGCGAGAAAGATAGACAAATAC480 
ATGTTGGTCGTTTTCTCG AGCCAGATCCGGCTGACTAACAAACATAGGAGGATGATAGCA540 
GACTTTGTTCTTCAAGAGCTCAGCTAGTTGTTTAAGTATATATATCGGTGGAGAGTTTTC600 
CTTCAAATCTAGCACTGCAAGAGCCCATCGTTTCTGGAAATGCAGGAGGGGTTTGCTATA 660 
GTCACGGCTATAGATTGCAAAAGCAAATCGGATCCCCTCGAATAGGTTTATCTGGCTCCA720 
TGCTGGAGTGAGATCTACTGGTTGAAATCGTGGAAGGAATAGCAATTTGGGATC774 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 537 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 1..537 
(D) OTHER INFORMATION: /label=ORF12 
/note="Sequence ORF12 corresponds to bases 12466 
through 13002 of Seq. ID No. 19. " 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
GCTGAAGACGACCTGTGTTCTCTCTTTTTCAAGCTCAAAGTGGAGGATGTGACAAGCAGC60 
GATGAGCTAGCTAGACACATGAA GAACGCCTCAAATGAGCGTAAACCCTTGATCGAGCCG120 
GGTGAGAATCAATCGATGGATATTGACGAAGAAGGAGGGTCGGTGGGCCACGGGCTGCTG180 
TACCTCTACGTCGACTGCCCGACGATGATGCTCTGCTTCTATGGAGGGTCCTTGCCTTAC240 
AATTGGATGCAAGGCGCACTCCTCACCAACCTTCCCCCGTACCAGCATGATGTGACTCTC300 
GATGAGGTCAATAGAGGGCTCAGGCAAGCATCAGGTTTTTTCGGTTACGCGGATCCTATG360 
CGGAGCGCCTACTTCGCTGCATTTTCTTTCCCTGGGCGTGTCATCA AGCTGAATGAGCAG420 
ATGGAGCTAACTTCGACAAAGGGAAAGTGTCTGACATTCGACCTCTATGCCAGCACCCAG480 
CTTAGGTTCGAACCTGGTGAGTTGGTGAGGCATGGCGAGTGCAAGTTTGCAATCGGC537 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 597 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Stain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 1..597 
(D) OTHER INFORMATION: /label=ORF13 
/note="Sequence ORF13 corresponds to bases 13723 
through 14319 of Seq. ID No. 19." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
GCTCGTTATTGCAGTGGTGGCTCTCAACGGCTTCATGTCGATGATTTTCGTTGGATCAAG60 
GAGCCCA CTCGACTGAAGGCTCAGCTTATTAATGTGGTGGAGACCTACAAGGCTGCACAA120 
ACAGAGACGTTAAAGTACTATATATCATCTGCAACTGAGCGTGTGGCTCATGTGGAGGCA180 
GCCGAGGTCAACAATGCGGAAATGGAGCTGCATCCTGCTGGGTTGAAGTACC CTCTGTCC240 
TTCGTCTTTACCTCCCTGGCCGTGGCTACAGCCTGCAAGGAGAACAAGCATCTCTTGTGC300 
GAGGAGCATTTGGAGGGGGACTTGATATCGTGCGTCGTTCCTCCCTATCAGACAAATGTC360 
TCACTCGCTGCTTTAAGGGAGCTCCACAAT TCCATTTCGGGAGGAGGGTACCAGGAACAA420 
GCAGACATGGATTATTTTGTGGCGATCATCCCAAATGATAATTTCGACTATCAGAGCTGC480 
GAAATCGACACACGAAGTTGCGGTAAAGGACTTTGCAAGATTTATAGTAGGGAACTGGGA540 
GGGCAGC CTCTAGCTTATGACGCCATACTGGCAATCGGCAAGGTGCTGCTGCTGGAA597 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 552 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 1..552 
(D) OTHER INFORMATION: /label=ORF14 
/note="Sequence ORF14 corresponds to bases 15659 
through 16210 of Seq. ID No. 19." 
( xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GCAGATGAGTTGGAGCGTCAATTGGAAGCCATTTCTCTCATTACAGTCCTGGGTCCGGAT60 
GTGAAGGCTGAGCTTGAGGCGGAGCTACGAGACTACTGCGAAGATCTCGACTTCTGGAAA120 
AGCCACGGTTTACCGGTGGCGGATCTCGATCAGACT GTGACTGTCGACAAGCTTCTATAC180 
ATGTATATGGATCGGGCAACAGCAGACCTGTGTGTGAAGAATCGCTGCCTCGTTTGCAAC240 
AGTGGCAATTCAGCCGCAAAAGTAACCTCGCTTCCACCATACCTTGCAGGCGTGACAAGC300 
GCCGAGGCCTATG AGAAACTCAACTCCATTGTTGATGGGAGTGTCGCCCCCCAATCTCGT360 
GGGCCTCCCTGCTATTTTGTGGCGTTCCTGCCCAGCAGCTGTTTCGAGAAAACCAGTGAG420 
ATATCGGTGCGCACAGTGGACGGCGAGTGTGGCCCCTTCGATGTCTTTACCCGGCAGCG T480 
CAGCCACAGGATCAGAGTGATATGTTTTTTAAATATGAAGGAGTTGTATGTGCTGGAAAG540 
AGTGTATTTATG552 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1029 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (1..1029) 
(D) OTHER INFORMATION: /label=ORF15 
/note="Sequence ORF15 corresponds to bases 16517 
through 17545 of Seq. ID No. 19. It is read 5'to 
3'on the complementary strand." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
ATGCCCGTGTTCCATCGGGCCAGCGAGTTTATTCAAAAGAGTTTCGTACACGTGGGCGGC 60 
GACGGCAACGTCAATGCTTGCTAGCCCTACCGGCGAGAAGTTGGCCGGCCCCTTCCATGC120 
CTTGAGGTCATTCATCAAGGCCTCGTCATCGAGAATTTCGGTGTAGTTCTTGATCCCATC180 
GCGCTTGCCGTGTTGGGTCAGTTTCATACCGCGCCTAGAA TAGTAGAGGGCAACGGCATC240 
AACGTTGCGGGCTTCCATCGCAACAAGGTCATCGGCGACAATTAGACCATCCGCAGATAG300 
GACATGCTCAATGTAATCCGGCGGCATGTCATCAATACCGAGTGACAAAGTGACTGCGTT360 
GGGGGCGATTTCAGCGG CTTCGAATACCGGTTTTCCGTAGTTGGTCGCCATGATGACGAA420 
TTGAGAATATGGCAAAAGGCTACGATCGCCGACAGCTTCAAGGCTAAAGGTTACGCAATC480 
ACGTAACTTTTCGACGAGCTCGAAATTGGATTTCTTACCGCGGCTGAGCACTGCTACCTT 540 
ACGAATTCTCTTAGCGGCACCATAGTTAAGTGAGAGAATTACAGCTTCGGCAACTTTTCC600 
AGCCCCAAACAAGAAAACGTCGATGTCCTCTCTGCCTTGCAACAGCAGGTTTACGCATGC660 
TAGCGAGAACCAACCCGTTCTTCCATTAGAAATTGCCACG CCCTCTACCGACATAAGGAG720 
CGTCCCGGACACCTTGTCGCGCAGGAAAATATCGGAGTGCTGGAGCGGCTTTCCGGTAGC780 
GGCGTTGGTTGGCGCGAAGTGGATGTCTTTGGTGCCGGAATATCTTCCGAAATAGCCAAT840 
GAGTGCTCCTTCAGTCC ATCCAGGAACATTCTTGTTGAACGTTAGGTAAGCTTTGACATG900 
TCCGGCTTTTCCTGCGGCAAACACCTCCCAATAGGACTTGAGAGCTTCGTCAACAAATGC960 
TGGTGTGATCTGGATATCGAGGTTTGATAGTGCAGATTCAGTCCAGTGTACCTCGCAAAG 1020 
TTGTTTGGC1029 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 853 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi ) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (1..853) 
(D) OTHER INFORMATION: /label=ORF16 
/note="Sequence ORF16 corresponds to bases 17737 
through 18189 of Seq. ID No. 19. It is read 5'to 
3'on the complmentary strand." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
TATCTTCCGAAATAGCCAATGAGTGCTCCTTCAGTCCATCCAGGAACATTCTTGTTGAAC60 
GTTAGGTAAGCTTTGACATGTCCGGCTTTTCCTGCGGCAAACACCTCCCAATAGGACTTG120 
AGAGCTTCGTCAACAAATGCTGGTGTGATCTGGATATCGAGGTTTGATAGTGCAGATTCA180 
GTCCAGTGTACCTCGCAAAGTTGTTTGGCCATCTGCCTTGTAGGTGCGAATTTTCTCTGC240 
TCAAATTGTTGAGGTTAGCGGATTTGTAAACGCGTTTATATGG GCTGCTTGGAGGGTACT300 
TTTGGATTAATTTTTTTCTGCCAGCGCATTCTGACGCGGCACCGCTTTGGAAAGTGCGCT360 
GTGGGTCCGCGTTTTCTACAATAATGTGCCGATCCGGTCAGAAAGTATATGGATGAGTTG420 
TGCCAGCCTCACCAACGTGC TGCAGGCCCATCATGACTACTTCAATGTTAATGGGGGTAA480 
TGAATAAATAGGCGAAATTGGGTTCACGGTGGGCCCAGGGAATATAATATTGCCGCAGAG540 
GTAGTCGGATGCCAAGGCCCGCAACTAATAGTTCACGAACAAATTCATTGTAGTGGGCGG600 
CCAACTCCAAAACCAATTGCCAGTTATTGTATTGCAATACATATATGAGTATTCGGATAC660 
AACTAATTTCATTAAATAATATTTTAAGTGTGGACAGAATAGCGCCTAATAAATTTGCGA720 
ATGTTGTCCAATTGACGTTTTTATAGGTAACTCGATAAATCGT GCTTTTGTGATATTCTG780 
ATGCGGACAATATACATTTAAACATAAAGATATAAGTTATTGAGGCATTTATGTATATTA840 
CAATAGTGGGGTA853 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 567 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (1..567) 
(D) OTHER INFORMATION: /label=ORF17 
/note="Sequence ORF17 corresponds to bases 18177 
through 18743 of Seq. ID No. 19. It is read 5'to 
3'on the compementary strand." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
CAATAGTGGGGTACATTTTTCACAGATGCTGTCACCCATGAAATATT GGCAAAATACTCT60 
TAAAATATGCAAGAAACTAAAGAGGATGCATGGGTTGGGCTGTAGGTACATGGATGCAAA120 
TGCTGTTTTGCAATAAGTCATATAGTCTCGTCTGTTGAGTGAGGCCCATTCAATCAGCAA180 
GTAGGACTGAGGTGCATGATCGAC ATATTTTTGAACCACAGTTTTGGCAAGTTTTTCATA240 
CAAATGCACGGCTACGGCCAAATCGTAGCTTGCAAGTCCAACTGCTGAAAAGTTAGCCGG300 
CCCGTTCCAAGAAATTAGCCTTTGCATAAGGACTGGATCGCGGAGAACTTCAGAGTAGTT360 
C CTGATCCCATTGTCCCTGCCGTGTTTTGTTAGCTTTAAATGGCGTCTTGAATAGTGCAG420 
CGCCAACGAGTCGATATTACGTGTTTCCATCGCATCCATATCATCTGCCACCACGATGCC480 
ACTCAGCTTCAACACGTGATCAAAATAGTCAGCTGGCAATTCGTCAA TTCCAAGCGTCAA540 
TGTAACGGCATTGTCTGTGATCTCCTT567 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 360 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
( ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: Strain A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: Convolvulus arvensis plant cells 
(B) CLONE: Clone 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (1..360) 
(D) OTHER INFORMATION: /label=ORF18 
/note="Sequence ORF18 corresponds to bases 19031 
through 19390 of Seq. ID No. 19. It is read 5'to 
3'on the complementary strand." 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
GTGCCAGCCAACCAGTTCTCCTCTCCGATATAGCCACCCCATCAACAGAGAAGAGACGTC60 
TACCTGTGAAACGATTGCGAAGCCAACGTCGATGTGAGAAGTCGGTTCTT TGTATCTCGC120 
GTTTGACGGATTAGAATGGATGCTTTTCACACCCGAATAGTCGCCGACGAAACCCACCAG180 
AGCTCCCTCCGTACAGCCCTCTCGATCAAGTGGAACGAAGACCTTGTTGTGGCCGAGCCG240 
CCCTTCAGCAAAGAGGTGCCAATAATCT TTCAAGGCATCCGCGACGAGTTCCGGTGTAAT300 
GTATATTCCAAAAGCCGATAGAGATTCCTCTGTCCAACATTGCTCGTGTATTTGATCGGC360 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21126 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Agrobacterium rhizogenes 
(B) STRAIN: STRAIN A4 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: CONVOLVULUS ARVENSIS PLANT CELLS 
(B) CLONE: CLONE 7 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (937..2262) 
(D) OTHER INFORMATION: /label=ORF1SUBSEQUENCE 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (2649..3458) 
(D) OTHER INFORMATION: /label=ORF2SUBSEQUENCE 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 3726..4799 
(D) OTHER INFORMATION: /label=ORF3SUBSEQUENCE 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (4041..4400) 
(D) OTHER INFORMATION: /label=ORF4SUBSEQUENCE 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (4607..4918) 
(D) OTHER INFORMATION: /label=ORF5SUBSEQUENCE 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 5143..6216 
(D) OTHER INFORMATION: /label=ORF6SUBSEQUENCE 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (5071..5643) 
(D) OTHER INFORMATION: /label= ORF7SUBSEQUENCE 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 6609..8888 
(D) OTHER INFORMATION: /label=ORF8SUBSEQUENCE 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (6576..6830) 
(D) OTHER INFORMATION: /label=ORF9SUBSEQUENCE 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 9748..10044 
(D) OTHER INFORMATION: /label=ORF10SUBSEQUENC 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (10509..11282) 
(D) OTHER INFORMATION: /label=ORF11SUBSEQUENC 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 12466..13002 
(D) OTHER INFORMATION: /label=ORF12SUBSEQUENC 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 13723..14319 
(D) OTHER INFORMATION: /label=ORF13SUBSEQUENC 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 15659..16210 
(D) OTHER INFORMATION: /label=ORF14SUBSEQUENC 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (16517..17545) 
(D) OTHER INFORMATION: /label=ORF15SUBSEQUENC 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (17737..18189) 
(D) OTHER INFORMATION: /label=ORF16SUBSEQUENC 
(ix ) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (18177..18743) 
(D) OTHER INFORMATION: /label=ORF17SUBSEQUENC 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: complement (19031..19390) 
(D) OTHER INFORMATION: /label=ORF18SUBSEQUENC 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
GGCCGCAGGATTTCGTTCGTCGTGCGTGATGAGATCGATAAATGTTTATCGACG AGGACA60 
AGATCGACGATGCGGTTCTTGCGCTGTTGTAGTGACGCTCCACAACGAGTGTTGCGCCGT120 
GAAAGGCTTTGACTGGGCCGCGACGGACCGCCTTTGCAGGAAGGGTTCGGTCGGCGATCC180 
CGTCAATAAATCGAAGCTATTGATCCTGACG GATAAAGGTCTGCGTCGATCGGAGGAGCT240 
ATTCCGACAGCTGTTTACGCGCTAGCCATTGGCCGACGGTCTTTGCGCCCTCCATTCCCA300 
CGGCGTAGTTAATGCCGGCGGGGACGGGAGTGTCTACTATGTGCAAGCACGTCGGCGAAC360 
CATGCCTTC GGATTAATGTCGTTCAGACGGGCGGTCGTAAGTTGAATGAGTATGACTGCC420 
GCATGGTCAGCGCCGCGTTGGGAGCCGGCAGATGTCCAGTCGCGGCGCCTCAAGGCCATC480 
ACATGTTCACTCTGTGGCCAGAAGGCGTCGCTCCTTGGGTGGCAGGATATATTG TGATGT540 
AAACAGATTAGATATGGACATGCGAAGTCGTTTTAACGCATGCTTTATCGAATATAAAAT600 
GTAGATGGGCTAATGTGGTTTTACGTCATGTGAATAAAAGTTCAGCATTCGTTTAATAAT660 
ATTTCAATATCGGTGTCTAGAGACCCGTGGA TTTGTATAGTCAGCACCATGATATGAATC720 
TATAAAATATTGTATCTCCAATTGCAATTCAATCGATATAAGAAATTAATACAAGCCGTT780 
CATATAGTAAGGTTGCCAATGGCATTCAATAACGACCGTACAGTTGCCGCTATATTAATC840 
TACGTGCCA TTTCTTAAATAAAGATAGGCGAATGACTATCGAAAATAAAACAATTATTAA900 
TGAGTGAAAACGTATTGCACAAATAAAGATTCATTATGGTTGGCTCAAATTTTGGCTCTG960 
GTGCTCGATGACGTCGAGATGAGGACAGTAGTGATCAACTTGGCGGTCGATACC TTGGTT1020 
ACGCCACTCCCAGAGTGCCATGTCGTCCTCCGAGCGGTCTGAGATAACCCAGTCGGCAAT1080 
TGCTGCTGCATTGCCGGGCGTTCCCCAACCACGACGAATATGCTTTCGTTCATCTAACTC1140 
GCGTCGCACTGCCCTCCCAGTCATGAAGTCA AAGCCAAATTCTACCCTCTCTCCATTTCC1200 
CAGCTCAGTCGAGAAATCGTAACACCTCGTGGCAGCTGACAGTTTCAGAAAGGGGCGTAT1260 
CCCTCGAACTCCAGGGTCCTCTTTCACATAGTTAGCAAGGCGTACTGCTGCATAATCTGC1320 
GTTGAAGGC TCTGATGACTACAGGATCCTCGGACAAGCCCAATTGATCAGGGCGAACCCT1380 
CGCGCTCATAATATGAATTGCGACGACCCTTGCTTCCTGTCGGAGCATCGAATCAATCCA1440 
AGCCTTCCCTGCGGCATAGAGGTCATCGACTGCGATGTCATCAAGATCGAGTAG CTTTGC1500 
CAACCTAGGAAGTTCTTGAGGAAAAATCACCGGCATGACAGCAACCGTCTCTCGCCAGTC1560 
AGTTGCCGGACTGGCTTCCCTAACGCCATCCACGAATGCCTCACCGCTTGCGTATTTGAA1620 
TGTGTAAAAGAGAAGGACCACTCTTTGGCGG TACTTCGGACGCCGGCTTAGCCACGCGGC1680 
AATAATGTGGGCCTCAAACTCACGACCATCCAAAAATATAGTCGCGCCTGGATTGACCTC1740 
GCTGGCCTTGTCGAGAAGAGGTTCCAAAAAGGGAACGGTGTCTTTCGTAATAGTACTTAA1800 
ATCTGTGAG TTCGCCATGCGAAACCTCTCGAACGATTATCGGCGTATCCCTGACATCAGC1860 
TGAATGAAATTCTCGGACGAGTTTGTCGGGCAAAGTGGAGACCCGCCACGTGTTGAAGTC1920 
GTGGGAAACGATGGGCACATCGTCGCCGGTGAGTGCGGCATCGAGCTCAGAGAG GTTCCG1980 
CCTGCCAACCTCACCGAGAGCAGCTAACAACGAAGTTTCGGTGCATTCCTGTATCCCTTT2040 
ACCCAGATTATACATGCCCCGGTGTTCGATAACTTGAAGAGGCAGTGGCTCCTCAAGATG2100 
TTCAAGGAGGTGGGGTACAGAGTGCCGGGCG AGGACCTCATCCACCGTGACACCAACCGG2160 
GAGATCCCATTCGAGTTTCCACTGGGGCCAGCATGTGCCCGCGACGGCGAAAGGTTTGCG2220 
CTGGCAAAGAACCCGGCTGCTGCAGGTGGACCTATCCTTACCCATGGCAATGGGGTTTTG2280 
CTAAAAAGT CAGGCACTTTACTGGGCAATTGATAGGGTGGGATTGCGTTATTAACTGTTC2340 
TCCAGCGGGAATCTTTATCTTTATTGAAATGCTAAAGCACTTAGATAAAATACAGCTGTA2400 
CCGCAATATAAAATAGTAGGATAATGTAATATGTGTATCGAGAATACGACAAGC TAATAT2460 
AATCTAGCGTCAAATTGCAATAATTTAAATCAAAACTACTGATGAAATAATAAAAGATGG2520 
TCAATTTTTATTGGTAGGAGTTGTCGAAAGATTCGACGGACGGCCATTACAATACATAGG2580 
TGCAAGAAGTAAAACAGGAAGGGAAACGGAA AACAGTGCTATAAAAAAGCGACAGATCGC2640 
GGCGATCACTGACTGCGATCGGGAAGAAGCTCGCCAAGTTCACCGAGAATAGCAGAGAGC2700 
GCATCCTCATCGGGTACTACGAACACATTCGTCCCAGAGGGCTTTGTTTCAGCTGCGCCA2760 
ACCCAGAAA GCAAGGCCATTTTCCAAGTTGCCGATGGCGGTCAGCATGTTTTGATTGTTG2820 
CTGCCGTTTCCACAAGCGATGTGAAGGCCGATCCCGTGAGAGAGGCCCTTGACGAAGGTG2880 
AAATAGCCTTTGGATTTTCCAACTGTTTCAACGGGCACTAGATATTGACCCTCT GGCGCG2940 
GCAACCACCTTGAATTTGCGAGATGACTGGTTGCCGATGAGCGAAGAAAGCATTTCTCCG3000 
GCTTCTTTGTAAGATTTGTGAGATTCCCACATTTGACAGCCGTAGAAATGCCCCATCGGA3060 
ATGTTGCGGATTCCCGGGATGCCACCAAATT TGTTCTCCATAGCCGCGTGAACGGCTTGC3120 
CAGTTGGGCAGGGAGAAAGAATCGAAGCGATCATCTTTGTAGATCGTGACCATTCCATCA3180 
TTTCCCTGGAATCCGATATTTTCAATGGCGCTGAAAACTGACCTTGCGATTTCTTCGCAT3240 
TCCCGTGCG GATGTGAGCAATTGATAATGGCCCTTGCAGGCGATCCTGGTCAAATTGGCG3300 
ATGATGTTGATGGCAGGATTAATATCCCAACACTGGTGATTTCGATCTTGCTTAAAGGTG3360 
GTACCATCGCCGTCGAAGGCGAGCAGGGCCCGGAGAGATGAATCGGCAAGACTG CGTCGG3420 
ACCCGCTCCGCGGCGTCGGGAATGAGGCTGATAAGAGACATATCCAAAGGTGTTTGTGGG3480 
TAACGGGCTGCTCAATGAAGCCTTAAATGCAACGCAACATATGTAAGGATGAGTTGACTT3540 
ATTGGAGAGAGAAATAGGAATGAGCTGGCCA GCCATTATCAACGTGGGGCCATGCTGACA3600 
ATGTTTACGTGAAAGGCTCAACTACCTCGAAGCAGACCTCTATATTCGTTGACTTTATTA3660 
CTGAACAAGAAGTTGCTTGCCACTCATTTTCTTAAATCTTGCCCTTTCTGCGCCTCGCTA3720 
TCATGCCCG CCAACGACGCGACATGCGCTGCCGCGATTGCCTTCCCCGAGGGCAACTGGA3780 
AGGAAGAACTTGATGCGCTCCGCACCTTGTGTGACCCCGTCGAGGTGGTTAAGGTCGCAG3840 
TCGGCAGAGGTCTTAGCGGCATATGTAATGTTGTTGCAGCAATGAATCCCACAA AGGTGA3900 
GGGGCCTCGGCGATGTCATCGGGCAGATGCCGGCTCTTAATCACCGTATTGCTGCCGCCG3960 
CCGGCGAAACTCCGGTGCGAGACCTTGGAATAGGTTACCAGTGCGCAATCTGCCACCCCG4020 
ACATAGCCAGTGCGATGTTAGCCACTTCTGA GGGGATCAGCCACGTTCTCCGTGAAAGGA4080 
TTGAGAAAGAAGTTGACCGGGACATTGGAGAAGGCGCCACCGTCTGCATTTTCGTTCAGC4140 
CGAGAATGAGCTCCAAGGGCTCTCCAGTTTCTGTCCATTTCACCCTCCAGTTTGCGAGAT4200 
CTGGAACTC TTGTCGATGCCAGAATGATGGAGAGTTACAATTTCATGAAAGGCAATGGCA4260 
CAGTGACCGCACCGGATTTGAAAAGTCATTGGAAGAAGCACGGTATTGACAGGCCAGGCC4320 
CACGTCCGCCCACGTCCAAGTTTGAACTCCTCTTCGCCGCTGTCCCCGACAACA GTAAAC4380 
TTGCCGCCACCGATTTTACCCATCTCGGCCCTGTCGAGCGTGATAAGGAACTACTCGGCA4440 
GCACGGTATTCGGGATTGCCGCTAAGAAACCTGGTACGATCGTTTATCCGTGCGAAAAGG4500 
TTCTCTGTTTGGAGGTCGACGTACACGCGCA TCGCGCCCTAGAAGTACTTCACCGCCTTG4560 
GGGAACAGGCTTATAGCAATGGCCGTGGCACTAGCTTCGGTCTTCACACCGGTCCGTCCT4620 
CTTGCCTTAATCTTTCCGCCGCCGCGCTCGCTACATTTTTCAAACGCTCGGATCTCTGTT4680 
CCCTTCCAT TGAGTGATGCTTTTGTCCTTTTCTGCGACCCGCCACCGCCTACAGCGCCAA4740 
GAAAGATGGCCTTCCGATCACTGCCTTCTCCCCCACGAGCACCAATCAGTTCGAACTCGT4800 
AGAGCCTCAGGTCGTCAAGGCATATGTTCTCGGACTTTTCGACGCGCCGACGAT GGTTAC4860 
GCCCCGCGACAAAACGCGAGCCAGCTTCTGCAGCCAATATGTACGTTTCCGTGAACCGCA4920 
TCCCTGTGAAGAGTTCAATGAAATTGGAGTTTTGATCCTCGATGCTGCTGCTAAAATGCT4980 
CGAACGTTATGCAAAATTTCTAGAAGATGGT GGAAGAGATGATGATGAAATGGCGAACAT5040 
AATAGATGTATTTGGGTTTTGTCTTAACTAGTGGATTGATTGAAACAAAGGAGTCCGAGT5100 
TGGGATTCCCTTTCGGTCTTCGTCGTGCAACGATATCGTATGCGTACAGGTATCACATTT5160 
AACGTTGCT GCGGCGGACCGAGCCCGCTTGGAAGCGATTGTTGCAGCTCCAACTTCTGCT5220 
CAGAAGCACGTGTGGCGAGCGAAGATCATCTTGATGAGCAGTGATGGCTCGGGAACGGTC5280 
GCGATCATGGAGGCAACCGGTAAATCCAAAACCTGTGTCTGGCGCTGGCAGGAG CGCTTC5340 
ATGACTGAGGGCGTCGATGGCCTTTTGCACGACAAGAGCAGACCGCCCGGCATTGCGCCG5400 
CTTGATGGCGAACTCGTTGAGCGTGTCGTCGCACTGACGCTTGAGACGCCTCAACAGGAA5460 
GCAACGCACTGGACTGTTCGTGCGATGGCCA AGGCCGTTGGGATTGCAGCCTCTTCGGTT5520 
GTGAAGATCTGGCACGAGCATGGTCTTGCGCCGCATCGCTGGCGCTCTTTCAAACTGTCG5580 
AACGACAAGGCCTTTGCCGAGAAGCTTCACGACGTCGTTGGCCTCTACGTCTCGCCACCG5640 
GCCCATGCC ATTGTCCTGTCCGTCGATGAGAAGAGCCAGATCCAGGCACTCGATCGGACG5700 
CAACCGGGACTCCCCTTGAAGAAAGGGCGCGCCGGCACAATGACCCACGATTACAAGCGC5760 
CACGGCACCACCACCCTATTTGCCGCCCTCAACATCCTCGACGGCTCGGTGATC GGCCGA5820 
AACATGCAGCGTCACCGGCATCAGGAGTTCATCCGTTTTCTCAACGCCATCGAGGCGGAA5880 
CTGCCAAAGGACAAGGCCGTCCACGTCATTCTCGACAATTACGCGACCCATAAGCAGCCG5940 
AAGGTCCGCGCCTGGCTGGCAAGGCATCCGC GCTGGACCTTCCACTTCGTCCCAACATCA6000 
TGTTCATGGCTGAACGCCGTCGAGGGATTCTTCGCTAAATTGACACGTCGACGTCTGAAG6060 
CACGGTGTCTTTCATTCCGTCGTTGACCTCCAGGCCACCATCAACCGCTTCGTCAGAGAG6120 
CATAATCAG GAACCAAAGCCGTTCATCTGGAGAGCAGATCCAGACGAGATCATTGCAGCC6180 
GTCAAACGTGGGCACCAAGCGTTGGAATCAATCCACTAGCGTATGAACAGTAATAAGAAA6240 
ATCCCGATTGTGAATAGTCCCAATTTCAAATGTGTCCGTGTGTAATTTGCGTGT CTTCAG6300 
TTGAATTTCCTTTAATAATATCAAATATTCAATTGTGAAAAGTTGTATTGGTTCAGGTTC6360 
AAGCTTTCCGAATTTGTTGAATTTTATTCCCTGTTTTCAATTTGTTGACTTGTTTGGGAG6420 
ACACCTTTTTTGTGTTTCGTGAACATGTCAC CCCTTCGGTATACATTAGCCTACAAAGTA6480 
AATAACGTTGATAAATGTCACTCATGTTGTAATAAAATTGAGCTTATTATGTATAACCAG6540 
ACCCTGTGTTAATCTAATTACAAAGAAATTCATCATTCTCCCAAGCAATCCTGAGTAGCT6600 
GCGTGATGG ATCTTCCATATCAGCGCCCACGTTTCACCCCGTTTGCCGTCACCCATCCAC6660 
GTAGTGGAGTCAACCTGAACCGTGCAATTTCTCAGGCCTTTGTCTGCTATGATCAGTTCT6720 
GCGAACGGCTCTTGCGATATCAGCAAAGCTGGACGGATTGGGTGTTCGACCACG GATTTG6780 
CAGAAGCCATTGAAGACGTGGCGCTGGTGTTCCAGGTTGCACCTTGCCTTCATGGCCCCC6840 
GAATAGGCGCGCTCGAAGTGTTGATACCTCGTCGCACCCAGGTCTTCATTTATATGTCGA6900 
ACAACCAATTGCAGCGCTTTGTTGCACACCA GTGCATTGCTCAACTTGGCGACGCCGTGC6960 
TTGCTTGCATGATCCCGCCCTACGCGAGTGACCTCTCGCTGCAGGAAATGGCTCGGGCGC7020 
ACAACAGATTTTGCCCAGGCAGTTACACGAGGTCCGCAGACGTACAGTGCTTTATCGCCA7080 
TCCAACTCA GCAGCCGATTCGTTGAGGAGGGCACATGTAACGTGCACGGGCGAAATGGCT7140 
TAAAAAGAACCTGCCGCTTCTTTCGTCGCCCTGCTGAGTTCTTCAGCCGTTATGACATCG7200 
TTGCCATTGGGCCGGTGCTCTTCCATGATGAACTGGATTGCCCAGCAAACTGCA ATGAGC7260 
CTCTTTCCTGCTTTGACCTGCGGTACGACTATCAGGTTTTCCTCCAGGAGTGCGATGCCC7320 
ATGATGGTGTGGGGCATTATCCGGAAGGCGCACCACTACCTAGTGTTGCCATCGTAGGAG7380 
GCGGGCTGTCTGGCCTTGTTGCTGCCACAGA ACTACTTGGCGCTGGCGTCAAGGAAATCA7440 
CTCTTTTCGATACCGTTGATGAGATCCGTAGTTTTGGGGCATCGCCGATGCCAAACGGCG7500 
ACGCTCACCAGGCCTTGACGTCGTTCGGTGTCATGCCTTTCTCCGCCAACCAACTTTGCC7560 
TGTCATACT ATCTGGATAAGTTTAGAATTCCGTCCAGCCTTCGTTTTCCTTGTGCCGGCA7620 
ACGACCACACAGCACTATATTTCCGCCAGAAACGCTACGCATGGCACGCGGGGCAAGCTC7680 
CGCCGGGGATATTTCAGCGGGTACATGTCGGATGGAAGACACTACTCTACCAAG GGTGTG7740 
AACGGAATGGCAGGAGACTGATGGCTCCGATGGATATCTCTTTCATGTTGAAAGAGCGTC7800 
GTCGTGATGAAGCCTCAGAAGCACGGCAGCTTTGGCTCCGAGAGTTCGGAAAATTCACTT7860 
TCCATGCCGTTTTGGTCGAGATCTTCAGCTG TGGTAATTCGAGTCCTGGTGGCAAGGCAT7920 
GGCAAACACCCCATGATTTCGAGGCTTTCGGGATACTGAGGTTGGGATACGGCCGAGTTT7980 
CGTCCTATTACAACGTGTTGTTTTCAACGATCCTGGACTGGATTATCAATGGCTACGAGG8040 
AGGACCAGC ATCTTTCTATTGGTGGGGTTCAACTTTTGCAGGCTCTGATGCGCATTGAAA8100 
TATTCCAGAAAAGCCATGCGAAAGCACGACTCTGTTTTGATCCCGTGCGTGGAATAGCCA8160 
AGGAGGGCGGGAGATTGAAGGTATGCTTGAAACACGGTCATTCGCGTGTTTTTG ACCAGG8220 
TCATCATTGGCGGCAGTGCTGAGGCCGCTACAGTTGATAACAGACTGGCCGGGGATGAGA8280 
CTTCCTTCAGCTACAATATCGAACCCGCCGTCGGAAACTCGTCTGCCGCTGTCAATTCAG8340 
CACTCTTCATGGTCACGAAGCAAAAGTTTTG GGTTAACTCCGGCATCCCAGCAGTGATAT8400 
GGACCGATGGGCTTGTCCGTGAGCTGTGTTGCATTGACATCGAATCGCCAGCTGGAGAGG8460 
GCCTTGTCGTTTTTCACTATGCTTTGGATGACTATCTATCCCGGCCGATCGAGCATCATG8520 
ACAAGAAGG GACGGTGCTTGGAATTGGTCAGGGAGCTTGCTGCTGCCTTTCCTGAACTGG8580 
CTTGTCACCTGGTCCCAGTCAACGAAGACTACGAACGATATGTCTTCGACGACCACCTAA8640 
CGGATGGTTTTAAGGGAGCTTTGTGGAGGGAAAATTCTCTGGAAAAAGGTCAGT ATATCC8700 
AGGATCTGCCTGGGAATAATTTTCCTATTGGGGATCACGGGGGAGCCTATCTGATTGACC8760 
GTGACGACTGCGTCACCGGAGCCTCGTTCGAGGAGCAGGTGAAGGCGGGCATCAAAGCGG8820 
CCTGCGCCGTCATCCGCAGCACCGGCGGGAC GCTCTCTTCACTCCAACCGGTGGACTGGA8880 
ATAAAAAATAGAAATTTCCTGATTAAGTTATAGTCAATGTACTATTGCGTGTTAATCCCG8940 
TAGGTATGCAAGCTGCACCGGCAGCATCATAATTTGATGTTCCATCAATAAATTAAGGTG9000 
CCCGTTCAT TGTGTATTACATTATGTATGTTTATCAAAAATATAATCGAAGTCCATTTTA9060 
AGTCTGATATTAATTGGAATTCCAAACGATTCCTTGATGCCTATCTTCGCTATGATTGTA9120 
TGGTAATAAAGTCTCCACATCTCCCGAAAAATGCTTTCGTGATTTACTTGTCTC TCACGT9180 
GCTTTCGCATCTTGACAGCCAAAAGTGGGCAACTTGAGAAGAGTATTAACTGGCCACGCA9240 
ACTCGAGATATTCCCACTAACCCCAATGACGTCATTGCACTCGTCACGGGTAGCAGCCCC9300 
ACTTGCCTTTGCCACTTTATTAATTCTTTGG CCCACTGGCCATTAATTGGCACCTACATA9360 
TATTAGTGGAGAAGATAAAGTGTCACTATCGTTTCCTGTTCAATTTTGAATTTTGCAAGG9420 
ATTTCATGTTGTCAACTACACAGCTTGAAAGGAAATCCGCAATCAACGGAGAAACGTCAA9480 
CATCTCGAC AAAAAAAGAATGCTTCATCATTGCGTAGACTGCATATTGACCGCTCCTTTC9540 
GGCGCTGGGCCTGCTTTTACTGTTGCCTAGCGTTCGGACAGCCACCAGAGAATGGGCTAT9600 
ATAGATCCTTTCATCAAACCAAAACATTACTAAGATCATGCTGTAACGCTTCAA TACGGT9660 
GAGTGTGGTTGTAGGTTCAATTATTACTATTTTTGAAGCTGTGTATTTCCCTTTTTCTAA9720 
TATGCACCTATTTCATGTTTCAGAATGGAATTAGCCGGACTAAACGTCGCCGGCATGGCC9780 
CAGACCTTCGGAGTATTATCGCTCGTCTGTT CTAAGCTTGTTAGGCGTGCAAAGGCCAAG9840 
AGGAAGGCCAAACGGGTATCCCCGGGCGAACGCGACCATCTTGCTGAGCCAGCCAATCTG9900 
AGCACCACTCCTTTGGCCATGACTTCCCAAGCCCGACCGGGACGTTCAACGACCCGCGAG9960 
TTGCTGCGA AGGGACCCTTTGTCGCCGGACGTGAAAATTCAGACCTACGGGATTAATACG10020 
CATTTCGAAACAAACCTACGGGATTAATACGCACGTGGCTGGCGGTCTTCGATTCATTTC10080 
CACGCCGGAGATGATATCGAATATGTTCTGTTAAGTTAAAATAAGCTGCGAGCC ATGGCG10140 
CGATTGTCCTGTTTTATTAATATAGTACTTTAACGTCTCTTTAGAGCGTTTGTGTAATGT10200 
CGTGAAAATGTTTTATGTCAAATGTACTGTTGAACTATAATATTATAAGTCCAGGTGTGT10260 
CGTTGTTGTTGATACTGCAATATATGTGTAG TAGATTAGATAGTCATATGAGCATGTGCT10320 
GTTTTTGGCAAAATTCAGCAGCAGGATCAACACAGAAGAAAATATTTAGTACAAGAAAAT10380 
AGGTCAACACATTACAACGTACGCTACAACTCCCAAGGTTCTGTGTCACAGACTGCGGGA10440 
GGGTACATA GAACTTATGACAAACTCATAGATAAAGGTTGCCTGCAGGGGGAGTTCAAGT10500 
CGGCTTTAGGCTTCTTTCTTCAGGTTTACTGCAGCAGGCTTCATGACGCCCTCCTCGCCT10560 
TCCTGATCAGGCCCCGAGAGTCGCAGGGTTAGGTCTGGCTCCGGTGAGGAGGCG GCCGGA10620 
CGTGATATCCCGAGGGCATTTTTGGTGAATTGTGTGGTGCCGCAAGCTACAACATCATAG10680 
GGGCGGTTTTCAGTCCCTCGCCGCAGAAAGAAGGTGCAAGCTACCTCTCTCCCGTAAACG10740 
TTGGTCACTTTTAACTCCAGCAAGTGAATGA ACAAGGAACTTGCGAAAATGGCGATGAAG10800 
CATTCTAAATCAGGTTCCTCCGTGCGGCTGTGCGGCCAAGCAAGGTTGTGAACACGGAGC10860 
ATCTCCTGGAGGGCGAGCTCGCTCCGATATGGTTGAATCGTTGTCGCCAGCACGGCCTCC10920 
ATTCCAAAT GTAATGGATTGTTCCTTCAGCACTTTCTGCATCTTCTCGCGAGAAAGATAG10980 
ACAAATACATGTTGGTCGTTTTCTCGAGCCAGATCCGGCTGACTAACAAACATAGGAGGA11040 
TGATAGCAGACTTTGTTCTTCAAGAGCTCAGCTAGTTGTTTAAGTATATATATC GGTGGA11100 
GAGTTTTCCTTCAAATCTAGCACTGCAAGAGCCCATCGTTTCTGGAAATGCAGGAGGGGT11160 
TTGCTATAGTCACGGCTATAGATTGCAAAAGCAAATCGGATCCCCTCGAATAGGTTTATC11220 
TGGCTCCATGCTGGAGTGAGATCTACTGGTT GAAATCGTGGAAGGAATAGCAATTTGGGA11280 
TCCATTGTGATGTGAGTTGGATAGTTACGAAAAAGGCAAGTGCCAGGGCCATTTAAAATA11340 
CGGCGTCGGAAACTGGCGCCAATCAGACACAGTCTCTGGTCGGGAAAGCCAGAGGTAGTT11400 
TGGCAACAA TCACATCAAGATCGATGCGCAAGACACGGGAGGCCTTAAAATCTGGATCAA11460 
GCGAAAATACTGCATGCGTGATCGTTCATGGGTTCATAGTACTGGGTTTGCTTTTTCTTG11520 
TCGTGTTGTTTGGCCTTAGCGAAAGGATGTCAAAAAAGGATGCCCATAATTGGG AGGAGT11580 
GGGGTAAAGCTTAAAGTTGGCCCGCTATTGGATTTCGCGAAAGCGGCATTGGCAAACGTG11640 
AAGATTGCTGCATTCAAGATACTTTTTCTATTTTCTGGTTAAGATGTAAAGTATTGCCAC11700 
AATCATATTAATTACTAACATTGTATATGTA ATATAGTGCGGAAATTATCTATGCCAAAA11760 
TGATGTATTAATAATAGCAATAATAATATGTGTTAATCTTTTTCAATCGGGAATACGTTT11820 
AAGCGATTATCGTGTTGAATAAATTATTCCAAAAGGAAATACATGGTTTTGGAGAACCTG11880 
CTATAGATA TATGCCAAATTTACACTAGTTTAGTGGGTGCAAAACTATTATCTCTGTTTC11940 
TGAGTTTAATAAAAAATAAATAAGCAGGGCGAATAGCAGTTAGCCTAAGAAGGAATGGTG12000 
GCCATGTACGTGCTTTTAAGAGACCCTATAATAAATTGCCAGCTGTGTTGCTTT GGTGCC12060 
GACAGGCCTAACGTGGGGTTTAGCTTGACAAAGTAGCGCCTTTCCGCAGCATAAATAAAG12120 
GTAGGCGGGTGCGTCCCATTATTAAAGGAAAAAGCAAAAGCTGAGATTCCATAGACCACA12180 
AACCACCATTATTGGAGGACAGAACCTATTC CCTCACGTGGGTCGCTAGCTTTAAACCTA12240 
ATAAGTAAAAACAATTAAAAGCAGGCAGGTGTCCCTTCTATATTCGCACAACGAGGCGAC12300 
GTGGAGCATCGACAGCCGCATCCATTAATTAATAAATTTGTGGACCTATACCTAACTCAA12360 
ATATTTTTA TTATTTGCTCCAATACGCTAAGAGCTCTGGATTATAAATAGTTTGGATGCT12420 
TCGAGTTATGGGTACAAGCAACCTGTTTCCTACTTTGTTAACATGGCTGAAGACGACCTG12480 
TGTTCTCTCTTTTTCAAGCTCAAAGTGGAGGATGTGACAAGCAGCGATGAGCTA GCTAGA12540 
CACATGAAGAACGCCTCAAATGAGCGTAAACCCTTGATCGAGCCGGGTGAGAATCAATCG12600 
ATGGATATTGACGAAGAAGGAGGGTCGGTGGGCCACGGGCTGCTGTACCTCTACGTCGAC12660 
TGCCCGACGATGATGCTCTGCTTCTATGGAG GGTCCTTGCCTTACAATTGGATGCAAGGC12720 
GCACTCCTCACCAACCTTCCCCCGTACCAGCATGATGTGACTCTCGATGAGGTCAATAGA12780 
GGGCTCAGGCAAGCATCAGGTTTTTTCGGTTACGCGGATCCTATGCGGAGCGCCTACTTC12840 
GCTGCATTT TCTTTCCCTGGGCGTGTCATCAAGCTGAATGAGCAGATGGAGCTAACTTCG12900 
ACAAAGGGAAAGTGTCTGACATTCGACCTCTATGCCAGCACCCAGCTTAGGTTCGAACCT12960 
GGTGAGTTGGTGAGGCATGGCGAGTGCAAGTTTGCAATCGGCTAATGGTTAGTC GATGGG13020 
CTGACGAGTTTGATGTCAGGAGAAGCTGAGTGTGTCACTTGTTTCCCTTTAAGAAGTATT13080 
AATGTAATAAAAATCAAGATCTGGTTTAATAACTGGATACTTGATTTCATCGCGCTTTTT13140 
TTGAATAAATGTTTGTTGTCTTGACTTTAAG ATATCCTTTGAAATTTGCGTTATTCGTAT13200 
TTCGCTTTTGGTTATTTCCAAAAGACTTTGCTCAGTAAGATCAAACGTTTGTATTTCTCC13260 
GGGCCACAATATTTGACCTATATGCACTGGCCCACGCGCCGCAATAGATGAAAATTGCCA13320 
AAATTAGCT ATCGGTCTTCTGAAAAGAAGGGCCGACATGTTTTCATAGACCATGCAAAGT13380 
CATACTACCTGAAACTGATAAATAACGACAAAGAAAGTAGCCTATTTAAAAGTCGCTATA13440 
GCATGAATTCAACACAAGGAAACCAAAAGTCGGAAGGAAGACTTTAATCCCGGA TTATTT13500 
GGACATGATAGGAGCTATGGGGCAACGTGTCATTTTCATGAGTGTTGAATGATTTTCTGT13560 
AGCAAATAGAAAACGTTTTTTAAAACGATGTGGCCTTGGAGTAATCAGCGGAAGAAATGG13620 
TCATGCTCAGATAATTTCCGTTGCTGACCTC GCAACCAACCCCTTTAAATACCTCTGCTG13680 
CCCATGCATTTTGCCAAGTTAACCTAAAGTGGCAGCTGAATGGCTCGTTATTGCAGTGGT13740 
GGCTCTCAACGGCTTCATGTCGATGATTTTCGTTGGATCAAGGAGCCCACTCGACTGAAG13800 
GCTCAGCTT ATTAATGTGGTGGAGACCTACAAGGCTGCACAAACAGAGACGTTAAAGTAC13860 
TATATATCATCTGCAACTGAGCGTGTGGCTCATGTGGAGGCAGCCGAGGTCAACAATGCG13920 
GAAATGGAGCTGCATCCTGCTGGGTTGAAGTACCCTCTGTCCTTCGTCTTTACC TCCCTG13980 
GCCGTGGCTACAGCCTGCAAGGAGAACAAGCATCTCTTGTGCGAGGAGCATTTGGAGGGG14040 
GACTTGATATCGTGCGTCGTTCCTCCCTATCAGACAAATGTCTCACTCGCTGCTTTAAGG14100 
GAGCTCCACAATTCCATTTCGGGAGGAGGGT ACCAGGAACAAGCAGACATGGATTATTTT14160 
GTGGCGATCATCCCAAATGATAATTTCGACTATCAGAGCTGCGAAATCGACACACGAAGT14220 
TGCGGTAAAGGACTTTGCAAGATTTATAGTAGGGAACTGGGAGGGCAGCCTCTAGCTTAT14280 
GACGCCATA CTGGCAATCGGCAAGGTGCTGCTGCTGGAATAGATAGTGGGCCGCTGATCC14340 
GAGTTTGATTTTGTCGTATTATGTTACGTGAACTTTTTATCATGCATGTTTCGCTTATGC14400 
TCCCGAGTGTCGGCCATGTTGTTGTGTTAAAATAAAAGGCTGATGTTAAGTCCT ATTGTA14460 
AAATACCTTTATAGATTAAATATATATAGTATAACTTCTGTATGCCGTCGATGAGCGGTT14520 
ATATGATTGTAATCTATACGTTGTTGCAATCAATCGTATTACAGTGAGCCGTGCTTAATG14580 
GGGGAAACGTAATAACATTGCGGTGGATACA GCGTTTATTGGGAGGTCCGCGGGCCGATA14640 
CACTTAAATAACATAGACAGAATTTGAGAGAGCACGCAGGTTGTAGCCAAGTTGAGCGAC14700 
TTGCCGGTAGCACGGAAGCTAAGCTCAGGTGTTACAAATAGACAGGCGTCGAGGCGACGA14760 
TTGCCGGTA GCACGGAAGCTAAGCTCAGGTGTTACAAATAGACAGGCGTCGAGGCGACGA14820 
GCACGACGACCTTGCCGGACATTGCGGTCGCAGGGGGCTCAAAGCGGTTGGCTTGTAACG14880 
GACCTTGTGTTTCTTGTTGTAGCTTTCATCGAGCATAACCATTGGGACGGTTGC TGAACA14940 
ACGGTAACGCACTTTTTTCACGGGAGCGAGGTAGAAGAACATATTTCCCCGTCGGCAGCC15000 
GGCGGTGAGCATGCCAATTCCTAAGGGATCAATGGACTCGTGCGAACGGTGAGCATGCCG15060 
TTCTGACCGTCGGTGCCCAATCAGCAGGCCA CTCCCAACATGTTTTCCAAGTCCTTAAAA15120 
CCAGTCTTTATAGCATTGATCTCCCAGCAATCTTTATTGAAGTCGATTTTAATATTCAAA15180 
AGAAGATTTTAGTGGAAAGGGAATATAATCGCGTGGCCGAAGAAGAGCCTTCAAAAATCA15240 
GAATCCACT AGGATAAACAATAATATCTGAAAAGCATTGAATTTGGGTTAGGCACGAGAG15300 
GCTGACGCGGATGCCACTCGATTGCTAGTGGAAGGATTCCCTTTTTTCTAGCGTATCGAA15360 
TTCACCGTTTCACTATATGTTTTCCTGATTGGTTGATCTGCGGGACCACCATTG ACTGCC15420 
ACTAATATCGAAAGTGGGTCTGCTTCGATTATGATGCTTTGTGAGAGGTTCTCTTCCCAA15480 
TGCATGCAAGCTGGCAGATTCGGATACTCTCAATAGAGATCTTATTTCGCGTCTCAAAAA15540 
GTTCCCAGAAATCAACAAAGGGGAGGGCAGG TCCTTTAAATACGTTGCAGCTGTCCTTTA15600 
AAATAGAAGAGAATTTACAGCTGGAGGCACAGACCACTAAACTGCGAAAGTAAGCATGGC15660 
AGATGAGTTGGAGCGTCAATTGGAAGCCATTTCTCTCATTACAGTCCTGGGTCCGGATGT15720 
GAAGGCTGA GCTTGAGGCGGAGCTACGAGACTACTGCGAAGATCTCGACTTCTGGAAAAG15780 
CCACGGTTTACCGGTGGCGGATCTCGATCAGACTGTGACTGTCGACAAGCTTCTATACAT15840 
GTATATGGATCGGGCAACAGCAGACCTGTGTGTGAAGAATCGCTGCCTCGTTTG CAACAG15900 
TGGCAATTCAGCCGCAAAAGTAACCTCGCTTCCACCATACCTTGCAGGCGTGACAAGCGC15960 
CGAGGCCTATGAGAAACTCAACTCCATTGTTGATGGGAGTGTCGCCCCCCAATCTCGTGG16020 
GCCTCCCTGCTATTTTGTGGCGTTCCTGCCC AGCAGCTGTTTCGAGAAAACCAGTGAGAT16080 
ATCGGTGCGCACAGTGGACGGCGAGTGTGGCCCCTTCGATGTCTTTACCCGGCAGCGTCA16140 
GCCACAGGATCAGAGTGATATGTTTTTTAAATATGAAGGAGTTGTATGTGCTGGAAAGAG16200 
TGTATTTAT GTAAGAATTATCTTTTATAGCCTGTGTTACGTTTGAACCCGGTCCGCGCGG16260 
TATTGTTTTCAATAAATGGTATGTGCGGAGGATATAATTGGTCTTTCATTGGTGTGATTT16320 
ACGTGTAACGCGGATAATAATAAAGTAAATTACAAAAGAGAAACGCATAATTTT ATTCCA16380 
GAATGATTGCGAGAAACGATGAAAATACATGAAAATGCATATTGTCGCCAGGGAAGGATG16440 
GCGCCGAAATAAACGAAACTGAGCCAATACAGTGACTTGCCAAGCGAGTTTGATCCTACC16500 
AAATTCGCGCAAATTAATGCCCGTGTTCCAT CGGGCCAGCGAGTTTATTCAAAAGAGTTT16560 
CGTACACGTGGGCGGCGACGGCAACGTCAATGCTTGCTAGCCCTACCGGCGAGAAGTTGG16620 
CCGGCCCCTTCCATGCCTTGAGGTCATTCATCAAGGCCTCGTCATCGAGAATTTCGGTGT16680 
AGTTCTTGA TCCCATCGCGCTTGCCGTGTTGGGTCAGTTTCATACCGCGCCTAGAATAGT16740 
AGAGGGCAACGGCATCAACGTTGCGGGCTTCCATCGCAACAAGGTCATCGGCGACAATTA16800 
GACCATCCGCAGATAGGACATGCTCAATGTAATCCGGCGGCATGTCATCAATAC CGAGTG16860 
ACAAAGTGACTGCGTTGGGGGCGATTTCAGCGGCTTCGAATACCGGTTTTCCGTAGTTGG16920 
TCGCCATGATGACGAATTGAGAATATGGCAAAAGGCTACGATCGCCGACAGCTTCAAGGC16980 
TAAAGGTTACGCAATCACGTAACTTTTCGAC GAGCTCGAAATTGGATTTCTTACCGCGGC17040 
TGAGCACTGCTACCTTACGAATTCTCTTAGCGGCACCATAGTTAAGTGAGAGAATTACAG17100 
CTTCGGCAACTTTTCCAGCCCCAAACAAGAAAACGTCGATGTCCTCTCTGCCTTGCAACA17160 
GCAGGTTTA CGCATGCTAGCGAGAACCAACCCGTTCTTCCATTAGAAATTGCCACGCCCT17220 
CTACCGACATAAGGAGCGTCCCGGACACCTTGTCGCGCAGGAAAATATCGGAGTGCTGGA17280 
GCGGCTTTCCGGTAGCGGCGTTGGTTGGCGCGAAGTGGATGTCTTTGGTGCCGG AATATC17340 
TTCCGAAATAGCCAATGAGTGCTCCTTCAGTCCATCCAGGAACATTCTTGTTGAACGTTA17400 
GGTAAGCTTTGACATGTCCGGCTTTTCCTGCGGCAAACACCTCCCAATAGGACTTGAGAG17460 
CTTCGTCAACAAATGCTGGTGTGATCTGGAT ATCGAGGTTTGATAGTGCAGATTCAGTCC17520 
AGTGTACCTCGCAAAGTTGTTTGGCCATCTGCCTTGTAGGTGCGAATTTTCTCTGCTCAA17580 
ATTGTTGAGGTTAGCGGATTTGTAAACGCGTTTATATGGGCTGCTTGGAGGGTACTTTTG17640 
GATTAATTT TTTTCTGCCAGCGCATTCTGACGCGGCACCGCTTTGGAAAGTGCGCTGTGG17700 
GTCCGCGTTTTCTACAATAATGTGCCGATCCGGTCAGAAAGTATATGGATGAGTTGTGCC17760 
AGCCTCACCAACGTGCTGCAGGCCCATCATGACTACTTCAATGTTAATGGGGGT AATGAA17820 
TAAATAGGCGAAATTGGGTTCACGGTGGGCCCAGGGAATATAATATTGCCGCAGAGGTAG17880 
TCGGATGCCAAGGCCCGCAACTAATAGTTCACGAACAAATTCATTGTAGTGGGCGGCCAA17940 
CTCCAAAACCAATTGCCAGTTATTGTATTGC AATACATATATGAGTATTCGGATACAACT18000 
AATTTCATTAAATAATATTTTAAGTGTGGACAGAATAGCGCCTAATAAATTTGCGAATGT18060 
TGTCCAATTGACGTTTTTATAGGTAACTCGATAAATCGTGCTTTTGTGATATTCTGATGC18120 
GGACAATAT ACATTTAAACATAAAGATATAAGTTATTGAGGCATTTATGTATATTACAAT18180 
AGTGGGGTACATTTTTCACAGATGCTGTCACCCATGAAATATTGGCAAAATACTCTTAAA18240 
ATATGCAAGAAACTAAAGAGGATGCATGGGTTGGGCTGTAGGTACATGGATGCA AATGCT18300 
GTTTTGCAATAAGTCATATAGTCTCGTCTGTTGAGTGAGGCCCATTCAATCAGCAAGTAG18360 
GACTGAGGTGCATGATCGACATATTTTTGAACCACAGTTTTGGCAAGTTTTTCATACAAA18420 
TGCACGGCTACGGCCAAATCGTAGCTTGCAA GTCCAACTGCTGAAAAGTTAGCCGGCCCG18480 
TTCCAAGAAATTAGCCTTTGCATAAGGACTGGATCGCGGAGAACTTCAGAGTAGTTCCTG18540 
ATCCCATTGTCCCTGCCGTGTTTTGTTAGCTTTAAATGGCGTCTTGAATAGTGCAGCGCC18600 
AACGAGTCG ATATTACGTGTTTCCATCGCATCCATATCATCTGCCACCACGATGCCACTC18660 
AGCTTCAACACGTGATCAAAATAGTCAGCTGGCAATTCGTCAATTCCAAGCGTCAATGTA18720 
ACGGCATTGTCTGTGATCTCCTTCATCTCAAAGACGGGCTTGTTTGAATTCGTC GCCGTA18780 
ATTATGAACTTGGATTTGCTGAGATATGCTCGATTGTTAACAGCCTTGAGTGAAATCTTG18840 
ACTTCCGGCTGAAGCCTTTGCACCAACTCATGGTTTGACTGGTTGCAGCGGCTGAGAATC18900 
GCGATTCGTTGAATTCTTCCAGATGCTCCCG AATTGAGGGCGAGGATGATGGCCTCGGCA18960 
ACTTTACCTGCTCCGAATAGGAAGACATTGATCTGGCTTCGGCCCTGCAATAGGAGATTC19020 
AGGCATGCTAGTGCCAGCCAACCAGTTCTCCTCTCCGATATAGCCACCCCATCAACAGAG19080 
AAGAGACGT CTACCTGTGAAACGATTGCGAAGCCAACGTCGATGTGAGAAGTCGGTTCTT19140 
TGTATCTCGCGTTTGACGGATTAGAATGGATGCTTTTCACACCCGAATAGTCGCCGACGA19200 
AACCCACCAGAGCTCCCTCCGTACAGCCCTCTCGATCAAGTGGAACGAAGACCT TGTTGT19260 
GGCCGAGCCGCCCTTCAGCAAAGAGGTGCCAATAATCTTTCAAGGCATCCGCGACGAGTT19320 
CCGGTGTAATGTATATTCCAAAAGCCGATAGAGATTCCTCTGTCCAACATTGCTCGTGTA19380 
TTTGATCGGCCATGTTTGTGTTTGATCAGCC TCCTTTCGAAAATTTCTTGAGTTTCGAAT19440 
AATTCTAAAATCGAAGGACGATTAATAGTGCCATACCAAGACAAGAAGGGTAGGTGGGCC19500 
ATCAATCCACAAGCCTAGCACATTTTGCTGTCTGCTCATGCAAGGTATCCAATGGAAGCC19560 
TGGATTGGT TAGCCGAACTTGGTGGGTTCAATTGGAGCGGGCAGGTCACTTTTTGTCTCT19620 
CAAATAACTGAAACTAAGTTTTGTTATTTGGTATGTGTTTGTCTGTTCTGCCGAAGGTGC19680 
CCGAATTTGCGCAAATTCCTTTCTAAAAAGGCTTACATCTAGCAAAAGGTGAGC CCTGTG19740 
CATCCCAGCATTTGGACAAAGCGCGCCAATTCGGACAGCGACTGGCTGCGTTGGAGGCTC19800 
GGATCTCAAAGAATAGAAAAGAGTTATGATCATGTTCAGAACCGCCAATTTTGTGCGGTA19860 
TGAGCTCTTTGATGAAAGTAATGGTTTCAAA AAAGCAACATCGTGGGTGAAAGGTACCTA19920 
CATATCTTCACAGACAATAACTACTGTTGCTGTTTGCTGATTGACTGACAGGATATATGT19980 
TCCTGTCATGTTTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTAATG20040 
TATAAGTTC GTGATGAAGGATGGTTGTTTTAAAAATAGTATGTTTGACTGAGGTTAAGTC20100 
ACTCACGTTTTGCACATCGACGGACCGTAAGCATTCTTTCGGTAAGACCGAAGCTCGTCC20160 
CAGATAATAGGCCCCGTGGAGGGAGGCCTTGTATGGGCCGACCGATGGGCGTGC TGAGCC20220 
GAGTACGGCGACGCCTGCGGCGATTGCGCGGGCGGCACTGCGCGCAGGGGCACGGGTTCA20280 
TACGAGGACGAGCGTAAGGGGCATAGAGCTTTCCGCCCGTCGGGTTTCAGCCATATTGCT20340 
TGATTGCGGCCGACTGGAATGCAGCCAGGTC GTGCTCGCCGGCGGCGCCTGGTCGAGCGG20400 
CATGCTGCGCACCTCAGTGTTCGGCTTCCTCAGCTCACGGTTACTGTGTCGGCGCTAAGA20460 
ACCGAGGCCTTTGATGGCGGACCTGGCCTTTTCAATCAAGGGATGCTGATTTTCCATCCG20520 
TAAGCGTCT CGACGGTGGTTACACCGTCGGCTTCGGCGCGACGATGAGAACCGAAATCGT20580 
CAGCGATAGCTTTCGTTTTCTGTCGGATTATTTCCTCCTGATGCGAGAGGAATGGCTTTC20640 
TATCCGGCTGCCGGCGGGTGCGCGCGTCCGGATTGACCCTCCCGTTCGTTGCTA CTTGGC20700 
TCGAGTGACGAAATAGCACGCCTGTGCCGCTGTATCATGTCCATCGGGCTCACAGGAGAT20760 
TCGCTCGTAGCGCGTTGGTGTCACTCACCAACACGCGTCGTCGCACCAAATTGGGGAGGA20820 
TGGTAGCGGAATCCTAAAATCCTAAAACCAT ACCGACGCGTCACGGCGCTCGTGACCCCT20880 
GCGAGCGACGCGGCACTCTCTCACCTGATCCGTGCTGCGGTTGCTCAATACGCAATGAGC20940 
ATTGTCACGGTTCTCAGGGTAAACGGCAATCTCTTCGTCATGCGGGCGTGGATGCTATCA21000 
CCGTTAGAA AGGGCCTGCCCCCATGGTGGGTCTCTAAGGTTCAGTCTGAGAAGGGGCAGC21060 
CAGAGCGGCACTGTTTGAAGAGCAGTCTGAACCGCTCAGATCGCTCGCATCGATGCTTGG21120 
GCGGCG 21126