PT78415B - Method for genetically modifying a plant cell - Google Patents

Abstract

A DNA vector comprises T-DNA having a plant structural gene inserted therein under control of a T-DNA promoter. The DNA vector is useful in genetically modifying a plant cell to introduce the plant structural gene thereto.

Description

Detailed Description of the Invention

The following definitions are given to render ambiguous the intent or purpose of its use in the specifications and claims.

T-DNA: A segment of DNA derived from a tumor-inducing principle (TIP) that integrates into the plant genome. As used herein, the term includes DNA originally derived from any tumor-inducing Agrobacterium strain including A.tumefaciens and A.rbizogenes. the inserted segment of the latter being sometimes referred to in previous works as R-DNA. In addition as used herein the term T-DNA includes any changes, modifications, mutations, insertions and deletions natural or inciped by laboratory techniques, the requirement being that

structural structure and limitation to such modifications that sufficient amounts of the right and left ends of the natural T-DNAs are present to ensure the expected function of stable integration into the transformed plant cell genome which is characteristic of T-DNA. In addition the T-DNA must contain at least one T-DNA promoter in a sufficiently complete form so as to control initiation of transcription and initiation of translation of an inserted plant structural gene.

Preferably the site of insertion will be downstream in the direction and the translation initiated by the promoter, thus located in relation to the promoter to allow a plant structural gene inserted therein to be expressed under the control of the promoter, either directly or in the form of a fusion protein.

Structural Gene of Plants.

As used herein includes that portion of a plant gene comprising a DNA segment encoding a plant protein or polypeptide or only a part thereof but without the functional elements of a plant gene which regulate the initiation of transcription and initiation of translation, commonly referred to as the promoter region. A plant structural gene may contain one or more introns or may constitute an uninterrupted coding sequence. A pltanta structural gene may derive from all or part of a plant genomic DNA, cDNA or DNA obtained by chemical synthesis. The structural plant gene may further comprise modifications in the coding or intron segments which may affect the chemical structure of the expression product, the expression rate or the expression control process. Such modifications may include, but are not limited to, "silent" meta-positions, insertions, deletions, and modifications that do not alter the chemical structure

of the expression product but which affect the intercellular location, transport excretion or stability of the expression product. The structural genes may be a segment composition derived from a variety of natural or synthetic diol sources, encoding a composite protein being in part a plant protein.

T-DNA promoter:

Refers to any naturally occurring promoters commonly associated with integrated T-DNA. These include, but are not limited to, promoters of the octopine synthetase gene, the nopaline synthetase gene, the tms, tml and tmr genes, depending in part on the TIP DNA source. Expression under the control of a T-DNA promoter can take the form of direct expression in which the structural genes normally controlled by the promoter are removed and replaced by the plant structural gene, the initiation codon being given by the T-DNA structural gene or belonging to the inserted plant structural gene, or by expression of a fusion protein in which part or all of the plant structural gene is inserted into the correct reading frame within which the existing T-DNA structural gene. In the latter case, the expression product is referred to as fusion protein.

Vegetable tissue

It includes differentiated or non-differentiated tissues of and derivatives of higher plants including roots, shoots, pollen, seeds, tumor tissue, such as crown galls and various forms of crop cell aggregates in culture, such as embryos and calluses

Plant Cell:

As used herein it includes plant cells in plant and plant cells and plant protoplasts in culture.

The production of a plant expressly genetically modified a plant structural gene introduced via T-DNA combines or the specific teachings of the present disclosure with a variety of techniques and resources known in the art. In most cases there are alternative resources for each phase of the overall process. The choice of resources depends on variables such as the choice of resources depends on variables such as the choice of the basic TIP, the plant species to be modified and the desired regeneration strategy, all of them present steps with alternative processes that those familiar with the matter to select and use to achieve a desired result. The fundamental aspects of the invention are the nature and structure of the plant structural gene and its method of insertion into the T-DNA. The remaining steps to obtain a genetically modified plant include transfer of the modified T-DNA to a plant cell in which the modified T-DNA is stably integrated as part of the plant cell genome, techniques for in vitro cultivation and eventual regeneration in whole plants, which may include selection and detection pathways of transformed plant cells and steps of transferring the introduced gene from the originally transformed strain to commercially acceptable cultivars.

A key feature of the present invention is the construction of T-DNA having inserted a plant structural gene under the control of a T-DNA promoter, as these terms have been defined supra.

The plant structural genes must be inserted in the correct position and orientation in relation to the T-DNA promoter.

The position has two aspects. The first relates to the side of the promoter on which the structural gene is inserted. Most promoters are known to control initiation of transcription and translation in only one direction along the DNA. The region of the DNA that is under the control of the promoter is said to be "downstream" or alternatively "behind" the promoter. Thus, in order to be controlled by the promoter, the correct position of the plant structural gene insert should be the "downstream" of the promoter, (some known promoters are known to exert a bi-directional control, in which case "either side of the promoter considered to be "downstream" thereof.) The second aspect of the position refers to the distance in base pairs between known functional elements of the promoter, for example the transcription initiation site and the translation initiation site of the structural gene The structural requirements in this regard are better described in functional terms As a first approximation a reasonable operability can be obtained when the distance between the promoter and the structural gene inserted is similar to the distance between the promoter and the T-DNA gene it normally controls. By convention, the portion of a structural gene encoding the amino acid end of the plant protein is designated the s' end of the structural gene, whereas the end coding for the amino acids near the carboxyl end of the protein is designated the end 3 * of the structural gene. The correct orientation of the plant structural gene is with its 5 'end proximal to the T-DNA promoter. A further requirement in the case of constructs leading to the expression of a fusion protein is that the insertion of the gene

The structural sequence of the T-DNA structural gene must be such that the coding sequences of the two genes are in the same reading step, a structural requirement that is well understood in the art. An exception to this need, of relevance to the present invention, exists in the case where an intron separates the TDNA gene from the first coding segment of the plant structural gene. In this case the excision sites. of the intron must be located such that the correct reading phase of the T-DNA gene and the plant structural gene are restored in phase after removal of the intron from post-transcription processing. The source of T-DNA may be any of the TIP plasmids. The plant structural gene is inserted by standard techniques well known in the art.

Differences in expression rates can be observed when a given plant structural gene is inserted under the control of different T-DNA promoters. Different properties including properties such as intercellular localization or excretion, antigenicity and other functional properties of the expressed protein itself may be observed in the case of fusion proteins depending on the insertion site, length and properties of the included T-DNA protein segment in the fusion protein, and mutual interactions between the components of the fusion protein that determine its coiled configuration all provide numerous opportunities to manipulate and control the functional properties of expression product depending on the desired purpose. Phaseolin structural gene expression was found when this gene was inserted under the control of p058 nopaline synthetase promoter in a nopaline plasmid of A. tumefaciens.

A convenient means for insertion of a plant structural gene into T-DNA involves the use of shuttle vectors as described supra, having a T-DNA segment (which segment the insertion is desired) embedded in a plasmid capable of replicating in E. coli The T-DNA segment contains a restriction site preferably one that is unique to the "shuttle" vector The plant gene can be inserted at this unique site on the T-DNA segment and the "V'-V vs" vector is transferred to cells of the appropriate Agrobacterium strain, preferably one whose T-DNA is homologous to the T-DNA segment of the "shuttle" vector.

The transformed Agrobacterium strain is grown under conditions allowing selection of a case of double homologous recombination that results in the replacement of a preexisting segment of the Ti plasmid by a T-DNA segment of the shuttle vector.

Following the strategy just described, the modified T-DNA can be transferred to plant cells by any technique known in the art. For example, this transfer is most conveniently achieved by direct infection of plants with the new Agrobacterium strain containing a plant structural gene incorporated into its T-DNA or by coculture of the Agrobacterium strain with plant cells.

The first technique, direct infection, results in time in the appearance of a tumor mass, or crown gall at the site of infection. The crown gill cells may be subsequently grown in culture and under appropriate circumstances, known to those skilled in the art to be regenerated for whole plants having the inserted T-DNA segment. Using the co-culture method a certain proportion of plant cells are transformed, ie have T-DNA transferred and inserted into the plant cell genome. In both cases, the transformed cells must be selected or tested to distinguish them from untransformed cells. The selection is

easily achieved having a selectable marker incorporated into the T-DNA in addition to the plant structural gene. Examples include dihydrofolate reductase or neomycin phosphotransferase expressed under the control of a nopaline synthetase promoter. These tags are selected by growth in methotrexate or canine mycin medium containing, respectively, or analogues thereof. In addition, the T-DNA has endogenous tags such as the gene or genes that control the hormone independent growth of the Ti-induced tumors in culture, the gene or genes that control the abnormal morphology of the Ri-induced tumors and the genes that control the resistance to toxic compounds such as amino acid analogues, such resistance being given by a synthetic synthetase. Trace methods well known to those skilled in the art include assays for the production of specific hybridizations with characteristic RNA or T-DNA sequences or immunological assays for specific proteins including "ELISA ¹¹ (enzyme linked immunosorbent assay" ), radioimmunoassays and Western blots.

An alternative to the "shuttle" strategy involves the use of plasmids comprising modified T-DNA or T-DNA in which a plant structural gene is inserted, said plasmids being capable of replication independently in an Agrobacterium strain. Recent results indicate that the T-DNA of such plasmids can be transferred from an Agrobacterium strain to a plant cell provided that the Agrobacterium strain contains certain trans acting genes whose function is to promote T-DNA transfer to a plant cell. Plasmids which contain T-DNA and are capable of replicating independently in an Agrobacterium strain are referred to herein as " sub-TIP " plasmids.

A spectrum of variations in which the "sub-TIP" plasmids differ in the amount of T-DNA they contain is possible. One of the

In the other end of the spectrum all of the DNA except for the minimal amount surrounding the T-DNA ends is removed, the The plasmid "sub-TIP" plasmids are advantageous in that they are small and relatively easy to manipulate directly, after which the desired gene has been removed from the host cell The introduction into a strain of Agrobacterium is conveniently accomplished by transforming the Agrobacterium strain or by conjugative transfer from a transgenic strain of the Agrobacterium strain. donor bacterial cell by techniques well known in the art.

Regeneration is achieved using known techniques. An aim of the regeneration step is to obtain an entire plant which grows and reproduces normally but retains the integrated T-DNA. Regeneration techniques vary according to principles known in the art, depending on the origin of the T-DNA of the nature of any modifications thereof and the species of transformed plants. Plant cells transformed by a Ri-type T-DNA are easily regenerated using techniques well known to those skilled in the art without undue experimentation. Ti-type T-DNA transformed plant cells can be regenerated, in some cases, by appropriate manipulation of hormone levels in the culture. Preferably, however, the Ti-transformed tissue is more easily regened if the T-DNA has been mutated in one or both of the tmr and tms genes. Inactivation of these genes restores normal hormonal balance in the transformed tissue and substantially increases the ease of manipulation of the hormone levels of the 33-

in culture, leading to a plant with normal hormonalynal physiology that is easily regenerated. In some cases the tumor cells are able to regenerate rebounds having integrated T-DNA and expressing T-DNA genes, such as nopaline synthetase, together with an inserted plant structural gene. The shoots can be vegetatively maintained by grafting on rooted plants and can produce fertile flowers. Sprouts thus serve as parent plant material for dairy plants

normal cells having TdNA and expressing the plant gene in it

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of.

Examples

The following examples utilize many techniques well known and accessible to those familiar with molecular biology and manipulation of TIPs and Agrobacterium; such methods are not always described in detail.

Enzymes are obtained from commercial sources and are used according to the recommendations of the vendor or following variations known in the art. Buffer reagents and culture conditions are also known to those skilled in the art. Reference works containing such Standard techniques include the following: R. Wu, ed. (1979) Meth. Enzymol. 68; JHMiller (1972) Experiments in Molecular Genetics; R. Davis et al. (1980) Advanced Bacterial Genetics; and RPSchleif and PCWnesink (1982) Practice Methods in Molecular Biology.

In the Examples, special symbols are used for better understanding of the sequences. Sequences that actually encode or could encode proteins are underlined and the codons are separated by rods (1). The positions of the cuts in each chain caused

by restriction endonucleases or otherwise marked by asterisks (*) (In one example a double-stranded DNA molecule is represented by a single line flanked by asterisks at restriction enzyme cleavage sites: the approximate position of a gene is in this case indicated by the single line "X" subheading). Except for plasmid IIc, plasmids and plasmids only, are preceded by a "p ⁿ , eg * p3.8 or pKS4. Plasmid-containing cells are indicated by cell identification and indicated in parentheses of the plasmid, egAtumefaciens (pTi 15955) or K802 (pKS4-KB). Table 1 provides a useful index for identification of plasmids and their interrelationships. Table 2 gives an index of the deposited strains.

Fig. 39 provides such a useful comparison of the constructs described in Examples 11, 12 and 14. Fig. 40 shows the genetic code and is useful for interpreting sequences. The nucleotide sequence of an important T-DNA gene, tml. although not used in the Examples, is shown in Fig. 41: it is useful in schematizing constructs which are not described herein.

Example 1

A gene for a fusion protein consisting of the octopine synthetase promoter, the 90 amino acids of the amino terminus of the octopine synthetase structural gene, a 3 amino acid overlap between the two genes, and the entire phaseolase were constructed except for the codons encoding their first 11 amino acids. Prior to the start of the construct, a T-DNA clone in pTi 15955? P233 (the sequences defined by p203 and p3O3 in pBR322, see Fig. 1) was sequenced between the BamHI site and the site

PvuII. This includes the entire octopine synthetase gene (Fig. 2). The octopine synthetase sequence and reading step near an EcoRI restriction enzyme cut site were found to be as follows:

EcoRI

5 '... ATG / GGC / CAG / CAA / GG * A / ATT / CTT ... 3' 3 '... TAC CCG GTC GTT CC T TAA * GAA ... 5' ... Met Gly Gin Gin Gly Ile Leu ... 84 85 86 87 88 89 90

Cutting with EcoRI gives a fragment with the following end:

• · · ATG / GGC / CAG / CAA / GG 3 '

... TAC CCG GTC GTT CCTT 5 »

The structural gene for the seed bean seed protein phaseolin (previously sequenced, Fig. 3) contains an EcoRI site near its 5 'end (amine terminus) as follows:

EcoRI

... CIG / TTG / CTG / GG * A / ATT / CTT / TTC ...

... GAC AAC GAC CC T TAA * GAA AAG ...

... Leu Leu Leu Gly Ile Leu Phe ...

9 10 11 12 13 14 15

Cutting with EcoRI gives a fragment with one end as follows:

5 '

3 '

A / ATT / CTT / TTC ... GAA AAG.

These two fragments, upon ligation, form the following structure:

84 85 86 87

CC T OK* The GAA THE AG. • · · Giy Ile Read Phe. • · 88 89 90 octopine synthetase 12 13 14 15 phaseolin

Not only are they preserved they are not generated any signs

stages of reading and stopping

Thus, the fragment obtained by EcoRI / BamHI restriction of the Phaseolin gene was ligated at the EcoRI site to the ptil5955 T-DNA octopine synthase gene. This fusion gene contains the ocs promoter, the first 90 amino acids of the octopine synthase, the phaseolin gene minus its promoter and its first 11 amino acids and a three amino acid junction identical to the sequences present in both parent proteins.

1.1 Removal of the EcoRI site from pBR322

The EcoRI site on pBR322 was removed by EcoRI digestion filled with the T4-DNA polymyrase, ligated by the erected ends and used to transform E. coli strain HB101. Transformants were selected from ampicillin and colonies were tested by isolation of small amounts of the plasmid DNA (D.Horowitz) (1982) in Molecular Cloning, CSH) and selection of a clone lacking an EcoRI site designated pBR-322 -R.

1.2 Cloning of the BamHI fragment of T-DNA into pBR322-R p203 (Fig. 24) was isolated and digested with BamHI. 0 frag

4.7 kbp of the T-DNA was isolated by agarose gel electrophoresis and ligated to the BamHI site of pBR322-R.

This plasmid was used to transform E. coli strain HB101 and selected for ampicillin resistance and sensitivity to tetracycline. A positive clone was selected and designated pk5169.

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1.3 Removal of the EcoRI sites and fragments of the octopine synthase gene

pk5169 was isolated and digested with EcoRI. An 8.6 kbp fragment was isolated by agarose gel electrophoresis and purified. This fragment has removed the 2 small fragments (0.36 kbp and 0.2 kbp) of the oos gene.

1.4 Isolation of the EcoRI fragment containing the DNA fragment of the phaseolin gene and the kanamycin resistance gene

pk54-kB (Fig. 7) was purified and digested with EcoRI. A 4.8 kbp fragment was isolated using the 3.0 kbp EcoRI / BamHI fragment of the phaseol gene bound at the BamHI site to a DNA fragment of 1 , 85 kpb containing the kanamycin resistance gene encoding neomycin phosphotransferase II (NPTII):

1.5 Binding of the phaseolin gene to octopine synthetase

The phaseolin / NPTIl fragment was then ligated into the EcoRI sites to the EcoRI fragment described in Example 1.3. The bound DNA was used to transform

HB101 and the colonies were selected ampicillin and kanamycin. A colony designated pks-B17-KB3.O (Fig. 43) was

selected and contained a plasmid with the correct orientation (ie the phaseolin gene linked to the ck gene in the correct reading direction and phase). This was found by making restriction maps of plasmids from a small number of colonies. The DNA sequence of the appropriate region was determined to verify the construct.

1.6 Transfer of the T-DNA fragment containing the BPIII DNA, phaseolin and ocs to pEE29O.

pE29O, a plasmid with a wide host range, was digested with BglII and ligated to a 9.1 kbp BamHI fragment containing the T-DNA, the NPTII gene and the pkS-B17-KB3.O phaseolin DNA. This was achieved by partial digestion of β5-B17-KB3 O with BamHI and isolation of a 9-well fragment from 6 other bands of an agarose gel electrophoresis. After ligation and transformation of E. coli strain 802. colonies were selected in kanamycin and tetracycline. A colony having the desired restriction pattern was selected and designated p-S-β-B3.0.

1.7 Substitution of the octopine synthetase in pTi 15955 by the octopine synthase fusion protein gene phaseolin.

Using triparental conjugation of A.tumefaciens (resistant to streptomycin), E.coli (pKS0SI-EB3.0). and E. coli (pR 2015), streptomycin, kanamycin and tetracycline resistant colonies were selected. A colony was conjugated to E. coli (pPH11). A kanamycin and gantamycin resistant colon was selected.

This was found to be A.tumefaciens with pl5955-12A, a pTil5955 having the phaseolin gene and the kanamycin resistance gene genetically engineered into the EcoRI site of the cc gene. making restriction and hybridization maps on restriction fragment filters separated by

electrophoresis. (Example 19). An analogous triparental conjugation is done with A.tumefaciens (pTiA66). Rebutations transformed by the resulting plasmid, pA66-12A, were found to contain phaseolin as described above.

1.8 Formation and expression of crown galls.

Sunflower plants were incubated with the genetically engineered Ti plasmid. They were established in tissue culture of crown galls. Expression was assayed ELISAse by hybridization in mRNA filters separated by electrophoresis. (Northern blots, Example 19) RNA of the expected size was detected with hybridization probes of the phaseolin and octopine synthetase genes and comprised about 0.5% of poly (A) 5 + 4 RNA. Poly (A) 5 + 4 RNA isolated from galls was translated in vitro giving a protein of the expected size precipitated by antibodies against phaseolin.

Example 2

A fusion protein gene similar to that constructed in Example 1 was constructed with phaseolin and nopaline synthetase, under the control of the promoter of the latter gene. It contains the nopaline synthetase promoter and encodes the first 59 amino acids of the nopaline synthetase (from which the latter residue was synthetically added), a junction of an amino acid and the entire structural gene of phaseolin with the exception of its first 12 amino acids. Prior to construction, a T-DNA clone of pTiC58 (pCF44A, Fig6) was sequenced from the BglII site on the far left to the medium HindIII site, which is outside the T-DNA region. This includes the entire gene in (Fig. 4). The nopaline synthase sequence

-40-

and the reading phase were found to be as follows near a site cut by the restriction enzyme ClaI.

Olal

5'AOOA / GGA / T * CG / ATC / TCA ... 3 '

3 '... GGT COT A GO * TAG ACT ... 5'

... Pro Gly Ser Ile Ser ...

56 57 58 59 60

The Ciai cut gives a fragment with the following end:

... CCA / GGA / T 5 '

... GGT CGT AGC 5 '

As was established in Example 1 the EcoRI site of the following phaseolin:

EcoRI ... CTG / GG * A / ATT / CTT / TTC ... GAC CC T TAA * GAA AAG ... Leu Gli Ile Leu Pen ... 1 11 12 13 14 15

may be cut to give the following structure:

5 ' A / ATT / CTT / TTC ... 3 ' GAA AAG ... The two adapters that follow: The) 5 'CGATCCC 3' B) 5 · AATTGGGAT 3 '

may be attached to form the following structure:

5 'CGATCCC 3' (a

3 'TAGGGTTAA 5' (b

which can bind all DNA fragments to form the following structure:

• · · COA / GGA / T ^X CG / ATC / GO * A / ATT / CTT / TTC ...

... GGT COT A GC * TAG GG T TAA * GAA AAG ...

... Pro Gli Ser Ile Pro Ile Leu Een ...

56 57 58 59 nopaline synthetase

13 14 15 phaseolina

It should be noted that the adapter serves several functions: a new amino acid is introduced; part of the sequence removed from the nopaline synthase gene is reconstructed; two incompatible restraint locations are made compatible and an open reading frame is preserved.

Summarizing the EcoRI / BAmHI restriction fragment of the phaseolin gene was ligated to the ClaI site of the nopaline synthetase gene after an adapter had converted the Eco RI site to a ClaI site. Ogene Fusion contained! the nopaline synthetase promoter, the first 58 amino acids ^;

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ethers of the nopaline synthetase, an adapter which rebuilt part of the nopaline synthetase sequence and a new inserted amino acid and the entire phaseolin gene with the exception of the first twelve amino acid residues.

2.1 Adapter Synthesis

The following two adapters were synthesized:

a) 5 'CGATCCC 3 *

b) 5 'AATTGGGAT 3'

These were synthesized by the methods of Example 17. The oligonucleotide a) and b) were

connected together to form the structure:

5 'CGATCCC 3' (a

3 'TAGGGTTAA 5' (b

2.2 Preparation of the "shuttle" vector

pks-nopIV, whose construction was des. found in Pig.6, is pEK29O with the nopaline type T-DNA grown at its BglII site. This nopaline-type T-DNA contains a single ClaI site resulting from the deletion between the ClaI site in nos and the site 01a downstream of the gene in (Figs. 5θ6). PKS4-KB (FIg.7) and digested with EcoRI. The resistance fragment with

4.8 Kpb., Was purified by gel electrophoresis. This fragment contains the EcoRI / BamHI fragment of phaseolin DNA (referred to as bean in the can / bean notation attached to the BamHI site of the BamHI / EcoRI fragment of the kanamycin resistance gene xcan) of Tn5 ·

Ciai-linearized pkS-nopIV was ligated with the purified can / bean fragment and the adapters of Example 2.1. E. coli k802 was transformed and colonies were selected for resistance to kanamycin and tetracycline. Two orientations were present, one with phaseolin DNA linked to the nopaline synthetase gene and the other with the kanamycin resistance gene attached after

nopaline synthetase gene. Restriction maps were determined to determine which cells contained a plasmid, pNNNI, having the desired orientation as shown in Fig.

2.3 Replacement of the nopaline synthetase gene in pTiC58 by modified phaseolin.

A triparental conjugation (see Background - vectors "going") with A.tumefaciens-str RC58 E.coli (pRK2O13) and E. coli (pNNN1) was used to insert the construct into a Ti plasmid. Cells from A .tumefaciens resistant to streptomycin, kanamycin and tetracycline. The selected transformants were conjugated to E.coli (pEH1) and selected and colonies resistant to kanamycin and gentamicin.

2.4 Formation and expression of crown galls.

Sunflowers were inoculated and crown galls established in tissue culture Expression was tested by ELISA and hybridization with nERA as described in Examples 17-20.

Example 3

The purpose of this example is to reconstruct the complete coding sequence of the phaseolin gene from the translation initiation signal to the EcoRI site which can then be ligated to the remainder of the structural gene. A Ciai site will be constructed at the 5 'end so that the gene can be easily recovered. The following two oligonucleotide sequences will be synthesized:

a) 5 'AÂTTCCCAGCAACAGGAGTGGAACCCTTGCTCTCATCAT 3'

b) 5 'CGATGATGAGAGCAAGGGTTCCACTCCTGTTGCTGGG 3 *

These may be attached to form the following structure:

Clal EcoRI

(A) TAC TAC TCT CGT TCC CAA GGT GAG GAC AAC GAC CCT TAA5 (b)

Met Met Arg Ala Arg Go Pro Leu Leu Leu Leu Gli IIe

1 2 3 4 5 6 7 8 9 10 11 12 13

As set forth in the Example, the following EcoRI site of the phase:

• · »CTG / GG * A / ATT / CTT / TTC ...

... GAC CC T TAA * GAà AAG ...

Leu Gli Ile Leu Fen.

11 12 13 14 15

may be cut to give the following structure:

5 'A / ATT / CTT / TTC ...

3 'GAA AAG ...

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Binding of this end to the double-stranded synthetic oligonucleotide described above results in a structural gene encoding a complete phaseolin polypeptide with cohesive ends Clal immediately ahead of the start of the coding sequence.

3 · 1 Synthesis of adapters

The following two adapters were synthesized by the method of Example 17:

a) 5 'AATTCCCAGCAACAGGAGTGGAACCCTTGCTCTCATGAT 3'

b) 5 'CGATGATGAGAGCAAGGGTTCCACTCCTGTTGCTGGG 3'

These were ligated to give the following structure:

5 'CGATGATGAGAGCAAGGGTTCCACTCCTGTTGCTGGG 3' (b

3 'TACTACTCTCGTTCCCAAGGTGAGGACAACGACCCTTAA 5' (a

3.2 Construction of the complete phaseolin gene and kanamycin resistance gene cloned in pKS-nopIV.

pKS-nopIV is ligated with the ligated adapter of Example 3 · 1β purified the EcoRI / bean fragment from K54-KB (see Example 2.2). E. coli K802 is transformed and selected colonies

resistant to tetracycline and kanamycin. Again, although two orientations are possible, only one is the phaseolin gene attached following the nopaline synthetase gene.

The correct orientation is selected after the restriction maps of the clones have been made.

3.3 Formation and expression of crown galls

Homologous recombination and isolation of crown galls in tissue culture is performed as described in Example 21 and detection of expression of the phaseolin gene in crown gill tissues is as in Examples 19 and 20.

Example 4

The purpose of this construction is. to construct a "shuttle" vector for use in the pTi system for expression of foreign genes in gill cells; in crown, the foreign gene is either under the control of the promoter in the part of which is synthesized chemically and lacking codons for the nopaline synthase gene. Prior to the construction, a T-DNA clone of pTiC58 (pCP 44A) was sequenced to detect the promoter in (fig. 4).

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This is digested with ClaI and SstII giving a linearized 22 kbp vehicle and a 555 bp fragment. These are readily separable by centrifugation through a salt gradient. After the small fragment is digested with Hinfl the 149 bp SstII / Hinfl fragments and 208 bp Clal / Hinfl fragments are isolated by gel electrophoresis.

4.2 Overview of adapters

The following two adapters were synthesized by the method of Example 17:

a) 5 'AGTCTGATACTCACTCTCAA.TCCAAATAATCTGCCATGGAT 3 *

b) 5 'CGATCCAIGGGAGATTATTTGGATTGAGAGTGAGTATGAG 3'

They were connected together to form the following structure:

5'AGTOTCATACTCACTCTCAATCCAAATAATCTGCCATGGAT 3 '(the

3 'GAGTATGAGTGAGAGTTAGGTTTATTAGACGGTACCTAGC 5' (b

This sequence has a local Hinfl on the left and local Ncol and Ciai on the right. An alternative sequence will have a Bell location between the NcoI and CiaI sites. The sequence is identical to that found in the T-DNA except for the underlined bases which replace a base pair AT by a base pair CG.

4.3 Formation of pNNN2

I

i

I

The Clal / SstII vehicle is ligated as shown in Fig. 9 to the 149 bp SstII / Hinfl fragment and [to the synthetic adapter, forming the following structure:

(I.e. · » (I.e. (I.e. * • • • * • (I.e. • , 0 (I.e. (I.e. g ω (I.e. γ 1 ι-1 (I.e. • • • • • • And (I.e. ΕΗ * (I.e. (I.e. (I.e. (I.e. <2> rt Ε4 << ί (I.e. <5 Ε4 or (i.e. (I.e. (I.e. <25 (I.e. (I.e. And "1 (I.e. <<! And 25 X <25 (I.e. <25 C5 (I.e. And (I.e. (I.e. <25 Hey <5 Εί & Η (I.e. & <J (I.e. Ε-1 (I.e. Ε4 +3 Aa Ε4 μΏ (I.e. <25 (I.e. or <25 Ε4 η (I.e. And 0) Ε4 & or <25 And <5 or (I.e. <25 τ) And (I.e. rt (I.e. <25 (I.e. <5 (I.e. ft (I.e. <25 rt And «3 -d (I.e. rt (I.e. Ej And <4 (I.e. EH (I.e. <25 And (I.e. (I.e. or (I.e. * (I.e. And or β (I.e. ي (I.e. <5 Ε4 (I.e. * or (I.e. "1 Ε4 (I.e. <1 • « • « * • ρ (I.e. g HERE (I.e. (I.e. (I.e. • • • • • • «

(I.e.

4.4 Insertion and expression of a phaseolin gene

pNNN2, the plasmid constructed in Example 4.3 (Fig. 9) is cut with ClaI, mixed with the Clal / EcoRI adapter synthesized in Example 4.2, and the EcoRI / Clal bean caB / bean fragment purified by electrophoresis from pKS4-KB, ligated to transform, isolated and made the restriction map. The appropriate plasmid, pNNN4, is transferred and tested for expression as described in Examples 21, 19 and 20.

4.5 Insertion and expression of an intron-free phaseolin gene.

The procedure outlined in Example 4.4 is repeated with the replacement of pKS4-KB by a pcDNA 31 or a pKS4-KB analog derived from pMC6 cDNA. (see Example 9).

4.6 Insertion and expression of a phaseolin cDNA

This construct is analogous to Example 10 with respect to the use of cDNA, a single chain PstI adapter, and the PstI fragment and is analogous to Examples 4.1, 4.2 and 4.3 with respect to the use of the promoter in the semi-synthetic. The characterization, transfer and expression detection is as described in Example 4.4.

pNN2, the plasmid constructed in Example 4.3 (Fig.9) is cut with Olal, mixed and ligated to the Clal / E.coEi adapter synthesized in Example 4.2, the 1.7 Kbp EcoEI / PstI bean fragment isolated from pKS4- EB and purified by electrophoresis, the PstI fragment of Tns with 0.93 kbp purified by electrophoresis and the 5'-GAATT3 'single stranded Clal / PstI adapter, previously synthesized by the method of Example 17

Example 5

Is the purpose of this construction simple? the phaseolin gene from the EcoRI site to the BamHI site to the active T-DNA gene that lies between the HindIII sites of p403 whose mRNA is designated 1.6 on the map, shown in Fig. The sequence was determined (see Fig. 10) from the HindIII site of p401 beyond the Clal site to its right (see Fig. 11). There is an open reading frame that begins between the HindIII and Clal sites going towards the Hind III site (see 1450 bp nRNA mapped in Fig. 11).

The Clal site is in the untranslated leader of the gene mRNA between the Hind III sites A promoter vehicle was created by excising the Clal fragment in the p403 medium This is possible because the internal Clal sites are not methylated in some E.coli strains, whereas the Clal site following the EcoRI site is methylated.

The phaseolin gene is now attached to the Clal site carrying an ATG. This can be !

achieved using pKS4-0KB. The sequence of bases from

the Clal site of pBR322 to the EcoRI site of the phaseolin is as follows.

Clal EcoRI

AT / GAT / AAG / CTG / CTG / TCA / AAC / ATG / AG * A / T / T / CTT / TTC. .3 '' ... TA GC * TA CTA TTC GAC GAC AGT TTG TAC TC T TAA * GAA AAG..5 »

Met Arg / Ile Leu Fen ...

13 14 15 |

derived from pBR322 / phaseolin

Note the open reading frame and the ATG. There are 18 bp between the Clal site and the translation initiation signal (ATG). This

is comparable to 12bp from the ClaI site until the initiation of the T-DNA gene.

Ciai

5 '... AT * CG A / TGG / ACA / TGC / TGT / ATG ... 3'

3 '... TA GC * T ACC TGT ACG ACA TAC ... 5'

Met ...

Again, the open reading frame and the ATG should be noted. Thus, binding to the Cla I site of the promoter clone should create a T-DNA active phaseolin gene. The phaseolin gene has a substitution of 2 amino acids for the 12 natural residues of the amino terminus.

5.1 Construction of a promoter vehicle

pKSIII which is a pE299 clone corresponding to the p403 T-DNA clone (see Fig. 1) is digested with 1α and then religated. The ligation mixture is used to transform K802 and made the selection for resistance to kanamycin. Plasmids are isolated by making mini-preps (plasmid preparations made from a small volume of cell culture) and resorption maps to verify the structure. The new vehicle, pKSprol, is not digested by HindIII but can be linearized by ClaI (Fig.12). pKS-pro is purified and linear molecules are produced by digestion with ClaI.

5.2 Binding of a partial phaseolin gene to a kanamycin resistance

A 3.03 t -b fragment containing extensive 3 * θ leader sequences all sequences

encodes of the phaseolin gene with the exception of the 5 'end was obtained by elution of an agarose gel after electrophoresis of the HindIII and BamHI digestion products of ρ7 · 2 (Fi.13), a pBR322 subclone of the phaseolin genomic clone 177 The construction of which was described in Example

6.1. This was mixed with and ligated to a HindIII / BamHI fragment with 3.0 kbp kanamycin resistance similarly isolated from pKS4 (Fig. 18) and pBR322 linearized with HindIII. After the restriction maps of the plasmids isolated from ampicillin resistant transformants were run, a plasmid having the structure shown in Fig. 7 was designated pKS4-KB.

5.3 Purification of the can / bean fragment of pKS4-3.OKB

pKS4-KB (Fig.7) θ was digested with 1α and the 4,9 kbp fragment purified by agarose gel electrophoresis.

5.4 Binding of the Can / Bean resistance gene to Clal-digested pKS-Prol.

pKs-pro is linearized by Clal digestion and the kanamycin / bean resistance gene fragment of Example 5,3 are linked to one another and used to transform K802. The myxin resistant transformants are selected and the plasmids isolated by mini-preps are made restriction maps to detect one with the appropriate orientation. The plasmid is designated pKS ProI-KB (Fig. 14). !

5.5 Transformation and expression

Cells containing pKS-proI-KB are conjugated to Agrobacterium cells containing pTI 15955 or pTi A60 or other appropriate TIP plasmids. After selection of the recombinants with kanamycin, the plants are inoculated and the crown galls are established in tissue culture. The assay for detection of phaseolin synthesis is as described in Examples 19 and 20.

Example 6

This example teaches the manipulations of a phaseolin gene, the main protein stored in the bean seed Phaseolus vulgaris, L., preparatory to other manipulations that insert the phaseolin gene into vectors described in other examples.

6.1 Subcloning of a phaseolin gene

A genomic clone of Choline 24AAG-PVP 177-4 (ar 177.4; SMSun et al (1981) Nature 289: 37-41, JLSlighton et al. (1983) Proc. Natl. Acad Sci USA 80, Pigl5 ) · Poi digested with BglII and BamHI. The 3.8 kbp fragment having the phaseolin gene and its linking sequences, isolated by agarose gel electrophoresis, was mixed with and ligated to BamHI linearized pBR322. The mixture was used to transform HB101 and selected the ampicillin resistant and tetracycline sensitive colonies. Restriction maps were made to the plasmids isolated from these clones. A plasmid having the structure shown in Fig. 16 was selected and designated AG-pPVPh3.8 (or alternatively, ρ 3.9). BglII and BamHI site binding inactivates both sites.

Another 177 * 4 subclone was constructed by EcoRI digestion. isolation of a 7.2 kbp fragment containing extensive 3 'flanking sequences and the entire phaseolin gene except for the 5' end and isolated after ampicillin selection of the HB101 transformants and the restriction map. A plasmid having the insertion so that the HindIII site of pBR 322 is adjacent the 5 'end of the phaseolin gene is distal; relative to the 3 'untranslated region, has been designated | AG-pPVPh 7.2 (or p * 7 * 2; Fig. 13, Sun et al. And Slightom et al., Supra) I

6.2 Cloning is the isolation of a myxin cane resistance gene

pRZ102 (RA Jorgenson et al. (1979) Molec Gen Gen 177, 65-72) a ColE-1 plasmid having a Tn5 transposon copy was digested with BamHI and HindIII. mixed with pBR322 (Fig. 17) previously linearized with those two enzymes, ligated and used for trans. forming K802. Plasmids, isolated from transformants selected for ampicillin and kanamycin resistance, were made restriction maps and one having the structure shown in Fig. 18 was designated pKS-4.

I

6.3 Binding of the phaseolin gene to a kanamycin resistance gene '

I

p 3-8 was digested with ClaI and BamHI and a 4.2 kbp fragment containing the phaseolin gene and some pBR322 sequences was isolated by agarose gel electrophoresis.

This was mixed with a Clal / BamHI fragment of Tn5 having a kanamycin resistance gene (neomycin phosphotransferase II) of pK54 (Fig, 18) and pBR322 (Fig. 17) which had been linearized with ClaI. The mixture was coupled and used to transform K802. After selection of the ampicillin and kanamycin resistant colonies, the plasmids were isolated and the restriction maps were made. A colony ^! having the structure shown in Fig. 19 was designated pKS-KB3.8.

I

!

Construction of another useful plasmid, pKS4-KB is described in Example 5 · 2.

Example 7

!

This example is analogous to the construction described in Example 5, except for the substitution of a phaseolin genomic clone by a clone j

of cDNA. This construct will result in a gene with no introes. ι

i

7.1 Construction of pKS4-KB2.4 (analogous to pKS4-KB). :

After digestion of pMC6 (Fig. 20) with EcoRI and BamHI, it is isolated to a 2.4 kPb phaseolin cDNA fragment by centrifugation through a salt gradient or gel electrophoresis. A fragment of 1.9!

kpb containing a kanamycin resistance gene is purified from the pKS4 digestion products with EcoRI and BamHI (Fig. 18), mixed with the EcoRI-ligated cDNA fragment and pBR322 bound to used to transform K802. The colonies are selected for cane resistance, mycin and after isolation of the plasmids and the restriction maps were made, a plasmid as shown in Fig. 21

was designated pKS4-KB2-4

7.2 Binding of the Ciai can / bean DNA to the digested pKS-Prol ClaI

pKS4-KB2.4 is digested with ClaI and linked to ClaI-linear pKs-pro (Fig. 12). After transformation, selection, plasmid isolation and characterization, the desired construct having the phaseolin sequences adjacent to the T-DNA promoter is transferred to a Ti plasmid. Inoculation and screening is as described in Examples 21, 19 and 20.

Example 8

This example teaches a method for removing introns from a gene. This is the same as putting a cDNA in a genomic environment. Restriction enzyme-sensitive sites are already found or are bound by site-specific metagenesis in the exons at both the 5 'and ¹ ' ends of the unprocessed transcription product. These sites exist both in the genomic clones and in the cDNA. The DNA contains introns and can be removed from the genomic clone and replaced by the corresponding non-intron cDNA clone delimited by the two sites. Reverse operation is also possible for genomic sequences containing introns can be placed in a cDNA environment. An internal fragment of the genomic clone is inserted into the corresponding space left by the cDNA clone. The latter strategy is analogous, though often technically more difficult, since the introns may contain sites susceptible to the enzymes chosen to create the exchange fragment. This difficulty can be overcome by careful selection of the partial digestion conditions and by purification of the desired fragment by electrophoresis on agarose gel. Further elaborations of this strategy include manipulation of individual introns within a gene leaving other introns and exons unaffected, and bit-by-bit sequencing when inconvenient restriction sites arise within introns as discussed above. I

8.1 Replacement of a fragment containing quadraphore introns by cDNA.

p 3.8, a plasmid clone of the phaseolin gene and its targeting sequences, was partially digested with EcoRI and completely SacI and a 6.4 kbp fragment containing the pBR322 vector and both the 5 '3' ends of the gene, was isolated by agarose gel electrophoresis. pcDNA 31, a clone in cDNA plasmid pBR322 made from the phaseolin mRNA, was partially digested with SacI and completely EcoRI and a fragment of 1.33 kbp containing the entire phaseolin cDNA except for the sequences of the terminations of the 5 'θ 3' ends, was isolated by agarose gel electrophoresis. These two fragments were ligated together and used to transform HB101. After selection of the colonies cell growth and plasmid isolation, restriction mapping, allowed to identify a sink, having the desired structure. This plasmid was described p3.S-cDNA (Fig. 22).

8.2 Use of p3.8-cDNA

It should be noted that p3.8-cDNA can replace the source of genomic DNA, eg ρ3 · 8, used in other Examples and that when so used results in analogous constructs differing only in the absence of introns.

Alternatively, this strategy can be used to remove introns from already constructed constructs.

Example 9

This example teaches expression

of a gene without introns. The phaseolin cDNA is prepared as described in Example 8, but a naturally occurring intronless gene can also be used.

A construction similar to

taught in Example 8. pKS4-K3 and pMC6 are digested with EcoRI and SacI as taught in Example 8 and as described therein the cDNA insert is attached to the pKS4-KB fragment containing the vector and the 5 'θ 3' ends of the gene of the phaseolin. The novel plasmid, pKS4-KBc, is used in constructs in a manner analogous to pKS4-KB.

Example 10

The purpose of this example is to teach the T-DNA placement of a cDNA of a Phaseolus vulgarus lectin under the control of the promoter of a T-DNA gene, the transfer of this construct, to a plant cell and the detection of the expression of this construct in the vegetable tissue. This construct utilizes a single-stranded adapter to bind the cohesive ends resulting from digestion with the restriction enzymes Pst I and Hind

III. When the PstI and HindIII sites

PstI

HindIII

5 '... C TCCA * G ... 3'

3 '... G * ACGT C ... 5'

5 '... A * AGCT T ... 3' 3 '... T TCGA * A ... 5'

are cut to form the following ends:

in the presence of an adapter

PstI

5 '... CTG0A

3 '... G

and mixed with each other in an appropriate sequence

HindIII

AGCTT ... 3 '

The ... 5 '

5 '... CTGCA AGOTT ... 3'

3 'G A ... 5'

+

3'ACGTTCGA5 '

may be attached to form the following structure:

5 '... C TGCA * AGCT T ... 3'

3 '... G * ACGT TCGA * A ... 5'

Note that the HindIII site is reconstituted.

The lectin cDNA is obtained from a plasmid clone, pPVL134, ATCC39181, which was constructed through the collection of polyC tails in double-stranded cDNA followed by insertion into a pBR322 with

polyG tails and cut with PstI. This clone is described by L. Hoffman et al (1982) Nucleic Acids Res.10: 78197828.

10.1 Adapter Synthesis

The 5 'AGCTTGCA3' adapter is synthesized by the method of Example 17

10.2 Construction of a clone containing the lectin cDNA and a kanamycin resistance gene.

pPVL134 is digested with Bell and

PstI and the intermediate size fragment containing the lectin coding sequence, the 3 'untranslated region and a C / G tail is isolated by elution of an agarose gel after separation by electrophoresis. pBR325 is digested with Bell and HindIII and the larger fragment is isolated after sedimentation through a salt gradient. The BclI / HindIII pBE325 vector is mixed with and connected to the lectin BclI / PstI fragment and the PstI / HindIII adapter prepared in Example 10.1. E. coli K802 is transformed, selected from drug resistance and presence of lectin sequence, and the plasmid isolated from such cells is designated as Ilo. The larger fragment resulting from the digestion of IIc with HindIII and BamHI is mixed with and ligated to the HindIII / BamHI fragment of pKS-4 having the kanamycin resistance gene and which was previously isolated by agarose gel electrophoresis (Fig. 23) K802 is transformed, colonies are selected for resistance to kanamycin, the plasmids are isolated and characterized by restriction maps. The desired plasmid is designated pL-B. !

10.3 Exchanging a Clal location for a BamHI location in pKS-pro

!

pKS-support, whose construction was discontinued ί crita in Example 5.1 (see Fig.12) is digested with Cla I _P 0;

The cut-off site is situated between the promoter and the ATG translation initiation signal of the transcription product with: 1.6 kbp (see Fig. 1). The cohesive ends are converted at the blunt ends by filling with DNA polymerase I. The BamHI adapters are bound to the range and chordate to expel bound BamHI ends and used to transform K802. Colonies having the desired plasmid, pKS-proIA (Fig. 24), are selected from

10.4 Insertion of lectin genes and kanamycin resistance in pKS-proIA

!

pL-B (Example 10.2) is digested; with Bell and BamHI and the fraction having the resistance gene.

i

to kanamycin and lectin sequences is eluted from an agarose gel after separation by electrophoresis. This fragment is mixed with and bound to the BamHI linearized pKS-proia. The bonding mixture is used to make sea E802. Plasmids are isolated from kanamycin-resistant colonies, characterized by restriction maps and the desired construct designated pLK-pro IA (Fig.25).

i

j

I

10.5 Expression in plants'

pLK-proIA is transferred to a Ti plasmid by triparental conjugation (Example 21) of K802 (pLK-proIA), E. coli (2013) and A.tumefaciens (pTil5955) (streptomycin resistant). After further conjugative transfer of pPH1 to Agrobacterium, double-homologous recombinants are selected by growth of the cells in kanamycin, streptomycin and gentamicin. The lectin is detected by E1ISA with the appropriate antibodies.

i

Example 11

The end of this example is to produce a Ti plasmid with a deletion from tms ("shoot induction" locus) to tmr (root induction locus) of the pTI

15955 and other octopine-like plasmids. This derivative is useful because cells transformed by it are easier to regenerate whole plants than cells transformed by pTil5955 with intact tms and tmr genes.

The p55955 with deletion in tms and tmr is finally modified in two ways by the activation of tms-tmr and the insertion of a foreign gene. These two changes are to be made at different points of the T-DNA each change is made independently by the other through different shuttle vectors. Each dependent change of a "shuttle" vector is selected independently as needed the use of at least two selectable tags in Agrobacterium In addition to the usual resistance to kanamycin, this example used a resistance to chloramphenicol derived from pBR325

11.1 Construction of a clone of the chloramphenicol resistance gene

pBR325 is digested with Hind III and ligated by cersed ends to HindIII linkers. The resulting preparation is digested with Hind III, performed selected for resistance to chloramphenicol (cam) and designated pKS-5 which will serve as the source of the HindIII / Bcl I fragment containing the gene (Fig. 26).

11.2 Construction of a T-DNA clone with a deletion and a cam gene in pBR322.

A 9.2 kbp linear DNA fragment is isolated from complete HindIII and partial BamHI digestion of p203. The fragment having the cam gene

is isolated from pKS-5, mixed with the bound 9.2 kbp linear fragment, used to transform E. coli, selected for resistance to chloramphenicol and desig

(I.e.

pKS-Octm. Cam 203 (Fig. 27).

pKS-oct. Cam 203 is a plasmid clone that can now be used to construct a series of TL-deletion pTi 15955 mutants. It contains the right arm of TL and a resistance gene for the left of the right folded. Several left TL arms can be ligated to the left of the cam gene (HindIII lcoal). For example if p02 is linked to deletion is 5.2 kbp and includes the totality of tms and tmr. If pl03 is connected to deletion it is 3.2 kbp and includes part of tms and all tmr. See Fig.

pKS-oct. Cam 203 is digested with HindIII. pl02 or p05 is digested with HindIII and the 2.2 kbp or 2.0 kbp T-DNA fragment is isolated and ligated to pES-oct. Cam 203 used to transform and isolate the pKS-oct.delll (Fig. 28), respectively. These constructs are transferred to A.tumefaciens by conjugation homologous recombination and selection of chloramphenicol resistant. Alternatively, the constructs for pEK290 are transferred using established methods by linearization of the plasmids with that of the Bam HI construct and binding to the BglII site of pEK29O (Flg3O). ;

Example 12

The TiO2 Ti column is annealed in this example by deletion of the T-DNA between the Hoal site and tmr

to the local Smal in tml. Plasmids Ti that may be j

i

The modified peptides include ptil5955, pTiB6, pTiA66 and the like. This construction is shown schematically in Fig.

12.1 Isolation of the cam gene

pKS-5 (Fig. 26) is digested with. Hind III and Bell. The minor fraction is isolated after separation on an agarose gel, as taught in Example 11.

12.2 Construction of a clone in pBR322 of the T-BRA with a deletion

The right arm of the I-DHA deletion is constructed by insertion of the BglII sites into the SmaI sites of p20f (See Fig. 1). p 'C ^r is digested by SmaI, ligated with BgIII adapter. digested with BglII, held and used pear transform K802. Ruma alternative construction, BamHI linkers may be substituted for Bgl II adapters and isolated suitable products of partial digestion with BamHI. 0 plasmid is designated p203-BglII and is digested with BglII and HindIII The BglII / HindIII fragment containing the vector is ligated to the chloramphenicol resistance fragment whose isolation was described in Example 12.1 After transformation of K802 the selection of resistant The resulting plasmid is designated p2f (Fig. 31).

12.3 Construction of the left arm of the clone with deletion

in T-BRA

HindIII sites are inserted into the HpaI site of p202 by Hpal digestion and binding to HindIII adapters. After the HindIII cohesive ends are exposed by digestion with this restriction enzyme, the 2 kbp Hpal fragment which is now HindIII ends is isolated. The Hpal fragment with HindIII ends produced by HindIII digestion is used for: transform K802. After isolation of a colony containing

-65-

the desired construct and its characterization the plasmid is designated p3e (Fig. 32).

12.4 Construction of clone with deletion in T-DNA

The left arm of the clone is obtained by purification of a 2 kbp fragment, pro-

I

by digestion of p3e with HindIII. eluting from an agarose gel after electrophoresis. p2f is cleaved with HindIII. treated with alkaline phosphatase, mixed with the 2 kbp fragment, bound and used to transform Î "802, followed by selection for resistance to chloramphenicol. Plasmids are isolated from individual colonies and characterized by restriction maps. A plasmid having the two arms in the desired tandem orientation is chosen and designated pKS-Oct. delIII (Fig.33). j

pKS-Oct.del III is transferred to A.tnmefaciens by conjugation and homologous recombinants are selected with chloramphenicol. Roots and shoots)

I

sunflower and tobacco are inoculated as described elsewhere

ι

Examples and tumors produced are tested for the production of opines.

Example 13

This example teaches a construct j with deletion of tmr and tml which constitutes an alternative to that taught in Example 12.

13 * 1 Construction of a chloramphenic-resistant fragment with a BglII site

pBR325 or HincII digested. ligm:

(Fig. 34). Fig. After conversion of £ 802 or GM33 the chloramphenicol resistant is selected. The resulting plasmid, pKS-6 serves as the source of BglII / BclI fragment having the cam gene.

13 · 2 Construction of clone with deletion of tmr and tml

p203 is digested with Hpal and Smal. After ligation by blunt ends to the BglII adapters is digested with BglII to expose the BglII cohesive ends. performed and used to transform K802. The desired construct is identified and designated p2 (Fig.35)

13.3 Construction of clone with deletion in T-DNA (pKS-oct.del. Illa) ·

The BglII fragment having the cam gene isolated from pKS-6 and ligated cut with BglII.

After conversion of  £ 802 chloramphenicol resistant are selected. The resulting plasmid is designated pESoct.del IIIa (fig.3θ) β is tested as described in Example 12.4.

Example 14

The purpose of this construction is to give an example of the mutation at the tmr locus only at the Hpal site by insertion of the chloramphenicol resistance gene. This gene is isolated as the BglII / BclI fragment from pKS-6 and is attached to the H20 site of p203 after this site has been replaced by a BglII site.

14.1 Conversion of the Hpal site to a BglII site

p203 is digested with Hpal. connected to BglII adapters. cut with BglII and reconnected. After transformation of K802, colonies and restriction maps are selected to detect the insertion of BglII sites (Fig. 37).

14.2 Isolation of the cam gene

pKS-6 is digested with BgHEe Bell:

The smaller fragment is isolated by agarose gel electrophoresis:

14.3 Construction of the T-DNA Mutated Clone

The modified p203 of Example 14.1 is digested with Bgl II, linked to the purified cam gene of Exeaplo 14.2 and used to transform K802. Baleooiona-sa! for resistance to chloramphenicol and after isolation of the resistant transformants and characterization by restriction maps, the plasmid is desired pKS-oct.tmr j (Fig. 38).

Example 15

Regeneration in this example involves carrot tumors induced by a TIF plasmid based on E1 and is done essentially as described by MD Chilton et al. (1982) Nature 295: 432-434. j

15.1 Infection with hair roots

Carrot discs are inoculated

with about 10594 bacteria in 0.1 ml of water. Segments 1 to 1.5 cm from the ends of the obtained roots are cut, placed on solid Monier medium (1-1.5% agar) without hormones (D. .Tepfer and JC Tempe (1981) CRHebd Seanc., Acad, Paris, 222: 155-156) and grown at 25-27 ° C in the dark. Cultures not contaminated with bacteria are transferred every 2 to 3 weeks and are subcultured in Monier medium without hormones and agar.

15 · 2 Regeneration of roots in plants

The root tissue in culture described

in Example 9.1 is placed in solid Monier medium (0.8% agar) supplemented with 0.36 æM 2,4-D and 0.72æM kinetin. After 4 weeks, the resulting callus tissue is placed in Monier medium without cormones. During one month of incubation at 22-25 ° C on a shaker (150 rpm), the callus dissociates to give a suspension culture from which embryos are differentiated, which when placed in Petri dishes containing Monier medium, without hormones, grow into seedlings. These seedlings are grown in culture and after "hardening" by exposure to atmospheres with progressively decreasing humidity, are transferred to the soil in an oven or on a bed.

15 · 5 Use of vectors that do not originate roots in hair

Ti-based vectors that do not

have functional tmr genes are used in place of R1-based vectors as described in Examples 15.1 and 15.2. Construction of suitable deletions is described in Examples 12, 13 and 14.

'ηΊΒΤΙ-Ο'

Example 16

The regeneration in this example involves tobacco tumors induced by a TIP-based TIP plasmid and is made essentially as described by KA Barton et al (1983) Cell.

16.1 Infection with crown galls

Tobacco tissue is transformed using an approach using inverted stem segments described by ACBraun (1956) Oan.Res.16: 55-56.

The stems are surface sterilized with a solution containing 7% commercial Chlorox and 80% ethanol, washed with sterile distilled water, cut into 1 cm segments, placed with the basal end up and the Petri dishes containing MS medium solidified with agar (T.Murashige and F.Skoog (1962) Physiol. Plant 15: 475-497) without hormones.

The inoculation is done by placing the basal surface of the stem cut with a syringe needle and injecting bacteria. The stems are grown at 25 ° C with 16 hours of light per day. The growing callus is removed from the upper surface of the stem segments, placed on solid MS medium containing 0.2 mg / ml carbenicillin and without hormones, transferred to fresh MS medium with carbenicillin three times at about of one month and are tested to see if the cultures contained with the bacteria. Axenic tissues are maintained on solid MS medium without supplements under the above described culture conditions (25 ° C; 16hr; 8h dark light).

jWm:

16.2 Culture of processed fabrics

From transgenic oxidative tissues obtained clones are obtained as described by A.Binns and P.Tteins (1979) Plant 145: 365-369 · Callus is converted into cell suspensions by culture in MS liquid medium having 0.02 mg / l naphthaleneacetic acid (NAA) at 25 ° C for 2 or 3 days with shaking at 135 rpm. and filtration through stainless steel filters first with 543 pm and then with 215 pm. The filtrate passed through the filters is concentrated, seeded in 5 ml of MS medium, having 0.5% molten agar, 2.0 mg / l NAA 0.3 mg / l 1 kinetin and 0.4 g / l Difco yeast extract having a density of about 8 x 10534 cells / ml . Colonies reaching a diameter of about 1 mm are peeled and grown on solid MS medium having 2.0 mg / l NAA and 0.3 mg / l kinetin. The resulting calluses are divided into pieces and tested for the transformed types.

16.3 Regeneration of plants

The transformed clones are placed in solid MS medium having 0.3 mg / l kinetin and grown as described in Example 16.1. The shoots that are formed create roots when placed in solid medium (1.0% agar) containing 1/10 of the concentration of the MS medium salts, 0.4 mg / l thiamine, without sucrose and hormones and had a pH 7.0. Root seedlings are grown in culture, hardened as described in Example 15.2 and wounds to the soil in an oven or bed.

16.4 Vectors used

The methods described in Examples 16.1, 16.2, and 16.3 are suitable for QL-based vectors

its functional tmr genes. Construction of suitable deletions is described in Examples 12, 13β. These methods are also effective when used with Ki-based vectors. The method described in Example 16.1 for infection of the inverted stem segments is often useful for the establishment of TIP-transformed plant cell lines.

Example 17

Techniques for chemical synthesis of DNA fragments used in these Examples utilize a number of techniques well known to those skilled in the art of DNA synthesis. Modification of nucleosides is described by H. Schallor et al (1963) J.Amer.Ohem.Soc. 85: 5820 and H. Buchi and HGEhorana (1965) J.Amer.Ohem.Soc. 82: 2990. The preparation of deoxynucleoside phosphoramidites is described by SLBeaucage and MH Caruthers (1981) Tetrahedron Let.22: 1859. Solid resin resin titration is described by SPAdams et al (1983) J.Amer. Chem.Soc. Hybridization processes useful for the formation of double-stranded synthetic linkers are described by JJossi et al (1982) J. Biol. Ohem. 257: 11070.

Example 18

Phaseolin is the most abundant protein reserve (approximately 50% of the total seed protein) of Phaseolis vulgaris. The transfer of the functional genes from phaseolin to alfalfa plants and translation of the m-BNA from the phaseolin in reserve phaseolin has significant economic importance at one time

which introduces a reserve protein in leaf material to be used as fodder. Alfalfa is an important plant for the transfer and expression of the phaseolin gene due to its acceptability as cattle fodder, its rapid growth, its ability to fix nitrogen through Rhizobial symbiosis, its susceptibility to infection by crown galls and to their ability to regenerate alfalfa plants from isolated cells or protoplasts. This example teaches the introduction of a sustaminable phaseolin gene to be expressed in whole alfalfa plants.

18.1 Construction of the "shuttle" vector.

Alfalfa plants are regenerated from crown-gill tissue containing engineered-modified Agrobacterium plasmids as described below. In the first step a "shuttle vector" is constructed containing a T-DNA mutant tmr 5-4 and another tms-5 bound to a phaseolin structural gene under the control of a T-DNA promoter. This construct is, in turn, linked to a nopaline synthetase promoter which has an adjunct functional neomycin phosphotransferase (NPTII) structural gene (published by MDChilton, et al. (January 18, 1983) ^{at the} Winter Symposium of Miami; also see JL Marx (1983) Science 219: 830 β R. Horsch et al (18 January, 1983) 15th Miami winter Symposium). This type of construction is illustrated in Example 1.

18.2 Transfer to Agrobacterium and plant cells

The "shuttle vector" is then used to transform by conventional techniques (Example 21) an Agrobacterium strain containing a Ti plasmid such as pTil5955. Bacteria containing recombi-73-

are selected and co-cultivated with alfalfa protoplasts which have regenerated cell walls (Marton et al (1979) Nature 277: 129-131, GJ Wullems et al (1981) Proc. Natl Acad Sci. 8: 4344-4348; and RBHorsch and RT Fraly (18 January 1983) 15th Miami Winter Simpésito).

The cells are cultured and the resulting callus tissue is tested for the presence of appropriate mENA by Northern blotting (Example 19) and presence of appropriate proteins after ELISA assays (Example 20) (see JLMarx (1983) Science 219: 830: RBHorsch and RTFraley (18 January 1983) 15th Miami Winter Simpésio).

18.3 Regeneration of plants

Alfalfa plants are then regenerated from callus tissue by methods similar to those previously used by AVP dos Santos et al (198)

Z. Pflanzenphysiol. 99: 261-270. TJ McCoy and ETBingham (1977) Plant. Know. Leters 10u 59-66 and KA Walker et al (1979) Plant. Know. Leters 16: 23-30. These regenerated plants are then propagated by conventional plant crossing techniques forming the basis of new commercial varieties.

Example 19

In all Examples the SNA was extracted, fractionated and detected by the following processes.

19.1 Extracting RIA

This process is a modification of Silflow et al. (! 981) Biochemistry 13: 2725-2731. Substitution of the GsCl centrifugation by LiO1 precipitation has been described by Murray et al (1981) J. Mol. 42.

The use of 2M LiO2 plus 2M urea to precipitate was taken from Bhodes (1975) J. Biol. 25: 8088-8097.

The tissue was homogenized using a polytron or glass ground homogenizer in 4-5 volumes of cold Tris-H01 5θ® (pH 8.0) containing 4% p-amino salicylic acid, 1% tri- isopropylnaphthalenesulfonic acid, 10mM dithiothreitol (made at the time) and 10mM Nametahisulfite (made at the time). To control foaming an octanol was used. An equal volume of Tris-saturated phenol containing 1% 8-hydroxyquinoline was added to the homogenate which was then stirred to emulsify and centrifuge at 20,000-30000 g for 15 minutes at 40 ° C. The aqueous upper phase was extracted once with chloroform / octanol (24: 1) and centrifuged as above. A concentrated solution of LiCl-urea was then added to a final concentration of 2M each and the mixture was allowed to stand at 20 ° C for several hours. The SNA precipitate was then centrifuged and washed with 2M LiO2 to disperse the pellet. The precipitate was then washed with 70% ethanol 0 3 M aa-acetate and dissolved in sterile water sufficient to give a clear solution. Half of the ethanol volume was acidified and the mixture put on ice for 1 hour, after which it was centrifuged; to remove various polysaccharides. The SNA precipitate was then recovered and redissolved in water or buffer! poly (U) without salt.

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19.2 Poly (U) / Sephadex Chromatography |

Two poly (U) sephadex (trade mark): Pharmacia, Inc. Upsala Sweden) buffers were used containing the first salt containing Tris 2mM, EDTA, 1mM and 0.1% J SDS β second with 0.1M NaCl added to the first. To obtain good results for A42605, a tam-;

2x stock bread and the salt added to one part. After adjusting the final concentrations, the buffers were autoclaved.

Poly (U) Sephadex was purchased from Bethesda Research Laboratories and 1 g of poly (U) Sephadex was used per 100 pg of poly (A) RNA. The poly (U) Sephadex was hydrated in poly-UU buffer without salt and poured onto a column with a jacket. The temperature was raised to 60øC and the column was washed with unsalted buffer until the baseline at 260 nm was horizontal. Finally the column was equilibrated with the poly (U) buffer salt containing 40 ° C salt. RNA at a concentration of less than 500 μg / ml was then heated in salt-free buffer at 65 ° C for 5 minutes, after which it was cooled on ice and NaCl added at a concentration of 0.1M. The RNA was then placed on the column which should have a flow of not more than 1 ml / min until the optical density has dropped to a stable baseline. The column temperature was then raised to 60øC and the RNA was eluted with unsalted poly (U) buffer. BM. Normally, it leaves three! column volumes. The eluted RNA was then concentrated with! butanol for a convenient volume after addition | NaCl to 1mM precipitated with 2 volumes of ethanol. The ethanol precipitate was dissolved in water and NH445-acetate to 0.1M, followed by reprecipitation in ethanol. Finally the RNA was redissolved in sterile water and stored! at -70 ° C.

1

19.3 Gels of RNA in formaldehyde;

!

The method used was Thomas (1980) Proc. Natl. Acad. (USA) 77: 5201 and Hoffman, et al (1981) J. Biol. Chem. 256: 2597

i

Gels of 0.75-1.5% agarose containing 20 mM Na-phosphate (pH6.8-7.0) were made. If bands of high molecular weight aggregates appeared then experiments were repeated with the addition of 6% formaldehyde or 2.2M (use of the 36% stock solution) to the gels. The formaldehyde was added to the agarose after cooling to 65 ° C. The addition of formaldehyde makes visualization with ethidium bromide more difficult. The electrophoresis buffer was 10 mM Na-phosphate (pH 6.8-7.0).

Prior to electrophoresis the ENA was treated with a denaturing buffer having the final concentrations of 6% formaldehyde, 50% formamide, 20 mM Na-phosphate buffer and 5 mM EDTA. The ENA was incubated in the buffer at 60 ° C for 10-20 minutes. The incubation was terminated with the addition of stop buffer. To a sample of 20 ^P-1 was added 4pl% glycerol at 5θ · EDTA lQmM Na-phosphate, 5 mM bromophenol blue.

Submerged electrophoresis was used. The ENA was applied prior to submerging the gel and entered the gel at 125 mA for 5 minutes. The gels were then submerged and the stream reduced to 3θ (overnight) or 50 mA (6-8 hours). The buffer was recirculated and electrophoresed in a cold chamber.

19.4 "Northern"

If the gel is used for transference to detect a specific ENA it is not stained; A separate strip is used with markers to blush. The staining is done with 5æg / ml of ethidium bromide in 0.1M Na-acetate, and the discoloration is for several hours in Na₂; 0.1M acetate. Treatment in water at 60-70 ° C for 5-10 minutes before staining helps visualization.

A gel to be transferred is soaked for 15 minutes in standard saline citrate (SSC)

10 χ -3% formaldehyde. If large SNA molecules are not eluted from the gel then a pretreatment in 5θ NaOH for 10-30 minutes helps to break the SNA.

If using the base treatment, the gel is neutralized and soaked in SSC-formaldehyde prior to transfer. The transfer of ENA to nitrocellulose-j was done by "Standard" methods.

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Prehybridization was done at 42 ° C. For a minimum of 4 hours in 5θ% formamide, 10% Indeed dextran, 5 ^x 550, 5 x Denhardt's solution, 100 ug / ml I denatured carrier DNA, 20 pg / ml of poly (A), 40mM Na-phosphate (pH 6.8-7.0) and 0.20% SDS. Hybridization was done by adding the probe to the same buffer with incubation overnight. The probe was not used at more than about 5 x 10554 cpm / ml. j

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After hybridization, nitrocellulose! was washed a number of times at 42 ° C with 2x SSC, 25 mM Na-phosphate, 5 mM EDTA and 2 mM Na-pyrophosphate followed by a final wash for 20 minutes at 64 ° C in 1 x SSC. The best results were obtained in case the filter was not dried prior to autoradiography and the probe removed by washing abundantly in 1mM EDTA at 64Â ° C.

Example 20

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Western blotting to detect antigens after polyacrylamide gel electrophoresis, SDS, _{Form I-} 00 described by, SP

Legocki and DPS Verma (1981) Analyt. Biochem.III: 385-392. ;

Micro-ELISA (enzyme-l _i inked immuno-assay assay) assays. ! were made using 96-well Immulon-2 type plates by the following steps:

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On day 1, the wells were coated with a 1: 1000 dilution of antibody (rabbit anti-phosphokinase IgG) in 200 μl / well coating buffer incubated at 37 ° C for 2-4 hours. The plates were covered with Saran Wrap. The plates were then washed three times with phosphate buffered saline-Tween buffer (PBS-Tween) leaving a 5 minute interval between each wash. 1% bovine serum albumin (BSA) was added to the lavage solution and, after addition to the well, allowed to stand for 20 minutes before being discarded. The wash was repeated five more times with PBS-Tween.

20.2 Homogenization of tissues

The tissue was cut into small pieces and then homogenized with polytron using 1 g of tissue / ml of phosphate buffer-Tween-2% polyvinyl pyrrolidone-40 (PBS-Tween-2% FVP-40) buffer. All samples were stored on ice before and after grinding and made standard phaseolin curves. A standard curve was made with tissue homogenates and another standard curve was also made in buffer to test for phaseolin recovery when comminuted in the tissue. After centrifugation of the homogenized samples, 100 μl of each sample was placed in a well and left overnight at 4 ° C. To avoid error: Duplicates of each sample were made. The plates were sealed during incubation.

20.3 Binding enzyme

After overnight incubation, the antigen was discarded and the wells washed five times with PBS-Tween leaving a 5 minute interval between each wash.

One conjugate (rabbit anti-phosphokinase IgG bound & alkaline phosphatase) was then diluted 1: 3000 in PBS-Tween-2% PVP containing 0.2% BSA and 1x 50æl was added to each well; followed by incubation for 3-6 hours at 37 ° C. After incubation the conjugate was discarded and the wells were washed five times with PBS-Tween, five minutes between each wash as before.

20.4 Testing

Immediately prior to the test, a 5 mg p-nitrophenyl phosphate tablet (purchased from Sigma and stored frozen in the dark) was added to each 10 ml of substrate and vortexed until the tablet was dissolved. Rapid addition was added. 200 æl solution at room temperature at each well. The reaction was measured at various times, eg t 0, 10, 20, 40, 60, 9θ, β 120 minutes, using a Dynatech micro-ELISA reader. When the colorless p-nitrophenyl phosphate is hydrolyzed by alkaline phosphatase to inorganic phosphate and p-nitrophenol, the latter compound gives the solution a yellow color which can be read on the spectrophotometer at 410 nm. The lower limit of detection is less than 0.1 mg.

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Triparental conjugations were generally achieved as described below; other variations known to those skilled in the art are also acceptable.E.coli K802 ("shuttle vector" based on pRK 290) was conjugated to E.coli (pKE2013) and to a streptomycin-resistant S.tumefaciens strain. 0 |

pEE 2013 was transferred to the carrier strain of the "shuttle vector" and mobilized the "shuttle vector" for transfer

-80-

for Agrobacterium. Growth in a medium containing streptomycin and the drug the "shuttle vector" is resistant, often kanamycin or chloramphenicol, resulted in the selection of Agrobacterium cells containing "vector" sequences. A conjugation of these cells with E. coli (pEH11) resulted in the transfer of pPH11 to the Agrobacterium cells. The "shuttle vectors" pPH1J1 and pEK290-based can not coexist for a long time in the same cell. Growth in gentamicin to which pPH1J1 has a resistance gene resulted in the selection of cells having lost the pEX290 sequences. The only cells resistant to streptomycin, gentamycin and kanamycin or chloramphenicol are those having Ti plasmids that have undergone homologous double recombination with the "shuttle vector" and which now have the desired construction.

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Claims (1)

over there. A method for genetically modifying a plant cell, characterized in that the steps of: (a) insertion of a plant structural gene into a T-DNA containing a T-DNA promoter, the promoter and the plant structural gene in position and orientation relative to one another, allowing the expression of the vegetative structural gene in a cell under the control of the T-DNA promoter thus forming a promoter combination of the T-DNA / plant structural gene and then (b) transfer of the T-DNA / plant structural gene promoter combination to a plant cell. 2ã. A method according to claim 1, characterized in that it further comprises, following the execution of step (b), the step of: (c) detecting the expression of the plant structural gene in a plant cell containing the combination of the T-DNA promoter / plant structural gene. A method according to claim 1, characterized in that the plant structural gene contains one or more introns. 4â. 2. A method according to claim 1, wherein the plant structural gene encodes a protein accumulated in seeds. D-. 2. A method according to claim 1, wherein the plant structural gene encodes phaseolin. -88 ' 6â. A method according to claim 1, wherein the T-DNA / plant structural gene promoter combination is maintained and replicated prior to step (b) as part of a shuttle vector, . 7 ^. A method according to claim 1, characterized in that the promoter of step (b) is selected from the group consisting of transcription promoters tmr, tml, tms, nopaline synthase, octopine synthetase and "1,6". 8ã. A method according to claim 1, wherein the plant structural gene is fused to a T-DNA structural gene, whereby the structural gene combination of the T-DNA / plant structural gene encodes a fusion protein comprising the sequences proteins encoded by T-DNA and the plant protein. 9a. 2. A method according to claim 1, wherein the plant cell is a dicotyledoneous plant. I 10ã. 2. A method according to claim 1, i characterized in that the dicotyledonous plant is a member of the Compositeae or Leguminoseae. III. Plant, plant tissue or a plant cell when produced according to the method of any one of claims 1-10. OFFICIAL OFFICE OR INDUSTRIAL PROPERTY Rua Vlotor Cordon, lOA-l.® LISBOA Kilo phases of Bases 25 L V O LO -Q r Q. T " O Q The r co O O H- O_ CM Q. O co Q. 1_1 CM -Oh T "" CM O V CO The co O co LU The T " rt co O rk CM to <-> ro co CM H- O r- CM O- π ffl õ " 't- CM O O) O "'jz' co LJL O Q K L- < iO- (I.e. ro z rt CM O co co O T * " The The co < H" O- co co Ο®S flL 00 0> oo LO LO σ> LO ίο. co LO t co Only «Ul •H halo (I.e. < z Q I Itis 00 O ct (I.e. Ro ro and c o frog O O •H -P T the (I.e. < * 3 icd O LU m CO fi (I.e. ω z <P> (I.e. frog m p (I.e. Ri 3 ra !O O A O O The Ctí O THE fi fi (I.e. PM -p 03 FIG. 2 TCAGGGATCCTTTTTACCGACAACTCATCCACATTGATGGTAGGCAGAAAGTTAAAGGATTATCGCAASTCAATACTTCCCATTCATTGATrTATTTAA .................................................. 1 £ V AGSTST5fiCCTCAAG6ATAATCSCCAAACCATTATATTT6CAATCTACCAAATSeCTAAAâTSfiCAATTTTSeeT8CSe6AAACeTfieCTCTTAfTCní ... ».............................« .......... JOfl MALAGA »ValAlall« LíuGlyA, aGlyA »flYa1A1aL« uT >> rL »uA CAeSTGATCTCGCCCGGAGGCTCGGCCAGSTGTCCTCAATCTGGGCGCCAATCTCCAACAGGAACAeCTTCAACTCTSTGAMTCecnGeCTCCnSÍA + ........................................ * ......... * JPO laGlyApLeuAlaArgArgLeuGlyeinValSerS «rIleTrpAlaProIseerAinArgA» nSerPhíA »nSirValArgS» rLl GCTAGTAGGGCCGGACTATGGAGGCGACTTTCAGCCGCAATTGGAGGATGATCTTGAAACAGCGATTTCAGGCGCGGCGTTCArTTTTCTTACGGTCCCG uLe 'V' lGlyProApTyrG1y61yA $ pPheGlnPro61nLeuGluAtpAjpLeuGluThrAlaneSerGlyA1aAHPhenePheLeuThr ', Pre ACCATGGGCCA6CAAGGAATTCnTGCGAGTTG6CGAACTTCAATCTGAGCAGCTCGGTCCTCGTAGCATTGCCCGGTAGCGCAACGTCTeTGGCAT6CA ThrHetGlyeineinGlylleLeuCyíGIuLeuAÍaAínPheAinLeStrSerSerValLíuValAlaLíuProGlySerAlaThrSírLêuAtaCyaL TGF Eur-lex.europa.eu eur-lex.europa.eu .................................................. 600 yjG1nThrLeuThrProA1aPheAiAaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa TGTGAAGA6AAC6TTCSAA6TT6C6TCAACTCAGGCTTT6A6CGAAGAGGTTAGGGGGGGCTTCGAGATTCTCTTTCCAAATCGGCTTCAGTGGTATCAA ..............................................- .. 700 R¥P »» »» Arg Arg »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» »» » AATCCCGCGTC6ATATTTTTTTCAAATACCAACCCT6TT6CTCACCCT6CTGGAATTCTCGCTGCAAAAGATACCATCGAGCAAG6GATATCGCC6ATCC ................................................. 800 AsnProA1a5erI1ePhePhIerAinThrA »nProYalA1aH1» ProAlaeiyIltLauA1iAlaLyíAjpThrIleG1uGlnG1yI1iSíProIleP CAAAATTCTACCGGAA6TTTGTCCCACAAeCCATCAC6CeT6TTACCGCAATA6ACGAGGAACGCCTTACGATTGTTAATGCGCTTGGACTT6AGTCCGA roLy $ Ph * TyrArgLyjPhtYalProeinA1in «ThrArgYalThrAlaIlíA» p61uGluArgLeuThrI1eVa1AsnAlaLeuG1yLeuGluSerGl 6ACG6ATTTC6CATACTeCAAAAAATGeTATGeTG66CACGCCA6CAACGCAAGAGAATTCTATGAGACCTTTGAGGGCTACGCTGATATCGAAACTCCA 9 .... 9 .... 9 .... 9 .... 9 .... 9 .... .............................. uThrA $ pPh «AlaTyrCy» Ly $ lyjTrpTyreiyeiyH1 »A1aS« rA »nA1aArg61uP» i <TyrG1uThrPheGluG1yTyrAlaAspIleGluThrPro AGAAACATeAACCATCeCTACCTTAeTGAGGACGTeAAeC * CATTTTeeTGCTCTGGGTTGAGATA6CTGAAGTGATCGGCGTGCAAGTTCCAGAAaTGA ApgA »nHitAanH1jArgTyrt» uS «reiuA» p »alLyíHUn« Leu »a1LwTrpValGluníAlaG1uVaineG1yVa1G1n ¥ alProGluNetL AATCT6TA6T6CA6GAGeCAAGC6ACeTGCTeAACeA6eACeTeTCTCATACT6GTA6GGG6CTATCGTCTCTCAACCTGGMGGTTCAAACGCGAATSC ......................... 1200 and, SerYalYalGln61uAlaS «rA» pYalLwA »neiuA» pt.euS «pH1» Thr61yAr9GlyííuSerSírLeuAsnLiuG1uG1ySerAsnAlaA nAlAl OATAGTCAGGGCTCTCAATGeAGTTTGAATCAAATCTTCCAGCTGCrrTAATGAGATATGCGAGACeCCTATGATCGCATGATATTTGCTTTCAATTCTG _________ 9 ......... 9 ......... 9 ......... 9 --- .......------- Eur-lex.europa.eu eur-lex.europa.eu alIeYalArgAlaLeuAtneiyVaÍEnd ........................, ... nGTGCACGneTAAAAAACCTGAGCATSTGTAenCAeATCCTTACCGCCfiGTTTCGGnCATTCTAATGAATATATCACCCGTTACTATCGTAnTTT 1400 FIG. 3-1 1ZZ.4 CATATGCeTGTCATCCCATGCCCAAATCTCCATGCATGTTCCAACCACCTTCTCTCTTATATAATACCTATAAATACCTCTAATATCACTCACTTCTTTC ......... 4 ......... 4 ..------ 4 .-------- 4 --------- 4- -------- 4 --------- 4 --------- 4 ------... 4 ______... 4> 1 177.4 ATCATCCATCCATCCA6AfiTACTACTACTCTACTACTATAATACCCCAACCCAACTCATATTCAATACTACTCTACTATSATGAGA6CAAGGGTTCCACT 4 ......... 4 ......... 4 ... --- ... 4 --------- 4 --------- 4 4 4 _________ 4 --------- 4 ------- .. 4 100 C0NA31 ATCATCCATCCATCCA6AGTACTACTACTCTACTACTATAATACCCCAACCCAACTCATATTCAATACTACTCTACTATGATGAGAGCAAGGGTTCCACT Z (5'-FHetHetArgAIaArgVa, Prole C * p 177.4 CCTGTTGCTGG6AATTCTTTTCCTGGCATCACTTTCTGCCTCATTTGCCACTTCACTCCGGGAGGAGGAAGAGAGCCAAGATAACCCCTTCTACTTCAAC ......... 4 ......... 4 ......... 4 ......... 4 -... 4 . 4 ......... 4 ......... 4 ......... 4 200 CDNA31 CCTGnGCTGGGAAKCTrrTCCTGGCATCACrrrCT & CCTCArrTGCCACTTCACTCCGGGAGGAGGAAGAGAGCCAAGATAACCCCTTCTACTTCAAC uleuLeuLeu61yI1eLeuPleleuAlaSerLaSerAlaSerPheAliThrSerLeuArgGluG1uGluGluSerGlnAspAsnProPheTyrPheA5n 177.4 TCTGACAACTCCTGGAACACTCTATTCAAAAACCAATATGGTCACATTCGTGTCCTCCAGAGGTTCGACCAACAATCCAAACGACTTCAGAATCTTGAAG ......... 4 ......... 4 .... ----- 4 --------- + _________ 4 .._______ 4 _________ 4 _______ .. 4 .________ 4. 4 300 CDNA31 TCTGACAACTCCTGGAACACTCTATTCAAAAACCAATATGGTCACATTCGTGTCCTCCAGAGGTTCGACCAACAATCCAAACGACTTCAGAATCTTGAAG SerAípAsnSerTrpAínThrLeuPhelysAsnGlnTyrGIytUsIleArgValleuGlnArgPheAspGlnGlnSerLysArgleuGlnAsnleuGluA 177.4 ACTACCGTCnGTGGAGTTCAGGTCCAAACCCGAAACCCTCCTTCTTCCTCAGCAGGCTGATGCTGAGTTACTCCTAGTTGTCCGTAGTGGTAAGTAATT 4 ......... 4 ......... 4 ......... 4 ......... 4 . ......... 4. CDHA31 ACTACCGTCTTGTGGAGTTCAGGTCCAAACCCGAAACCCTCCTTCTTCCTCAGCAGGCTGATGCTGAGTTACTCCTAGTTGTCCGTAGTG jpTyrArgLeuValGIuPheArgSerLysProGluThrleuleuLeuProGlnGlnAlaAspAlaGluLeuLeuValValArgSerG 177.4 GCTACTGGTATCACnGTTTCTTCTTGCAGAAATAATGGTAATGAGTTTTTTATATCTACTCGTCTTGGTGAAACCTGATGATCGC ......... 4 ......... 4 .....- .. 4 .......... 4 ..................... 4 _________ 4 _________ 4 _________ 4 _________ 4- 4 500 C0HA31 (IVS 1.72 bp) GGAGCGCCATACTCGTCTTGGTGAAACCTGATGATCGC JySerA1 aIleteuVaILeuVaILysProAspAspArg AGRICULTURE ......... 4 ......... 4 ......- .. 4 ......- .. 4 ......... 4 Eur-lex.europa.eu eur-lex.europa.eu 600 CDHA31 AGAGAGTACTTCTTCCTTACGAGCGATAACCCGATATTCTCTGATCACCAGAAAATCCCTGCAGGAACCATTTTCTATTTGGTTAACCCTGATCCCAAAG Arg61uTyrPhePheLeuThrSerAspAsnProI1ePheSerAspms61nLysIleProAlaGlyThrIlePheTyrLeuValAsnProAspProlysG AGRICULTURE ......... 4 ......... 4 --------- 4 --------- 4 --------- 4 --------- 4 --------- 4 --------- 4 --------- 4 ......... 4 700 C0HA31 AGGATCTCAGAATAATCCAACTCGCCATGCCCGTTAACAACCCTCAGATTCAT (IVS 2, 1uAspLeuArgI1eIleGInLeuA1aMetProValAsnAsnProGlnI1eH1s 177.4 CAATTTCTCTCCAAATSTGATGATAAATGTTTGTCCTGTAGGAATTTTTCCTATCTAGCACAGAAGCCCAACAATCCTACTTGCAAGAGTTCAGCAAGCA --------- 4 --------- 4 --------- 4 -------- 4 --------- 4- -------- 4 --------- 4 --------- 4 --------- 4 --------- 4 800 CDNA31 88 bp) GAATTTTTCCTATCTAGCACAGAASCCCAACAATCCTACTTGCAAGAGTTCAGCAAGCA GluPhePheleuSerSerThrGluAlaGInGInSerTyrleuGlnGluPheSerLysH, 177.4 TATTCTAGAGGCCTCCTTCAATGTAAGAAAGAAAACAGCATCTAACTACATAnTGCGTTGCCATTTAGCTAGTACTTTGTCTAAATGTCACACTTGTTG 4 ------------------ 4 ------ 4 ---- 4 --------- 4 ......... 4 ......... 4 ......... 4 900 C0NA31 TATTCTAGAGGCCTCCTTCAAT (IVS 3, 124 bp, sIleLeuGluAlaSerPheAsn 177.4 AATTTGTTGAATGATATCATTATATATGTTTGCATGATTTTTATAGAGCAAATTCGAGGAGATCAACAGGGTTCTGTTTGAAGAGGAGGGACAGCAAGAG --------- 4 -------- 4 --------. 4 ... ------ 4 --------- 4- -------- 4 ......... 4 --------- 4 ......... 4 ......... 4 1000 CDHA31 AGCAAATTCGAGGAGATCAACAGGGTTCTGTTTGAAGAGGAGGGACAGCAA6AG SerLysPheG1uGluI1eAsnArgValLeuPheG1uGluGluGlyG1nG1nGlu 177.4 GGAGTGATTGTGAACATTGATTCTGAACAGATTAAGGAACTGAGCAAACATGCAAAATCTAGTTCAAGGAAATCCCTTTCCAAACAAGATAACACAATTG ......... 4 ......... 4 ......... 4 --------- 4 ......... 4 ......... 4 ......... 4 ......- .4 ......... 4 ......... 4 HOO CDHA31 GGAGTGATTGTGAACATTGATTCTGAACAGATTAAGGAACTGAGCAAACATGCAAAATCTAGTTCAAGGAAATCCCTTTCCAAACAAGATAACACAATTG GlyValIleValAsnlleAspSerGIuGlnneLysGluLeuSerLysHIsAlaLysSerSerSerSerArgLysSerLeuSerLysGInAspAsnThrlleG 177.4 GAAACGAATTTGGAAACCTGACTGAGAGGACCGATAACTCCTTGAATGTGTTAATCAGTTCTATAGAGATGGAAGAGGTAAATACAAAGAAAAACCATAT .4 _________ 4 _________ 4 --------- 4 --------- 4 --------- 4 ....----- 4 .------- 4 ......... 4 --------- 4 1200 C0HA31 GAAACGAATTTGGAAACCTGACTGAGAGGACCGATAACTCCTTGAATGTGTTAATCAGTTCTATAGAGATGGAAGAG IyAsnGluPheeiyAsnLeuThrG1uArgThrAspAsnSerLeuAsnVa, LeuIleSerIleGluHetGIuGlu 177.4 agacaaactcagcaattgagttctattattcactgtcgtcttggttagaaaatcttagtattgagactataattaaataatggttttttttgtataaaaa ----------------------------- 4 ......... 4 --------- 4 --------- 4 --------- 4 --------- 4 --------- 4 Π00 (IVS 4, 128 bp) 177.4 tttagggagctctttttgtgccacactactattctaaggccattgttatactagtggttaatgaaggagaagcacatgttgaacttgttggcccaaaagg -... 4 ......... 4 ......... 4 ......... 4 --------- 4 4 ......... 4 ......... 4 ......... 4 ......... 4 HOO CDNA31 GGAGCTCTTTTTGTGCCACACTACTATTCATAGTAGTGGTTAATGAAGGAGAAGCACATGTTGAACTTGTTGGCCCAAAAGG GlyAlâleuPheVa, 177 4 AAATAAGGAAACCTTGGAATATGAGAGCTACAGAGCTGAGCTTTCTAAAGACGATGTATTTGTAATCCCAGCAGCATATCCAGTTGCCATCAAGGCTACC --------- 4 ......... 4 --------. 4 ......... 4 ......... 4 4 ......... 4 ......... 4 ......... 4 ......... 4 1500 C0NA31 AAATAAGGAAACCTTGGAATATGAGAGCTACAGAGCT6AGCTTTCTAAAGACGATGTATTTGTAATCCCAGCAGCATATCCAGTTGCCATCAAGGCTACC yAinLysG1uThrLeuG1uTyrG, uSerTyrApgAlaG1uLeuSerLy $ AspAspVaIPheVaineProA1aA1aTyrProVa1A, aIleLysAlaThr 177 4 TCCAACGTGAATTTCACTGGTTTCGGTATCAATGCTAATAACAACAATAGGAACCTCCTTGCAGGTATATATATTTATTATATATGACCATGAATTTGAA --------- 4 --------- 4 ......... 4 --------- 4 ......... 4 ......... 4 ......... 4 --------- 4 ......... 4 --------- 4 1600 C0NA31 TCCAACGTGAATTrCACTGGTTrCGGTATCAATGCTAATAACAACAATAGGAACCTCCTTGCAG SerAsnValAsnPheThrGIyPheGlylleAsnAlaAsnAsnAsnAsnArgAsnLeuleuAlaG 177.4 tatagggttgttgatggaattttttatttataattggtaatgcgtgattgtgattgtaaatatgaaggtaagacggacaatgtcataagcagcatcggta _________ 4 ......... 4 ......... 4 ......... 4 ......... 4 --------- 4 -....... 4 ......... 4 ......... 4 .......... 1700 CDNA31 (IVS 5, 103 bp, GTAAGACGGACAATGTCATAAGCAGCATCGGTA lylysThrAspAsnValIleSerSerlleGlyA FIG. 3-2 177.4 6AeCTCTG6ACGGTAAAGACGTGTTGGGGCTTACGTTCTCTGGGTCTGGTGACGAAGTTATGAAGCTGATCAACAAACAGAGTGGATCGTACTTTGTGGA F -------- + 1800 C0NA31 GA6CTCT & GACGGTAAAGACGTGTTG & GGCTTACGTTCTCTG & GTCTGGTGACGAAGTTATGAAGCTGATCAACAAACAGAGTGGATCGTACTTTGTGGA r9Alal.euAspeiyl.yiAspValt.eiieiyt.íuThrPheSerG1ySerGlyAspG1uValHetlysleuIleAsnLyíGlnSerG1ySerTyrPheValAs 177.4 T6CACACCATCACCAACAGGAACAGCAAAAGGGAA6AAAGGGTGCATTTGTGTACTGAATAAGTATGAACTAAAATGCATGTAGGTGTAAGAGCTCATGG Eur-lex.europa.eu eur-lex.europa.eu Eur-lex.europa.eu eur-lex.europa.eu C0NA31 TGCACACCATCACCAACAGGAACAGCAAAAGGGAAGAAAGGGTGCATTTGTGTACTGAATAAGTATGAACTAAAATGCATGTAGGTGTAAGAGCTCATGG pAlaHfsH1sH1sGlnGln6luGlnGlnLysGlyArgLysGlyAlaPheValTyrTER AGRICULTURAL 2000 CDHA31 AGAGCATGGAATATTGTATCCGACCATGTAACAGTATAATAACTGAGCTCCATCTCACTTCTTCTATGAATAAACAAAGGATGTTATGAT --- (A) k TTACGTTTGGAACTGACAGAACCGCAACGTTSAAGGAGCCACTGAGCCGCGGGrrrCTSGAGTTTAATGAGCTAAGCACATACGTCAGAAACCATTATTG - + - .-- + - .-- + .- ♦ 200 CGCGTTCAAAAGTCGCCTAAGGTCACTATCAfiCTAGCAAATAITTCTTGTCAAAAATGCTCCACTGACGTTCCATAAATTCCCCTCGGTATCCAAGAGA --.-- + -, - ♦ -, - + 300 eTCTCATATTCACTCTCAATCCAAATAATCTGCAATGGCAATTACCTTATCCGCAACrrCÍTTACCTATTTCCGCCGCAGATCACCATCCGCTTCCCTTG -.- + .___.-- +.-....- + -. Eur-lex.europa.eu eur-lex.europa.eu - + -. - + - + -. - + 400 HetAl »IleThrLeuSerAliThrSerl.euProlleSerA1» AlaAspH1smsPreleuProLeu GC - + -. - + -. - - - - + -, - + -. - + -. - + -. - ♦ 500 ThrValGlyVaKeuG1y5erGlyH1 $ A1a6lyThrAlal.euAlaAlaTrpPheAlaSerArgH1sValProThrAlaLeuTrpAlaProAlaAspH1sP CAGGATCGATCTCAGCAATCAAGGTTACCACCACCGAGGGAATGATTAACGGTCCATTTAGGGrCTCAGCCTGTGATGACCTTGCCGC - - - - - - - - - - - - - - - - - - - - - - - - - - - -. - + ----. 500 roGlySerlleSerAlalletyiAlaSereiueiyValIleThrThrGluGlyMetlleAsnGlyProPheArgValSerAlaCysAspAspLeuAlaAl A6TTATTCGCTCCAeCCGTGTACTGAnAnGTAACCCSTGCGGACGTTCACGACAGCTTCSTCAACGAACTCGCCAACTTCAACG6CGAACTCGCAACA 700 aValI1eArgSerSerSerArgValLeuI1eIleVa1ThrArgAlaAspValHhAspSerPheVa1AsnG1uLeuAlaA $ nPheAsn61yG1uLeuAlaThr AAGGATATT6TCGTCGATGGCTTCTCCATCAAGTACGAGAGACAGCTGCGATTCAAGCGAATATTCGAGACG6ATAATTC6CCCATACGT ..... -. + - - - - - - - - - - - - - ¥ al ¥ aCys6lyH1sGlyPheSerneLysTyrGluArgGlnLeuArgPheLysArgIlePheGluThrAspAsnS «rProIleThrS CTAAGCTATCG6ATCAAAAAAAATGTAACGTCAACATCAAGGAAATGAAAGCGTCTTTCGGACTGTCATGTTTCCCAATTCATCGCGATGATGCTGGCGT. - - - - - - - - - - - - - - - - - - - - - - - - 900 erLysLeuSerAspGlnlysLyíCysAsnValAsnlleLysGluMetLysAlaSerPheGlyLeuSerCysPheProIleHIsArgAspAslaGlyVa GATTATATTACCCGAAGATACCAAGAACATCTTTGCCCAGCTATrrTCCGCTAGAATCATCTGCATCCCGCCGTTGCAAeTGCTAnCTTTTCCAACTAT. +.-.-- + 1000 lIleAspLeeProG7ttAípThrtysAínIlePheA1a61nLeuPheSerAJaArgneneCysIleProProLeuG] »¥ a) LeiiPhePheSerAsnTyr ATCACTCAT6C6GTTCCG6CAGTCATGAACATCGGAAGACTCCGCGACCCAGCCAATTCTCnACTAAAAGAGCTGAGAAGTG6CTTCTT6AACTAGACG. -... +. Eur-lex.europa.eu eur-lex.europa.eu - ♦ .-. - + 1100 IleThrHIsAlaValProAlaValMetAsnlleGlyArgLeuArgAspProAlaAsnSerLeuThrLysArgAlaGluLysTrpLMLeoGluLeuAspG AGC6AACCCCACGAGCC6AGAAGGGCTTTTTCTTTTATGGTGAAG6ATCCAACACTTACGTTTGCAACGTCCAAGAGCAAATAGACCACGAACGCCGGAA - - - - - - - - - - - - - - - - - - - - - Eur-lex.europa.eu eur-lex.europa.eu luArgThrProArgAlaGluLysGlyPhePhePheTyrGlyGluGIySerAsnntrTyrValCysAsnValGlnGluGlnHeAspHIseiuArgArgLy GGnGCCGCAGCGTGTGGACTGCGTCTCAATTCTCTCTTGCAGGAATGCAATGATGAATATGATACTGACTATGAAACTTTGAGGGAATACTGCCTAGCA The CCGTCACCTCATAACGTGCATCAT6CATGCCCTGACAACATGGAACATCGCTATTTTTCTGAAGAATTATGCTCGnGGAGGATGTCGCGGCAATTGCAG Eur-lex.europa.eu eur-lex.europa.eu ProSerProH1sAsnValH1sH1sAlaCyíProAspAsnMetGluH1sArgTyrPheSer61u61uLeuCys5erLe «61iiAspValAliAlalleA1aA CTATTGCCAAAATCGAAATACCCCTCACGCATGCATTCATCAATAnArrCArGCGGGGAAAGGCAAGAnAATCCAACTGGCAAATCATCCAGCGTGAT Eur-lex.europa.eu eur-lex.europa.eu + 1500 lalleAlaLysIleeiuIleProLauThrHIsAlaPheneAsnllelleHIsAlaGlytyaGlytyalleAsnProThrGlytyíSirSerSertaUl TG6TAACTTCAGTTCCAGCGACTTGATTCGTTTTGGTGCTACCCACGnTrCAATAAGGACGAGATGGTG6AGTAAAGAAGGAGT6C6TCGAA6CAGATC Eur-lex.europa.eu eur-lex.europa.eu .-- + - .-- 1600 y61yAsnPheSerSerSerSpLeuIleArgPheGlyA1aThrHhValPheAsnLysAtpGluMetValGluEnd GnCAAAMnJGGCAATAAAGTTTCTTAAGATTSAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGnAAGCATGTAAT ______- ♦.-.-- + - .-- + - .-- + - .-- + --.-- + --..... + --.-- ♦ - Eur-lex.europa.eu eur-lex.europa.eu aattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgc ______- + .-- + --.-- + --.-- + --.-- + --.-- + --.-- + --.-- * --.-- ♦ 1800 FlG. 5 Eur-lex.europa.eu eur-lex.europa.eu ρ. Ρ Τ3 Ρ Ρ CQ cd ο a ρο the rap κόρη 3 θ '* - * - * ω (I.e. (I.e. <s 1-1 co AND ω The iH + J P cd C co o P o a cd h lop cd cd o β Pa O Ή · Η Η Ο Η τ! Ed ο Ρ4 -ρ S Η , -I δ cd cd (I.e. co ε V) (I.e. C0 e Your I co ω-1 (I.e. C0 And ω co o (I.e. 1- ο. (I.e.  * * ': <*. · · '.' (I.e. < (i.e. □ I (I.e. (I.e. (I.e. to η (I.e. "Β ω ρο Τ3 Ρ ρ or Τ The β PrH οο ρ Μ & 0 Ρ (I.e. (I.e. θ β Η (I.e. or (I.e. Ό '-1 the ο -ρ ρ (I.e. Ρ Ρ} rO γ r (I.e. (I.e. (I.e. Ip · Η Ip (I.e. β Ss , (I.e. 1 (I.e. 1 (I.e. I - -στ CQ < ν ν ι LL (i.e. α Μ 4 (I.e. ^σ): m _ (I.e. Ω. (I.e. (I.e. ω Ω. (I.e. co ο χ: 7th / ι Ω I (I.e. <0 t "w c = ± 1 with l-r + ti (i.e. Oh ho e C0 CQ C0 (I.e. C0 + ω ed ω P cd • Η +3 Η Ρ _Λ Ρ +5 | Ρα Ρ Ο · Η S 03 3 (I.e. W X Σ W X ' t =? 4- * ω ω £ α W X ' 1 = 1 σ) ' CD (I.e. -.W X F2 ο-ι £ or ϋ-1 LL1 t = f + ω ω 9 and W pKS Νορ Ί2 ΚΒ 3.8 σ> CQ ιωω And C0 ω (I.e. CL (I.e. co (i.e. ρ (I.e. (I.e. -I Ο C Ρ Ρ Ρ · « (I.e. (I.e. And l Ll x · ΆΑ: ί · ίέ Cia I I \ Hinf T Adapter Synthetic (-¾ SHEETS ' FIG. 9 I U L 1 1 SOO TGGGAGTCCAAGGTTTCAGGGAGCTTCGCAAATGAGGGCGCCGACGATTTTAAGGACTTCTGGGTCTATGCCCGAAGTGTCGAAATCCTTGGTATCAAGC - + ...., - + -, - + ACCCTCA6GTTCCAAAGTCCCTC6AAGC6TTTACTCCCGCGGCTGCTAAAATTCCTGAAGACCCAGATACGGGCTTCACAGCTTTAGGAACCATAGTTCG ρ a β c 1 1 a g ¥ 1 k 1 ¥ e p d 1 9 t t d f SN NSH AND s Ps CCP W W Hp WILL R R 1C 112 2 1 / // d k t d 1 Η Α Τ Η I Τ Ρ Ν U Η Α 3 12 2 ACGTGAAAGCTTACCGGATAGTTTTTGACGGTTTGTTCAAAAAAAGTCTTtATATCCAGCATGCTACCCCGGCTCCCATCTCCTGGGACGGTGAGAATAA Eur-lex.europa.eu eur-lex.europa.eu TGCACTTTCGAATGGTATCAAAAACTGCCAAACAAGTTTTTTCAGAAATATAGGTCGTACGATGGGGCCGAGGGTAGAGGACCCTGCCACTCTTATT γ h f 1 V p n k v tqefftk ⁽ 1 d 1 n s g r s g HTH RE s H H THE 8H NS T HAH SC W G G W 81 SA THE A CA AIR R THE THE Y VN PRAÇA Q 111 12 1 1 1 1 12 C3 1 / AeCGCGCCTTCGTACCAGGACeCACTGTCTCCACGACACTGTTCGAAAAAGTCAGTGGCGTCAACAACATGCACGTCGAACCCGAACCTTTCCTGGAGAAC ...... + + - + - 700 TCGCGCG6AAGCATGGTCCTGCGTGACAGAGGTGCTGTGACACCTTTTTCAGTCACCGCAGTTGTTGTACGTGCAGCTTGGGCTTGGAAAGGACCTCTTG ey ¥ s h f f d 1 : ad ¥ ¥ h ¥ dfgfreq ' 1 HHHXM HO M H AANHB ONLY B N Hello YOU 0 L 13121 21 2 1 / / / gcaaagcccgtaagcgtgcgggacgagcaggccagatcctgaggctatgaaataaaggggggtatcactcttgaagtcttcagcaaatgcctctccagcc Eur-lex.europa.eu eur-lex.europa.eu 800 CGTTTCGGGCATTCGCACGCCCTGCTCGTCCGGTCTAGGACTCCGATACTTTATTTCCCCCCATAGTSAGAACTTCAGAA6TC6TTTACGGAGAGGTCGG a h p I 1 9 sec gs i f 1 P t d s k f F 8 H HHS H E S F THE N B P Haf G C C 0 L U V THE AEA U R R K U H 1 2 12N 2 2 1 1 1 AACACACACTTCCTTGCAGCATGGAACAACGCCACCGGTAGCGCTTTCAGGGATGATGCCAGGGCTGGCATAAAGCTCCAACTTTCTTTGA ^T TTCCAAAA. + .....-.-, .- + .-, .- --..- + +. + .....-.-.-- +.-..... + + - + - + - + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - lvckraahflavplaklssalapmfswseklel E S C C R R 2 1 NSH CCP WILL 112 // TACCGCCAGTAAATGACGCCATTAGCAT6TGGGTAGCATGGAAAGCCTGGGTTGTTTCCCCGGTGAAAAACGCACTGTGACCATATGAGGCGAGCGGAGT Eur-lex.europa.eu eur-lex.europa.eu CT Iggtfsamlinhtahfaqttígtffashgysalpt 1000 HH F HA N AE U 12 H GTCTTGGGACTTCGTAAAGACGACTGTTTTACAAGGCTTGTTTTTCAGGCGCTTCGCTGCTTCTACAGTTTCTGGAGTTTTCCCAGACTTTGAAGACAGG Eur-lex.europa.eu eur-lex.europa.eu + + - CAGAÂCCCTGAAGCATTTCTGCTGACAAAATGTTCCGAACAAAAAGTCGCGGAAGCGACGAAGATGTCAAAGACCTCAAAAGGGTCTGAAACTTCTGTCC dqsktfrrtkepknklrkaaeyteptkgskssl s AA H T F N THE H MH F YS N THE N S L N SH THE AU F Q U P U L OK N 21 1 1 Η B 1 1 11 / acaatcaatgtgtctggagcatcagcgattgatggtttggctttcattaggtccacgaatctcgaagcggcgtagctaatgaaacggaggttttgcgcaa TGTTÃGTTACACAGÃCCTCGTAGTCGCTAACTACCAAACCGAAAGTAATCCAGGTGCTTAGAGCTTCGCCGCATCGATTACTTTGCCTCCAAAACGCGTT v1ltdpada1spkakmld »frsaays1fr1nqa 1200 FIG. 10-2 S NA Upon H D H F B HABHHBSH BC I L B D B B S 6CAHACCP The LC B U 0 and L U P IYRAEIRA 1 11 3 1 2 1 1 Η B C1112U2 / / / / CCTCGTCA6TATAC6CATTCAAGCTTTTTCCAATT6CCAACCCAGCACCAGCA6ATACGAAGAACACCTGCCTCAeTCCA6TG6CG6CTACAC6GC6CCC ...., .... + .... ,, .... + ......, .- + ......- + .-, .- + .- Eur-lex.europa.eu eur-lex.europa.eu CGA 1 to k g ι 1 to 1 9 4 9 1 s V f f V q r ι 1 g t a v r r AF H T N H N HM F B CT LB B THE B G B PB 0 s OVER THERE UU F 0 0 U 0 AL K P * 0 1H 1 1 1 2 1 21 1 W 11 (I.e. e6CTTCTGCGACATTTTCAAGCT6CCT6ATTGTA6AGTC6AGTTGTTGAGCGATCTCGGATGATCCG6TTGA6AGGGSTGCATACAGCATGTCCATC6AT -4 + -, + +, + +, + +, - + + 1400 CCGAAGACeCTGTAAAAGTTCGACGGACTAACATCTCAGCTCAACAACTCGCTAGAGCCTACTAGGCCAACTCTCCCCACGTATGTCGTACAGGTAGCTA • atvne1qr1tsdlqqa1essgts1payl @ d {1í) It is CF BB Q OA 0 F 1 BB 5 'RBA 21 TTGGTGTATCGA6ATYG6TTATGAAATTCAGATGCTAGTGfíXTStATTGGTAATTTGGeAAeATATAATAGGAAGCAAGGCTATTTATCCATTTCTGAA Eur-lex.europa.eu eur-lex.europa.eu (I.e. AACCACATAGCTCTAACCAATACTTTAA6TCTACGATCACATTACATAACCATTAAACCCTTCTATATTATÇCTTCGTTCCGATAAATAGGTAAAGACTT 1500 H H AH T Τ H T R N 6 P CG THE A H T s B THE H Already W C A H THE L 1 1 11 1 1 1 2 1 1 AAGGC6AAATGGCGTCACCeCATAGCGTTCTTGCTGTAAAGCGTTGTTTGGTACACTTTTGACTAGCGAGGCTTGGCGTGTCAGCGT. + 1600 TTCCGCTTTAÇÇGCAGTGGCGCTCeCAeTGCGCGTAAGGCAAGAACGACATTTCGCAACAAACCATGTGAAAACTGATCGCTCCGAACCGCACAGTCGCA "CAVf" O LO Q. anAdj ΙΟΟΝ " n nAdAd. Bioo V Q. 04 O V α r υιό _ I RS _ H JSS - n6a - £} Sd Q. Ha003g £ jog ^w _ in °° 3 α n O O 03 - XO 'm puiH ^m.p. 'οοε. IO Q. n O LO v f-1 O v. Q. Iiss I ^b Ouih -j-ia 003 α n O O co LO LO 03 LO H O. O Ll O tt +5 03 O & 0 •H O w 05 (i.e. EC O LU d Ll The d frog > d frog frog frog d frog x) frog g (I.e. frog •P O d The frog (I.e. frog σ frog halo O (I.e. frog d frog -P frog d P frog w O LHA 14 EYES) 12 O Ul < "W H" O _O CM O ' ¹ * ira / d CCI- = - ' P / G •H CC-- V * iiCCLC-- O OC-- Tj O _O CM O d 1 frog frog The O -frog frog The You < go) T -P CM O sz frog O rH frog frog G frog O na • frog O fd Hind HC P 3.8 FIG. 16 FIG. FIG. 18 NPTI FIG. 19 Bam Η I 11 ω Q. Cloning of the phaseolin cDNA in the genomic environment H CM CM O Ll 4 * FIG. 24 '{ind III Βθ4Τ} Hl EcoRI (I.e. THE li] (3ΐ * ϊβΒΗΗΒ) FIG. 29 EXAMPLE 3 (32 SHEETS) Tet ^R FIG. 30 ω SHEET 25 (3 * JUL3S5) • -w Bam HI FIG. 34 or W X , " co CQ (I.e. SHEET 28 (32 SHEETS) FIG. 37 pKS-oct.tmr. FIG. 38 • X CO (I.e. CM Ω. i a 003 fflPUIH d 0030 it's the ρ202 CO ' or CM (I.e. leuusIII pu! H I H tueg · ΙΠ. PU! H. Id 003. mp ^uh Id 003 'ffl PUH ffl PU! H X AND 05 CQ £ T5 W H X EJ 05 E ^m 05 F- The E co O (I.e. W X H 05 O AND 05 O 13 "O-, C t-H 05-1 AND And and 05 O 0 W X (I.e. ro O Lu s Cx The "D-, W X CQ O d β > Rl CQ-P-O η β ω β Ο Η ε 'ε or d β (I.e. or (I.e. β β φ d or • 0 (I.e. (I.e. The (I.e. (I.e. ω β or β β Γ β CD Ρ α Ο ι-1 β ω • σ 4-1 or or (i.e. χ: Ω. ι (I.e. BCM Ζ. (I.e. φ ι 00 (I.e. ι C0 (I.e. λgcM Ω. ω'Ω. CM α CM CM Ω. CM (I.e. (I.e. ι ω Ω. • κ * ...: 30 ^ Ê-TCESAS) eur-lex.europa.eu eur-lex.europa.eu 2nd phase Genetic Code u W THE G U Phe To be Tyr Cys U. Phe To be ^Tyr or Cys W Read To be Norr Non ^d THE Read To be NorJ Trp G W Read Pro His Arg U Read Pro His Arg W Read Pro Gin Arg THE (I.e. CQ Read Pro Gin Arg G S A Ile Thr Asn To be U • He Thr Asn To be W r j He Thr Lys Arg THE Met Thr Lys Arg G G Go Allah Asp Gly U Go Allah Asp Gly W Go Allah Glu Gly THE Go Allah Glu Gly G FIG. 40 [Ίίΐ 31 £ 32 TOMMY) FIG. 41 CAT6TC6A66CTCA6CAGCTGAAAATTCAAAC6C6CTA6TCAAT6CTATCAATCTGTCGT6TTCACACGGCATCAAAC6GTCACCAATGACGTCAAT6GG .-, - - - - - - - - - - - - - - - - - - - - - _- - + -------- ♦ 100 TTTCCTTAfiCTAATATAAAAACCAACSGCTCAGACTTACCAGCGGCAGGTATTTGTASTACATCCAACACAGCGATGACGGTAGCTAATTGGCAGGrrCG -, ...... -. Eur-lex.europa.eu eur-lex.europa.eu Eur-lex.europa.eu eur-lex.europa.eu - + 200 MetThrVa1A1aAsnTrpG1ηV »1Ar A6ATTTCAC6CnATCCT6C6CACCGGCGA6ATGAAGAGTCGCTT6GAACAG6C6AGAAC6GATTTCGGAGCGnACTGTCC6AAACTGTATACTTTCAG -. Eur-lex.europa.eu eur-lex.europa.eu Eur-lex.europa.eu eur-lex.europa.eu gAjpLevThrLevIld.e »ArgThpGlyeiu (1etLyiSerArgLeveiuG1nA1iArgThrAspPheGlyAlaLevLevSerG1uThr ¥» 1TyrPheGln CCTTCC6CGATTAGACTTG6T6A6TTT6AT6ACGAGTACATCCACTCTCGACAAGAGCT6GCnATGTATACCnCGTGAAGATAn6CACGCCAATGCG --- + .- ..- + .-- + .- - + ................- + .-- ♦ 400 PreSerA1iIeArgLevG1yGliiPheAspAspGluTyrIleH1iSerArgG1neiuLe * AlaTyrValTyrLe »ArgGluApI1eAlaArgGlnCyiA CCTTGCGTC6AAACCTACCGTCCAACTCCTCTAACTTC6GAACAATGGCAACT6CAATACC6CCGT6GCT6ATGAATGCACGCTGCCTGAATC6AGTTAT -. Eur-lex.europa.eu eur-lex.europa.eu + Soo liLevArgArgAsnLerProSerAjnSerSerAjnPhe61yThrt1etAlaThrAlaneProPr () TrpLevMetAsnAlaArgCy $ LevAsnArgValMe GCAG6AAA6GTGC6ATCAAGGTG6CCTC6TCAACTACTATCAAGGCCCACATACAAATCAGTTCTTTTTGGCGATTATGCCAAGCAACTGCTTTGTTCGG ......- + - ..- + -. - + - + -. - - - - - - - - - - - - - - - - + 600 tGln61uArgCyjA $ p61nG1y61yLev ¥ alAínTyrTyrGlnG1yProH1sThrA $ nG1nPhePeLe »A1aI1eMetPpoSerAsnCysPheVa1Arg TTCG6GACCGACATAATCAACAAT6AAMCTAC6GTTTTAA6CCCGGGGAG & AATACACTAGAGGAAGGAGAAGATGAC6ACGATGAGAT6GAC6ATGAA .-.-- +.-..- +. ....,. + + ..........-..- + _r + .-- + .- -..- .. ♦ 700 PheG1yThrApIleI1eAsnAsnG1uAsnTyrGÍyPheLyíProGly61uGluTyrThrArgGlyArgArgArgEnd 6GeGAG6CT6GTG6AfiCGGAACCAAGAGAGT6TCAGATCGGAAACCTTATCAATTATCC6ATCATTGCTTTAGGGTCATGCGATCTnCCGCATAATTCC --..- + -. - +. Eur-lex.europa.eu eur-lex.europa.eu + 800 CGTCGCCSACACCTAATAAAGTCGGCTAATCTATGTGAACTTTGTTATTTTGCATGrTTCCAATGTCATTTAGTAACSAAATAAAC - + - + .-- + .-- + - .. - + - + - + - .. + + .-- +. 900 GTTATCCTCTTCTAAAAGCAGGCTGTGTTTTCGGCAAACATCGCCACCCATCGCTAGTnTTCTAAAAGTGTTCTAAGCTAGCATGGTAATAATCTATAC.- + - .. + + .-. Eur-lex.europa.eu eur-lex.europa.eu or (I.e. W ω ct you (I.e. < (I.e. Ω H" V And co m oj έ or Ll \ .ζ νητ.ττΛ ζο (32 SHEETS) it 5i (I.e. Ll