PL176936B1 - Method of fighting against insects on plants - Google Patents

Abstract

Patatins control insects, primarily by stunting growth of larvae, thereby preventing maturation and reproduction. Genes encoding for one or more of these proteins may be cloned into vectors for transformation of plant-colonizing microorganisms or plants, thereby providing a method of controlling insect infestation.

Description

The present invention relates to a method for controlling insects on plants and a method for producing genetically transformed plants. The invention is practiced thanks to the protein which can be applied directly to the plant or can be produced there by microorganisms or by the plant itself by genetic modification.

The use of natural products, including proteins, is a well-known way to combat many harmful insects. For example, Bacillus thuringiensis (B. t.) Endotoxins have been used to control both harmful moths and heavy moths. Genes that produce these endotoxins have been introduced and expressed in a variety of plants, including cotton, tobacco, and tomatoes.

However, there are several economically important pests that are insensitive to B. t. endotoxins. Examples of such important pests are weevil (BWV), Anthonomus grandis and Diabrotica species (CRW). Moreover, the possession of other genetic products to control insects susceptible to B.t. endotoxins is important, and even significant, from the point of view of immunization problems.

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Several other known insecticidal proteins are found in plants. These include lectins, amylase inhibitors, and protease inhibitors that can affect the growth and development of insects when ingested at high doses [Boulter et al., 1989; Broadway and Duffey, 1986; Czapla and Lang, 1990; Gatehouse et al., 1986; Heusing et al., 1991; Ishimoto and K. Kitamura, 1989; Murdock et al., 1990; Shukle and Murdock, 1983), but they are not the cause of the acute deaths caused by B.t.

It is therefore an object of the invention to produce such proteins that would be capable of combating BWV, CRW, or other harmful insects, and genes useful for the production of such proteins. Furthermore, the object of the invention was to generate genetic constructs and a method for inserting genetic material into microorganisms and plant cells.

Patatin, the major storage protein of potato tubers, was found to control a variety of insects, including Diabrotica virgifera (WCRW), Diabrotica undecimpunctata (SCRW) and weevil (BWV), Anthonomus grandis. Patatins kill some larvae and inhibit the growth of others, thus preventing their maturation or delaying them severely, preventing reproduction. These proteins, showing known esterase (lipidactlohydrolase) activity, can be applied (SCRW), applied directly to plants, or otherwise introduced, e.g. by using plant-colonizing microorganisms transformed to produce enzymes, or by subjecting the plants themselves to a similar transformation.

Patatins are a family of proteins found in potatoes (Gaillaird, 1971; Racusen, 1984; Andrews et al., 1988) and other plants, especially nightshade [Ganal et al., 1991; Vancanneyt et al., 1989]. In potatoes, patatin is found mainly in tubers, but also in a much smaller amount in other plant organs [Hofgen and Willmitzer, 1990]. The esterase specificity of several patatin isozymes was investigated [Hofgen and Willmitzer, 1990; Racusen, 1986].

The genes encoding the patatin were previously isolated from Mignery et al., 1984, Mingnery et al., 1988, Stiekema et al., 1988 and other researchers. Rosahl et al., 1987, transferred them to tobacco and observed the expression of patatin. This shows that the patatin genes can be expressed heterologously in plants.

Patatin genes can be similarly isolated and inserted into appropriate transformation vector cassettes, which are then (1) used to transform plant-colonizing microorganisms that, when placed on plants, allow expression of patatin-producing genes and thus control insects, or (2) introduce into the plant's genome, which then protects itself from insect attack by gene expression and patatin production. In addition, the plant may also be engineered or bred to co-express one or more B. t genes encoding insect control proteins. Thereby, plants will be obtained which (1) are protected against many pests and / or (2) have a dual action of controlling certain pests, which is an important tool in view of immunization problems. Examples of plants transformed to express B. t genes are described in European Patent No. 0385962, corresponding to United States Patent Application Serial No. 476,661, filed February 12, 1990, [Fischhoff et al.], Incorporated herein by reference. Furthermore, the plant may also be transformed or bred to co-express a proteinase inhibitor gene, such as that encoding a potato papain inhibitor [Rodis and Hofff, 1984] or a soybean trypsin inhibitor [reviewed in Ryan, 1990], since proteinase inhibitors have shown to potentiate the activity of other insecticidal proteins. .

The present invention relates to a method of controlling insects on plants which comprises using an effective amount of an insecticidal patatin. The method preferably uses patatin produced by the plant colonizing patatin producing microorganisms when applied to the plant. Plants are preferably transformed to exhibit patatin expression produced by expression of a patatin gene inserted into the plant by prior genetic transformation of the plant parent cell.

In a preferred embodiment of the method according to the invention, the plant is cotton, corn, tomato or potato.

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The invention also relates to a method for producing genetically transformed, insect-resistant plants expressing an insecticidally effective amount of patatin, in which the host cells are transformed with a gene encoding the desired protein, transformed plant cells are obtained, and plants that are insecticidally expressed are regenerated from these transformed plant cells. an effective amount of patatin by inserting into the genome of the plant cell a recombinant, double-chain DN A molecule containing a promoter causing the formation of an RNA sequence in plant cells; structural, patatin coding sequence; a 3 'untranslated region for causing the addition of polyadenylation nucleotides in a plant cell to the 3' end of an RNA sequence, the promoter being heterologous to a structural coding sequence and operably linked to a structural coding sequence which is in turn operably linked to said untranslated region; wherein the promoter is heterologous with respect to the structural coding sequence, and the plants are selected from cotton, corn, tomato and potato.

Preferably, sequence no. Id is used as the structural coding sequence. 30 or · sequence no. 31. When a maize plant is used as a plant as a preferred embodiment of the invention, the preferred sequence is the structural sequence SEQ ID NO. 33.

Preferably, the plant expresses patatin in an amount of about 0.1-0.5% of the total protein.

As used herein, the term "insect control" means reducing the number of insects that reduce plant productivity through increased mortality, retardation of larval development (inhibition of development), or decreased reproductive capacity. As used herein, the term "insecticide" means the ability to reduce the number of insects that reduce the productivity of plants through increased mortality, retardation of larval development (inhibition of development), or reduced reproductive capacity.

As used herein, the term "structural coding sequence" means a DNA sequence encoding a polypeptide that can be obtained in a cell by sequentially transcribing DNa into mRNA, followed by translation into the desired polypeptide.

As used herein, the term "patatin" means a vegetable protein having 75% or more homology to the protein encoded by SEQ ID NO: 31, shown hereinafter, more preferably at least 80% homology, and most preferably at least 85% homology. The term also includes proteins produced with synthetic DNA sequences designed for improved expression in monocotyledons.

Patatins are a family of esterases found in potatoes (Gaillaird, 1971; Racusen, 1984; Andrews et al., 1988) and other plants, especially nightshades [Ganal et al., 1991; Vancanneyt et al., 1989]. In potatoes, patatin is found mainly in tubers, but also in a much smaller amount in other plant organs [Hofgen and Willmitzer, 1990]. The esterase specificity of several patatin isozymes was investigated [Hofgen and Willmitzer, 1990; Racusen, 1986] and their broad substrate specificity was found, indicating limited enzyme requirements for the substrates. The use of all plant-derived patatins and their equivalents, both described in detail herein, and homologous proteins, whether derived from natural or synthetic DNA sequences, for the purposes of controlling insect infestation on plants are within the scope of the invention.

Raw potato patatin preparations are commercially available. For example, Sigma Chemical Company, St. Louis, MO, offers potato protein preparations referred to by the company as acid phosphatase (P-1146 and P-3752) or apyrase (A-9149). Protein extracts of potato tubers can also be prepared by methods described in the literature (Racusen and Foote, 1980; Park et al., 1983).

Biological effectiveness tests

Biological tests in an artificial diet

Activity tests were carried out against SCRW larvae, BWV, Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the European species Ostrinia nubilalis (ECB) by applying the test sample to food agar, as described for SCRW by Marrone et al., 1985. Test samples

176,936 was prepared by dissolving the protein in 4-5 ml of 10 mM HEPES, pH 7.5, and dialyzing in the same buffer using a cut-off tube system at a molecular weight of 3500. Freshly hatched larvae are fed with the sample at 26 ° C and tested for mortality and inhibition of development after 5 or 6 days. The results of the tests P-3752 (Sigma) are given in Table 1. This crude potato preparation showed a broad spectrum of insecticidal activity.

Table 1

Quantity % Mortality / Inhibition of Development ' SGRW BWV CPB ECB 0.01X 0 11 0 0 0.03X 0 * twenty' 0 13 0.10X 19 ** twenty"" 6 0 » 0.30X * »_» »» 6 46 »u 13 » 6 » 1.00X ··· 6 73 "» 13 »» 6 »»

= slight development inhibition (approx. 30-40% size reduction) _{t (} = moderate development inhibition (approx. 50-80% size reduction) = acute development inhibition (more than 90% size reduction)

The exact weightings of the SCRW (table 2) after 5 days of testing and the ECB (table 3) after 6 days of testing were performed and are summarized below. The SGRW larvae developing on the food containing P-3752 showed a 92% weight reduction compared to the controls, and the ECB larvae showed a 62% weight reduction compared to the controls.

Table 2

Treatment average mass surviving (SEM) % weight reduction % mortality Tris 4.00 mg (0.60) ' —Controller level— P-3752 0.30 mg (0.03) " 92 6

the mean mass of survivors was significantly different at the level of 95% (one ANOVA factor).

Table 3

Treatment average mass surviving (SEM) % weight reduction % mortality Tris 5.39 mg (0.49) ' —Controller level— P-3752 2.05 mg (0.27) " 62 7

the mean mass of survivors was significantly different at the level of 95% (one ANOVA factor).

The protein nature of the insecticide component P-3752 active against SCRW and BWV was determined by heat instability, ammonium sulphate precipitation, molecular weight fractionation and protease susceptibility experiments.

To confirm that the effect of P-3752 is directly related to the consumption of patatin and not an indirect effect of the food reaction, a food selection study with ECB and SCRW was performed. The results of this study indicated that the larvae consumed food from both P-3752 and Tris, with no apparent preference. Avoidance of food from P-3752 compared to food from Tris was not observed.

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The long-term (25 days) test of P-3752 for SCRW utilized 2 instar larvae and repeated transfer of surviving insects to freshly treated food. At the end of the study, all control larvae became pupae. In contrast, 50% of the larvae after the procedure were dead, and the remaining 50% increased their body weight by only 16% of the initial weight (2.48 mg versus 2.14 mg). This indicates a halt, not just a slowing down of the development of the larvae. This is very important from the insect control point of view as the larvae do not reach maturity. The number of Diabrotica undecimpunctata will be lower in future generations.

Diabrotica virgifera (WCRW) larvae can be used in laboratory experiments only in the 2-moulted state. In order to test the effect of P-3752 on WGRW, a parallel test was designed using SCRW larvae after 2 instars. The action of P-3752 caused only 13% and 11% weight gain of SCRW and WcRw larvae after the 2nd instar, respectively. The control SCRW larvae increased weight by 474% and the WCRW larvae by 200% in 7 days. This indicates that »the activity of patatin against WCRW and SCRW is approximately the same.

P-3752 showed little activity against Heliothis virescens (TBW), Spodoptera exigua (BAW), Helicoverpa zea, Pectinophora gossypiella and Manduca sexta, giving a growth inhibition index of 1 to 1.5 at those low concentrations at which SCRW showed an inhibition index 3. P-3752 gave a growth inhibition index of 2.5 for Agrotis ipsilon. (Growth inhibition indices are defined above in Table 1.) He showed no activity against Myzus persicae at the tested concentrations.

Plant-based bioassay (1) Potato: 1 g of crude P-3752 was dissolved in 4 ml of 25 mM Tris, pH 7.5 buffer, then dialyzed and filtered through a 0.2 gm membrane. Triton®X-100 was added to form a 0.1% solution. Potato leaves were dipped in the enzyme preparation and placed on a moist filter paper in a petri dish. CPB larvae were added and the dishes were incubated at 27 ° C for 3 days. The treatment of the potato P-3752 leaves inhibited the development and slowed down the feeding of CPB larvae. At the end of the test, significantly less leaf tissue remained on the control leaves compared to those treated with P-3752.

(2) Corn and Cotton: Callus of black Mexican sweet corn (BMS) or cotton was removed from agar plates and transferred to 50 ml centrifuge tubes. The callus was centrifuged in an IEC clinical centrifuge for 5 minutes at setting 8 and the supernatant decanted. 30 ml of liquid 2% agar was added to a 50 ml tube containing 15 ml of calf granules. After thorough mixing, the food was pipetted onto the insect bioassay site. The food was covered with dialyzed P-3752 (20 vol.%) And the test was performed as described above.

The cut corn roots and shoots were soaked under reduced pressure (sat) crude P-3752 or 25 mM Tris, pH 7.5 buffer. A control sample was immersed in Tris buffer. Approximately 10-15 pieces of root or 3 pieces of shoot were placed in the wells in a 24-well culture plate which was repeated 4 times. The four newly hatched SCRW larvae were added to each well. The test plates were incubated at 26 ° C for 4 days, and the mortality and average larvae weight were determined. The test results are shown in Table 4.

Table 4

tissue us. the roots insect % mortality % weight loss 1 2 3 4 corn SCRW 90 44 us. shoots corn SCRW 51 52

176,936 cont. table 4

1 2 3 4 callus BMS after processing SCRW 24 51 callus BMS after processing WCRW 0 23 callus BMS after processing ECB 0 33 callus cotton after treatment BVW 60 no data

Thus, the insecticidal activity against all four species (SCRW, BWV, CPB, and ECB) is preserved when P-3752 is ingested with plant tissue. The above nutritional studies show that patatins are insecticidally active when tested with foods whose ingredients are purely plant tissue (roots, shoots, callus or leaves).

Mode of action studies

The following studies suggest that patatin, the insecticidally active ingredient of P-3752, acts directly on the insect and that the effect shown in the experiments described above cannot be attributed to the effect of the active ingredient on the insect's food before ingestion.

Food effect study

1 g of P-3752 was dissolved in 10 ml of 25 mM Tris, pH 7.6 buffer, then dialyzed in a MWCO 12-14000 tube system against the same buffer. After 0.2 µη filtration, 50 µl aliquots were added to the insect food wells of two plates 4 days prior to the addition of the insects. Both plates were incubated at 27 ° C for 4 days. After incubation, one plate was heated to 80 ° C for 1 hour to deactivate the enzymes. 50 µl portions were applied to the third plate. Thus, for the SCRW test, incubated, incubated and warmed as well as non-incubated plates were used.

Activity against SCRW in the pre-incubation study was as follows: unincubated P-3752 - 6 *** incubated P-3752 - 0 ** incubated, heated P-3752 - 0

Although some activity was lost during food incubation, heating resulted in total loss. These data are consistent with the theory of direct effect upon ingestion, and when considered in conjunction with plant tissue testing and variability in action against different insects, indicate that the action of the protein on SCRW and other insects is not based on the food effect.

Protein identification

The insecticide active ingredient from P-3752 was purified, partially sequenced, and characterized. The active factors were identified as patatin, a family of potato lipid acylhydrolases.

Isolation of protein

Four different protocols were used to purify the SCRW bioactive component P-3752.

Purification of SCRW active component by anion exchange chromatography. The SCRW active component from P-3752 was purified by anion exchange chromatography on Q-Sepharose (Pharmacia) followed by MONO-Q (HR 5/5, Pharmacia). The amounts of protein in the SCRW-active fractions indicated that the observed ** growth inhibition was achieved at a dietary protein concentration of 31 ppm. SDS-PAGE chromatography indicated that 3 major protein bands (M _r 42,000, ~ 26,000, and ~ 16,000) were present in the active fractions.

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Five Step Purification of SCRW Active Component - 5 successive steps of purification of SCRW active component from P-3752 were used. These were membrane sieving, ammonium sulfate precipitation, Q-Sepharose ion exchange chromatography, S-Sepharose ion exchange chromatography, and P-200 size exclusion chromatography. SDS-PAGE chromatography of the purest SCRW active fractions showed Mr 42,000, ~ 26,000, and ~ 16,000 protein bands.

Purification of the bioactive component by isoelectric focusing - Potato proteins bioactive against SCRW were purified in two successive runs on an RF3 protein fractionator (Rainin) according to the manufacturer's instructions. The SDS-PAGE profile of the SCRW active fractions was very similar to that observed for the active fractions from the 5-step purification and anion exchange purification. The IEF gel method showed that proteins were fractionated at a pH of 4.6 to 5.1, consistent with the published pl range for patatin (Racusen and Foote, 1980).

Successive isoelectric focusing on RF3 was used in a narrow pH range (4-5) in an attempt to separate patatin isozymes. As expected, the peak of bioactive fractions was observed in proteins with a pl of 4.6-5.1. These fractions have a separate system of isozymes and different contents of the bioactive component. The bioactivity in the fractions ranged from zero mortality to **** growth inhibition at doses of 80-512 ppm. Some of the bioactive fractions have only 2 major isozymes, indicating that bioc activity does not require a complex isozyme pattern.

Cleansing with the native method. PAGE - P-3752 was electrophoresed under native conditions and a triplet of bands with esterase activity was isolated (using acetate-naphthyl as substrate). The gel-purified esterase material showed activity against SCRW giving a growth inhibition of 1.5 *. SDS-PAGe chromatography of the substance showed major bands for Mr 42,000, ~ 26,000 and ~ 16,000, which is the profile already observed in other purifications. This further confirms that patatin is an insecticidal substance from potatoes.

Amino acid sequences

The NH2-terminal amino acid sequence was obtained for all protein bands (Mr 42,000, ~ 26,000 and ~ 16,000) in the SCRW-active fraction from anion exchange purification and five step purification. Overall, sequence data was generated for all bands of active fractions. Most of the bands showed> 85% homology with the 15 amino acid sequence at the NH2-terminus (SEQ ID NO: 1) or with the inner sequence (SEQ ID NO: 7) of the patatin isozyme (Stiekema et al., 1988). One of the 7 kD bands showed 75% homology with the initial eight amino acids of the published NH2-teminal patatin sequence. The other 7 kD band showed> 85% homology with the starting eight amino acids of the published internal sequence. These bands represent the proteolyzed patatin products. The presence of isozymes is clearly indicated by the variation of amino acids at positions 1 and 3 for both the NH2-terminal and the internal sequence.

published Seq: (Seq ID No. 1) Band 1 (42 kD):

(sequence ID no. 2) band 2 (28 kD):

(sequence ID no. 3) band 3 (26 kD):

(SEQ ID No. 4) band 4 (24 kD):

(SEQ ID NO 5) 5a band (17 kD) (SEQ ID NO 6)

N-terminal amino acid sequence of KLEEMVTVLSIDGGG

XLGMVTVI ^ SIf ^ (] (^ G

T L G Μ V Τ V L SID G G G

T L G Μ V Τ V L SID G G G

KLXMVTVLSIDGGG

XXEEMVT

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Internal sequence (amino acid position 224) published SEQ: (SEQ ID NO. 7)

Band 5b (17 kD) (SEQ ID No. 8) Band 6 (16 kD):

(SEQ ID No. 9) Band 7 (15 kD):

(SEQ ID No. 10)

SLDYKQMLLLSLGTG KLDYKQML SLXYKQMLLL SLGTG SLNYKQMLLLSLGTG

Transcription activity

Several experiments were performed to test the esterase activity in SCRW active fractions.

Substrate - α-naphthyl acetate: SDS-PAGE chromatography (10-20%) was used to determine if the fractions active against SCRW (from the 5-step purification) showed esterase activity [Racusen, 1984]. Heated and unheated fractions active against SCRW were applied to two equal portions of the gel. A single esterase-positive band was observed in the unheated sample at Mr 55000.

The heated sample showed the original Mr 42,000 band and the simultaneous absence of the 55,000 band. This result is consistent with the literature reports on the electrophoretic mobility of patatin esterase active fractions (Racusen, 1984). The Mr 55,000 band was not observed in the heated sample, indicating that the SDS heat treatment eliminated the esterase activity. In the absence of the Mr 55,000 band in the heated sample, the original Mr 42,000 band was observed by coomassie staining.

Up-Nitrophenyl Substrate Specificity Study: A series of p-nitrophenyl esters (C-2, C-4, C-6, C-8, C-10, C-12, C-14, and C-16 esters) were tested to determine substrate specificity. The C-8 and C-10 nitrophenyl esters were consistently the best substrates for the esterase activity of the majority of the patatins tested, compared to the other consistently.

Substrates - Lipid Esters: The purified SCRW-active fraction (from 5-step purification) was tested for the ability to hydrolyze several lipids. Each lipid was dissolved and incubated with an aliquot of the purified SCRW active fraction. The samples were analyzed by thin layer chromatography by developing with a three-solvent system (Pemes et al., 1980). The four lipids showed significant modifications in this analysis. It was oleylolysolecithin, dioleyl-L-α-phosphatidylcholine, 1-monolinoleyl-rac-glycerin and diolein (Sigma). The new spot in the thin layer analysis at Rf 0.37 for the organic extract of the lipid / active fraction reaction mixture was identified as free fatty acid by comparison with linoleic and oleic fatty acid standards. Thus, the substance active against SCRW has an esterase effect on these four lipid esters.

Thin WCRW elites were removed from the third instar larvae fed with corn roots. Intestinal lipids were extracted, dissolved and incubated under gut pH conditions (pH 6.55) with the purified SCRW active fraction. Samples were analyzed by thin layer chromatography as described above. The purified SCRW-active fraction showed esterase activity against WCRW gut lipids at gut pH. This is illustrated by the presumptive mode of insecticidal activity of patatin.

Alternative sources of patatin

Since all initial experiments were conducted with P-3752, a commercial enzyme preparation (Sigma) from potato tubers Minnesota Russet var. Kranz, it was desirable to demonstrate that the insecticidally active patatins could be recovered from fresh potato tuber tissue. Tuber extracts were prepared exactly as described in the literature (Racusen and Foote, 1980; Park et al., 1983). Three commercially available varieties of S. tuberosum (Russet, Desiree, and LaChipper) and seven wild species (S. kurtzianum, S. berthaultii, S. tarijense, S. acaule, S. demissum, S. cardiophyllum and S. raphanifolium, all from Inter-Regional Potato Introduction

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Station, USDA, ARS, Sturgeon Bay, WI). All extracts contained patatin as tested by SDS-PAGE and Western blot; all contained esterase according to the C-10 esterase test and all showed insecticidal activity against SCRW, that is, a growth inhibition index of 2-3. See Table 5. It follows that insecticidally active patatins can be isolated from tubers of several species and many members of this entire class of proteins may exhibit insecticidal properties.

Table 5

species [Protein] (mg / ml) ΔΟ. D./minml SCRW " IX 0.1X S. acaule 26.6 82500 2 1 S. berthaultii 23.1 29 3 1 S. cardiophyllum 14.6 89 2.5 1.5 S. demissum 35.3 375,000 3 1.5 S. kurtzianum 25.0 2700 2.5 1 S. raphanifolium 33.7 3725 3 2 S. tarijense 27.2 1008 2.5 1

“Activity against SCRW was expressed in terms of inhibition of larval development:

= mild growth inhibition (size reduction 30-40%), = moderate growth retardation (size reduction 50-80%), = acute growth inhibition (size reduction> 90%).

Extracts of S. berthaultii, S. kurtzianum, and S. tarijense were biologically tested against two other insects, CPB and ECB. The test data are summarized in Table 6 below. These extracts showed a weak effect on CPB and moderate to severe growth inhibition in ECB larvae at the 1X dose. However, ECB larvae are slightly less sensitive to these potato extracts compared to SCRW larvae as indicated by a complete lack of activity on EBC at 0.1X.

Table 6

Type CPB " ECB " IX 0.1X IX 0.1X S.berthaultii 0 0 3 0 S. kurtzianum 1 0 2.5 0 S. tarijense 0 0 3 0

“Inhibition of larvae development

Genomic DNA from nine different plants was tested by Southern analysis for proteins homologous to patatin. Specks Southern probe labeled with a- ³² P Sequence ID NO. 11 showed the existence of homologous sequences in several other plant species. Strong signals were obtained for maize, tomato, sugar beet, rice and potato. The individual bands were not separable in this experiment, however the size of the spots and their intensity were similar for all these species. Weaker signals were also obtained for zucchini, soybeans, and canola, and these appeared as a small number of single strands in the DNA of each species. Cucumber and Arabidopsis showed no detectable hybridization with the patatin probe under the conditions

This experiment, perhaps due to the small amount of DNA used, as indicated by the staining of the gel with ethidium bromide.

One skilled in the art can easily generate the DNA sequences of these homologous proteins and introduce them into plants or other organisms by known means. The insecticidal properties of such proteins can best be tested after heterologous expression, e.g. from rod virus or E. coli. Thus, other proteins that can be used in the method of the invention can be obtained after the usual number of experiments using known methods and then used to provide protection to plants against insect infestation.

Genetic identification

Several researchers have cloned the patin genes. The sequence described by Mignery et al., 1984 was named GM203. It contained an incomplete signal sequence. Mignery et al., 1988, identified a genomic clone, designated P520, comprising GM203 and containing a complete signal sequence. Sequence no. 11 was constructed with the signal sequence owaP520 and encoding the DNA portion of GM203, hereinafter referred to as PatA +. It also contains an NcoI restriction site and an EcoRI site immediately downstream of the translation termination codon.

Solanum tuberosum variety Russet Burbank

Twenty cDNA strands were isolated from potato tubers of Russet Burbank cv. And sequenced. The deduced amino acid sequences show that these cDNAs code for eleven different patatin isozymes. Eleven proteins share from about 82% to 100% identity with PatA +, SEQ. id no. 11, with differences at different positions along the length of the cDNa. The sequences for eleven different representative cDNAs encoding eleven different patatin isozymes are named as given in Table 7. The cDNA sequences were processed by PCR procedures using sequences id. 26 and no. 27, corresponding to the 5 'nucleotides encoding the first few codons of the signal sequence and the 3' end of the coding sequence, for further cloning purposes. The symbol "+" indicates that it contains a native signal sequence. Some cDNAs do not contain the complete native coding signal sequence and only the mature protein coding sequence was obtained by a similar PCR procedure using primers from sequence id. 32 and no. 27. They are marked with the subscript "m".

Table 7

Izozym id no. sequence PatA + seq. id no. 11 PatA _m seq. id no. 14 PatB + seq. id no. 16 PatC + seq. id no. 17 PatD _ra seq. id no. 18 PatE + seq. id no. 19 PatE _m seq. id no. twenty PatF _m seq. id no. 21 PatG + seq. id no. 22 PatH _ra seq. id no. 15 Pati _m seq. id no. 23 PatL + seq. id no. 24 PatM + seq. id no. 25

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Solasram berthaultii

Patatin cDNA sequences from the S. berthaultii diploid potato were isolated by reverse transcription of tuber mRNA followed by PCR with primers SEQ ID NO. 26 and no. 27, described above. Multiple independent PCR reactions were performed to avoid shedding of duplicate clones due to the amplification process.

A total of 14 patatin cDNAs were partially sequenced. All cDNA sequences (named Pat1 to Pat14) appear to have unique nucleotide sequences, suggesting that at least 14 different patatin mRNAs are expressed in S. berthaultii tubers. The sequence for Pat3 + is sequence ID No. 28. The sequence for Pat10 + is sequence ID NO. 29. The deduced amino acid sequence shows that 14 cDNAs coding for at least 11 different proteins. Overall, the cDNA sequences from S. berthaultii tubers were very similar. Only 12 amino acid positions out of 367 residues (3%) showed sequence variation. The amino acid residues present at each of these positions are shown in Table 8. Out of 5 of these positions, only a single clone variant with a unique residue exists. These changes may reflect actual mRNA differences or may be due to errors in the PCR process. In the remaining 7 items the volatility was greater; at least two cDNAs had an alternative amino acid. Each of the 9 different groups of amino acid sequences had a different pattern of residues at these seven positions. In some cases, the changes were conservative, such as changing from Tre to Ser at position 164. In other cases, there were much more significant differences, such as introducing proline at position 148.

Table 8 cDNA Positions of Amino Acid Differences

89 96 106 113 120 123 148 164 187 200 PAT3 + GLN LEU GLN TYR GLU SHAFT ALA ALA TRE ASP ASP PAT4 + GLN CHEESE ASP HIS GLU SHAFT ALA PRO CHEESE ASP SHAFT PAT5 + GLN CHEESE ASP HIS GLU SHAFT ALA PRO TRE ASP ASP PAT7 + GLN LEU GLN TYR GLU SHAFT ALA ALA TRE ASP ASP PAT8 + LIZ CHEESE GLI TYR LIZ SHAFT ALA PRO TRE ASP ASP PAT9 + LIZ CHEESE ASP TYR LIZ SHAFT ALA PRO TRE ASP ASP PAT10 + GLN CHEESE ASP HIS GLU SHAFT TRE PRO TRE ASP ASP PAT11 + GLN CHEESE ASP HIS GLU ALA ALA ALA TRE ASP ASP PAT 12+ GLN CHEESE GLI HIS GLU SHAFT ALA ALA TRE ASP ASP PATA + HIS - CHEESE - TYR GLU SHAFT ALA ALA TRE GLU ASP

Solanum cardiophyllum

Ten cDNA clones were generated by PCR using mRNA isolated from Solanum cardiophyllum tubers as described above. A nucleotide sequence of at least 75% of the length of each clone was obtained. Full-length sequence. the clone named Pat17 + is sequence id. 30. Sequence no. 31 is an engineered mature form, Pat17 _m . The S. cardiophyllum clones were nearly identical, only with random nucleotide sequence changes that could be real differences or PCR errors. However, at positions 54 and 519, several clones with identical changes were observed, suggesting that they are not related to the enhancement process. The nucleotide patterns at these positions indicated that they represent at least 4 different mRNAs. The mRNA of two of these groups was isolated several times and the mRNA of the two remaining only once in this set of cDNA clones.

The deduced amino acid sequences of S. cardiophyllum clones were also remarkably similar. There were 8 unique groups of amino acid sequences, each one differing from other 936 sequences by one residue. The cDNA clones encoding the amino acid sequence identical to the Pat 17+ sequence were obtained two & rotaie, af ^ c ^^ c ^^ 'cDNA 7 (Pat18 +, 19+, 20+, 21+, 22+, 23+ and 24+) contained one unique residue .

An embodiment of a method for producing genetically transformed plants

Genetic transformation

As described above, the patatin genes can be isolated from a variety of plant sources. One or more of these genes can then be used to transform bacterial or plant cells to allow the production of patatin.

An example of doing this with different patatin sequences is given below.

Production of patatin cDNA

In order to incorporate the patatin gene into vectors suitable for expression of patatin in heterologous host cells, it was necessary to introduce appropriate restriction sites near the ends of the gene. The goal of mutagenesis was to create a cassette containing the protein coding sequence with minimal non-coding flanking sequences and to include useful restriction sites to mobilize the cassettes. The cassettes were designed to allow the mobilization of the intact coding sequence together with the signal peptide or only the mature coding sequence. _M designed for PATA two mutagenesis primers in order to create such tapes. Mutagenesis by sequence no. 12 resulted in the substitution of two amino acids (methionine-alanine) for lysine at the N-terminus of the mature protein and the introduction of an NcoI site, and sequence id. 13 added a second termination codon and an EcoRI site.

The resulting modified sequence was identified as PatAm SEQ. id no. 14. For all other cDNAs, similar modifications were made and restriction sites were introduced using the PCR method and either the primers in sequence ID NO. 26 and sequence no. 27, or the primers of SEQ ID NO. 32 and 'sequence ID no. 27 as previously described.

Expression of patate in E. coli

The DNA coding sequence for PatAm (SEQ ID NO. 14) was inserted into pMON5766, an E. coli expression vector derived from pBR327 (Soberon et al., 1980) with the recA promoter and G10 start sequence (Olins et al., 1989). The resulting vector, pMON 19714i, was mobilized into the E. coli JM 101 strain, which was then produced by PatAm, as confirmed by Western blot analysis and the esterase effect on C-10 p-nitrophenyl ester. The DNA coding sequence for Pat 17m and also for PatAm was inserted into an E. coli expression vector derived from pMON6235 with the AraBAD promoter (inducible when cells were grown in arabinose), a G10 start sequence and an ampicillin resistance marker gene. The resulting vectors, pMON25213, containing Pat17m, and pMON25216, containing PatAm, were introduced into E. coli strain JM101.

Patatin is expressed in transformed E. coli; however, light refracting bodies (RB) accumulate. Intact cells and dissolved RB were used in the SCRW assays. The results are shown in Table 9.

Table 9

A sample (pMON) Rep intact cells SCRW activity ¹ dissolved RB activity of SCRW1 19714 A _m 1 2.5 not tested 2 1.5 1.0 25216 Am 1 1.0 0 2 0 1.0 25213 17m 1 3.0 3.0 ² 2 1.5 0.5

'Activity against SCRW was expressed in terms of inhibition of larval development:

= mild growth inhibition (size reduction 30-40%), = moderate growth retardation (size reduction 50-80%), = acute growth inhibition (size reduction> 90%). the mortality in this sample was 81%.

Expression of patathins in plant colonizing bacteria

In controlling insects, it may be desirable to express one or more patinates in the plant colonizing bacteria and then apply these bacteria to the plants. When the insect feeds on the plant, it absorbs a toxic dose of patatin produced by the plant's colonists. Such bacteria can inhabit the surface of plants, such as Pseudomonas or Agrobacterium species, or be endophytes inhabiting the plant vascular system, such as Clavibacter species. In the case of surface-colonizing bacteria, the patatin gene can be engineered into many vectors capable of replication in gram-negative hosts. Examples of such vectors are pKT231 from the IncQ mismatch group [Bagdasarian et al., 1981] or pVK100 from the IncP group [Knauf, 1982]. In the case of endophytes, the patatin gene can be introduced into a chromosome by homologous recombination or by incorporation of the gene into an appropriate transposon capable of chromosomal insertion in endophytic bacteria.

Expression of patatin in baculovirus

The patatin genes were cloned into the baculovirus donor vector pMON4327 described in co-pending US patent application No. 07/941363, filed September 4, 1992, incorporated herein by reference as NcoI / EcoRI fragments. The donor vector pMON14327 contains an ampicillin resistance gene, the left and right arm of the Tn7 transposon, and, between the arms, a gentamicin resistance gene, a strong polyhedrin baculovirus promoter and a polylinker. The baculovirus shuttle vector, or bacmid, consists of a miniattTn7 site in frame inside the lacZ gene and a kanamycin resistance gene recombined in the AcNPV viral genome. With the help of the tetracycline resistance helper plasmid, pMON7124, recombinant AcNPV virus was generated by transposing the patatin or GUS genes and marker genes into the viral genome (Lucków et al., 1993). The following genes are included in pMON14327: the genes listed in Table 7 and Pat3 +, Pat10 + and Pat17 +.

In accordance with the procedures of United States Patent Application No. 07/941363 and Luckow et al., Some amounts of patatin were produced from the above genes. The presence of patatin was confirmed by Western blot analysis and the esterase action of the C-10 p-nitrophenyl ester. Except for PatA _m , which isozyme was amplified at least twice for the SCRW biotest. While the PatE + and PatEm cultures consistently showed little or no expression of patatin, all other isozymes appeared to be expressed in a satisfactory amount for a bioassay. However, the nature of the post-translational processing of patatin in baculoviruses as compared to potato has not been established. Bioactivity of the baculovirus expressed isozymes against SCRW was observed for Pat 17+, PatB +, PatD _m , PaII _m , PatL + and Pat3 +.

The effects of multiple isozymes (produced by baculovirus) on the growth and development of insects were determined. Samples of 11 Russet isozymes were pooled into one for a bioassay against SGRW, ECB, farmer caterpillar, and TBW. 10 to 15 mg of each Q-Sepharose purified isozyme except PatDm, available in only 1.7 mg, was combined. The resulting mixture showed 100% lethality in TBW tests and 93% weight reduction versus ECB. Thus, each isozyme was separately tested against TBW and ECB. Compared to control larvae, TBW and ECB larvae fed food treated with PatC +, PatL + and PatIm areozymes showed significant inhibition of development (> 75%) and / or mortality. Pat B + and PatDm also inhibited TBW development by 69 and 78%, respectively.

Plant gene construction

Expression of a plant gene as double-stranded DNA requires the transcription of messenger RNA (mRNA) from one DNA strand by the action of RNA polymerase and subsequent processing of the primary transcript in the nucleus. Such processing involves the 3 'untranslated region adding polyadenylating nucleotides to the 3' end of the RNA. DNA transcription in mRNA is regulated by a region of DNA commonly referred to as a "promoter". The promoter region contains a base sequence signaling RNA polymerase to bind to DNA to initiate mRNA transcription from one of the DNA strands as a template to form the appropriate RNA strand.

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A number of promoters active in plant cells have been described in the literature. Such promoters can be obtained from plants or plant viruses and include, but are not limited to, promoters such as nopaline synthase (NOS) and octopine synthase (OCS) (plated on Agrobacterium tumefaciens tumor-inducing plasmids), cauliflower mosaic virus (CaMV) 19S and 35S promoters, inducible light, the promoter from the small subunit of ribulose 1,5-bis-phosphorane carboxylase (ssRUBISCO, an abundant plant polypeptide), and the 35S promoter Fig-wort mosaic virus (FM ^ '). All these promoters were used to generate various types of DNA constructs that have been expressed in plants (see, e.g., PCT publication WO 84/02913). It may also be necessary to limit expression to certain parts of the plant susceptible to insect attack. For example, a root-specific promoter can be used to limit expression to the root region or a promoter to facilitate expression in the root to increase the level of active protein in the roots. This method is beneficial for plants attacked by root-eating insects.

Certain plant promoters are also more effective in monocots. For example, the rice actin promoter described in WO 91/09948 is effective for expression in maize. The maize ubiquitin promoter described in EP 0342926 can also be used in monocotyledonous plants.

The promoters used in the DNA constructs (i.e., chimeric plant genes) can be modified as needed to affect their driving characteristics. For example, the CaMV35S promoter can be ligated to the portion of the ssRUBISCO gene suppressing the expression of ssRUBISCO in the absence of light to produce a promoter active in leaves but not in roots. The resulting chimeric promoter can be used as described above.

For the purposes of this specification, the term "CaMV35S promoter" includes variants of the CaMV35S promoter, eg, promoters ligated to operational regions, random or controlled mutations, and the like. In addition, promoters may be altered to contain multiple "enhancement sequences ⁴¹ to enhance gene expression." Examples of such improvement sequences are provided by Kay et al. (1987).

The particular promoter selected should be capable of causing sufficient expression of the enzyme coding sequence to result in the production of an effective amount of patatin. A preferred promoter is the CaMV E35S (enhanced CaMV35S) promoter.

The RNA resulting from the DNA construct also contains a 5 'untranslated starting sequence. This sequence may be derived from a promoter selected for expression of the gene and may be specifically modified to enhance translation of the mRNA. The 5 'untranslated regions can also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence. The invention is not limited to constructs in which the untranslated region is derived from the untranslated sequence adjacent promoter sequence.

As noted above, the 3 'untranslated region of a chimeric plant gene contains a polyadenylation signal which serves in plants to cause adenylate nucleotides to be added to the 3' end of the RNA. Examples of preferred 3 'regions are (1) transcribed untranslated 3' regions containing the polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene and (2) plant genes such as the soy 7s storage protein genes and the ssRUBISCO gene E9 peas [Fischhoff et al.]

Location

The vectors containing the patatin cassettes described above allow the expression of the active protein in the cytoplasm or vacuoles of plant cells. It may be desirable to direct most or all of the patatin into the plant's secretory system. To achieve this, it may be advantageous to use a signal sequence derived from a bacterial or plant gene, but a plant gene is probably preferable. Examples of such signal sequences are those derived from the endoproteinase B gene (Koehler and Ho) and the tobacco PR1b gene (Comelissen et al.). pMON 10824, described in EP 0385962, is a plant transformation vector designed to express the butterfly-active B. t. kurstaki protein. In pMON10824, the B. t. K. Coding sequence is aligned with the signal sequence of PR1b and the 10 amino acids of mature

176,936 of the PR1b coding sequence. To create a vector in which the PR1b signal is fused to the patatin gene, pMON10824 is cleaved with BglII and NcoI and the small BglII-NcoI fragment containing the PR1b signal is isolated. In the ligation reaction, the small BglII-NcoI fragment of pMON 10824 is mixed with the 1000 bp NcoI-EcoRI fragment of pMON19714 and the BamHI-EcoRI digested pMON19470 (Brown et al.). The reaction produces a plasmid in which the patatin coding sequence is fused to the secretion signal from the PRIb gene and driven by the CaMv35S promoter and the intron for expression in monocots. For expression in dicots, a similar reaction can be performed. The NotI-NotI fragment of the dicotyledon expression vector can be inserted into the dicotyledonous transformation vector as described below and mobilized in a disarmed Agrobacterium host to transform dicotyledons. Thus, plants can be obtained that produce patatin secreted into the intercellular space.

The NotI-NotI fragment of this monocot plasmid can be inserted into a maize transformation vector (such as pMON18181 described above) to generate patatin secreting maize.

It might be beneficial to direct the localization of patatin to another cell space, the chloroplast. Proteins can be targeted to the chloroplast by introducing at the N-terminus of the chloroplast transit peptide (CTP). One of the CTPs developed which directs heterologous proteins to the chloroplast is the CTP obtained from the small gene subunit of RUBISCO Arabidopsis, designated ats1A. A variant of this transit peptide encoding a transit peptide, 23 amino acids of the mature RUBISCO sequence, plus a reiteration of the transit peptide cleavage site was constructed for the preferred chloroplast localization of the B. tk pMON19643 protein, described by Brown et al., Contains the Arabidopsis ats1A transit peptide fused to the GOX gene, and can be used as a basis for constructing a vector for the chloroplast localization of patatin. Complete EcoRI digestion and partial NcoI digestion of pMON19643 are performed and the large fragment (4000 bp) is isolated. In a ligation reaction, the NcoI-EcoRI fragment from pMON 19714 is mixed with the large fragment of pMON19643. The reaction results in a plasmid in which the patatin coding sequence is fused to the Arabidopsis 23 amino acid mature RUBISCO transit peptide and driven by the CaMV E35S promoter. Alternatively, a similar plasmid can be constructed to replace the promoter with the FMV35S promoter. Such plasmids are mobilized in a disarmed Agrobacterium host to transform dicots. Alternatively, the NotI-NotI fragment is cloned into the maize transformation vector as described above. Plants which produce localized patatin in chloroplasts can thus be obtained.

Transformation and expression in plants

A chimeric plant gene containing a structural coding sequence may be inserted into the plant genome by any convenient means. Suitable plant transformation vectors include those derived from the Ti plasmid Agrobacterium tumefaciens as also described, for example, by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and EPO publication 0120516 (Schilperoort et al.). In addition to plant transformation vectors derived from Ti plasmids or inducing in the Agrobacterium root (Ri), alternative methods for inserting the DNA constructs of the invention into plant cells can be used. Such methods may include, for example, the use of liposomes, electroporation, chemicals to increase free DNA uptake, delivery of free DNA by microbombardment, and transformation with viruses or pollen.

Transient expression of patatin in tobacco cells

A particularly useful plasmid cassette transformation vector for a dicotyledonous plant is pMON11794. The pMON11794 expression cassette consists of the CaMVE35S promoter, the untranslated Hsp70 initial 5 'petunia sequence and the 3' end containing polyadenylation signals from the NOS gene. pMON11794 includes NcoI and EcoRI sites for insertion of coding sequences and NotI-NotI sites flanking the plant gene expression cassette.

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PatA + (SEQ ID No. 11), PatB + (SEQ ID No. 16), PatC + (SEQ ID No. 17) and PatG + (SEQ ID No. 22) were inserted in pMON11794 to generate pMON 19745, pMON19742 respectively , pMON19743 and pMO'N19744. Each of the vectors was electroporated in tobacco protoplasts. Patatin expression in transformed tobacco cells was confirmed by Western analysis.

Permanent transformation of dicotyledons

The permanent transformation of dicots with the patatin gene is described by Rosahl et al. The tobacco was transformed with the patatin gene under the control of a leaf and stem specific promoter. Patatin is expressed.

The NotI-NotI fragment from pMON19745 was inserted into pMON17227, a Ti plasmid vector described by Barry et al. in WO 92/04449, incorporated herein by reference, to prepare pMON22566. This vector contains the glyphosate resistance gene described by Barr for selection of transformed plants. Sequences ID no. 16, 17 and 22 to generate the corresponding vectors MON22563, 2564, and 22565.

The vector was introduced into a disarmed Agrobacterium ABI host to transform explanted tomatoes into tissue culture. After selection for glyphosan resistance and plant regeneration, whole plants expressing the patatin gene were obtained. The expression of the patatin gene was confirmed by Western blot analysis, and preliminary results show an expression of 0.1 to 0.5% of the total protein. Insect larvae bioassays are underway.

Transient expression of patatin in corn cells

The 1000 bp NcoI-EcoRI fragment of pMON19729, described above, was introduced into pMON19433 described in WO 93119189 and co-pending U.S. Patent Application No. 07/855857, filed March 19, 1992 (Brown et al.), Incorporated herein by reference . The resulting plasmid, pMON19731, was digested with NotI and the resulting fragment was inserted into pMON10081, also described by Brown et al., Yielding pMON 19740. This plasmid was electroporated into a maize leaf protoplast according to Sheen, 1991. Expression of patatin in transformed maize protoplast cells confirmed by Western analysis.

To achieve cytoplasmic expression of patatin, the NcoI-EcoRI fragment of pMON 19714 was introduced into pMON19433 to give rise to pMON19730. The NotI fragment of pMON19730 was introduced into pMON10081 and the resulting plasmid, pMON 19739, was electroporated to protoplast of corn leaves that produced patatin, as confirmed by Western blot analysis.

Pat17, in the presence and absence of locating signals, was also expressed in maize protoplasts. pMON19761 was constructed by inserting a 1100 bp fragment of NcoI-EcoRI (SEQ ID NO. 30) encoding the Pat17 + protein (mature Pat17 protein and its own signal sequence for vacuole localization) into pMON19648. Thus, pMON19761 contains the CaMV E35S promoter, the Hsp 70 intron, the Pat17 + gene and the NOS terminator for expression in maize cells.

In order to obtain a vector for cytoplasmic expression, the Pat17 + sequence in pMON 19761 was replaced with the NcoI-EcoRI fragment encoding the PatI7 _m protein (SEQ ID NO: 31) from pMON25213 to obtain the pMON25223 construct.

pMON25224 was obtained by inserting two fragments, a 300 bp XbaINcoI fragment containing a chloroplast transit peptide (CTP) from the SSU 1 a Arabidopsis thaliana gene (Timko et al.) from pMON19643 (Brown et al.) and a 1000 bp NcoI-EcoRI fragment for Pat17 _m from pMON25213, inserted into pMON19761 (XbaI-EcoRI). Thus, pMON25224 contains the CaMV E35S promoter, the Hsp 70 intron, the CTP / Pat17m coding sequence and the NOS terminator.

For extracellular localization, the 5 'end of endoproteinase B cDNA (Koehler and Ho) encoding the extracellular signal peptide of secreted protein was fused to the Pat17m gene from pMON25213. The BgUI-EcoRI fragment containing the chimeric gene was obtained by the splicing technique18

176,936 overlapping ends (Horton et al.) And inserted into pMON19761 (BamHI-EcoRI) to give pMON25225.

All of these constructs were electroporated into corn leaf progenitor and _m Pat17 expression was confirmed by Western blot.

Persistent transformation of maize with the patatin gene

The maize transformation vector, pMON18181, was constructed from pMON19653 and pMON19643 (Brown et al.). This construct contains the CaMV E35S promoter cassette, the Hsp70 intron, the glyphosate selection marker CP4, and the NOS terminator; CaMV E35S promoter cassette, Hsp70 intron, GOX glyphosate selection tag and NOS terminator; and a single NotI site for insertion of a gene expression cassette containing the patatin gene. Sequence no. 11 and sequence no. The 30 were introduced as NotI-NotI fragments into pMON 18181 to generate pMON 19746 and pMON 19764, respectively.

These vectors were introduced by bombarding the embryonic tissue culture using the biolistic launcher described by Brown et al. Transformed cells were selected for glyphosate resistance and whole plants were regenerated. The insect resistant plants express 0.1-0.5% of the total protein gene by Western blot analysis, esterase treatment and / or insect resistance test.

Synthetic genes for improved expression of monocots

Coding sequence modifications have been obtained to improve the expression of other insecticidal protein genes such as the Bacillus thuringiensis delta endotoxin sequence (Fischhoff and Perlak; WO 93/07278, Ciba-Geigy). The coding sequence modified in this way was designed to improve the expression of patins in plants, especially in maize. The modified sequence Pat17 + is shown as sequence ID No. 33. A DNA fragment containing the sequence ID NO. 33 can be synthesized and inserted into a vector corn expression cassette such as pMON19470 (Bown et al.). The maize expression cassette is then inserted into pMON18181 or other maize transformation vector containing a selectable tag gene for maize transformation, yielding complete maize to produce ąPat17 +.

All publications and patents mentioned in the specification are hereby incorporated by reference as if each had been separately described as being incorporated herein by reference.

Literature

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Bagdasarian et al., Gen, 16: 237-47, 1981.

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Cornelissen, B. J. C., et al. EMBO Journal, 5: 37-40,1986.

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Fischhoff, D. A. and Perlak, F. J. European Patent Application, No. 0385962, 1990.

Fischhoff et al., Bio / Technol. 5: 807,187.

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Gaillaird, T. Biochem. J. 121: 379-390, 1971.

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Horton et al., Gen, 77: 61-68, 1989.

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Luckow, V. A., et al., J. Virol. 67: 4566-4579, 1993.

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Olins, P., et al., J. Biol. Chem., 264: 16973-16976, 1989.

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Racusen, D., Can. J. Bot., 64: 2104-2106, 1986.

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Sheen, Jen, Plant Cell, 3: 225-245, 1991.

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SEQUENCE LIST (1) GENERAL INFORMATION:

(1) APPLICANTS:

CA) NAME: Monsanto Company (B) STREET: 800 North Lindbergh Boulevard (C) CITY: St. Louis (D) STATE: Missouri (E) COUNTRY: St. US Americas (F) ZIP CODE (ZIP): 63167 (G) TELEPHONE: (314)694-3131 (H) TELEFAX: (314)694-5435 (ii) NAME OF THE INVENTION: Insect Control Method (iii) NUMBER OF SEQUENCES: 33 ( iv) FORM FOR COMPUTER:

(A) MEDIA TYPE: floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentIn Release 1.0,

1.25 (EPO) (vi) PREVIOUS NOTIFICATION DATA:

(A) APPLICATION NUMBER: US 08/031146 (B) FILING DATE: MARCH 12, 1993 (2) INFORMATION FOR SEQUENCE ID NO .: 1:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 15 Amino Acids

Version a

176 936 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) THYi PARTICLES: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1

Liz Leu Glu Glu Met Wal Tre Wal Leu Ser Ile Asp Gli

Gli Gli

10 (2) INFORMATION FOR SEQUENCE ID NO .: 2 (1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids (B) TYi: amino acid (D) TOPOLOGY: linear (ii) THESEi PARTICLES: protein (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO. : 2. ·.

Xaa Leu Gli Glu Met Wal Tre Wal Leu Ser Ile Asp Gli Gli Gli

10 (2) INFORMATION FOR SEQUENCE ID NO .: 3:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 15 Amino Acids

176 936 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) THYi PARTICLES: protein (xi) SEEKNEJI OPIP: SEEKEECCA ID NO .: 3:

Tre Leu Gli Glu Met Eal Tre Eal Leu Ser Ile Asp Gli

Gli Gli

10 (2) IRFORMATION FOR SEQUENCES jr id .: 4:

(1) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 15 amino acids (B) TYi: amino acid (D) TOPOLOGY: linear (ii) THESE MOLECULES: protein (xi) SEEEWEECTION OPTIONS: EJ ID .: 4:

Tre Leu Gli Glu Met Eal Tre Eal Leu Ser Ile Asp Gli Gli Gli

10 (2) INFORMATION FOR SECTION CR ID .: 5:

(i) CHARACTERISTICS OF THE SECTION:

(A) LENGTH: 15 Amino Acids

176 936 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 5:

Liz Leu Xaa Glu Met Wal Tre Wal Leu Ser Ile Asp Gli

Gli Gli

10 (2) INFORMATION FOR SEQUENCE ID NO .: 6:

(1) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 6:

Xaa Xaa Glu Glu Met Wal Tre Wal

5 (2) INFORMATION FOR SEQUENCE ID NO .: 7:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

176 936 (ii) PARTICLE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 7:

Leu Asp Tyr Cheese Liz Gln Met Leu Leu Leu Cheese Leu Gli

Tre Gli

10 (2) INFORMATION FOR SEQUENCE ID NO .: 8:

(i) CHARACTERISTICS OF THE SEQUENCES:

(AND) LENGTH: 8 Amino Acids (B) TYPE: Amino Acid (D) TOPOLOGY: linear (ii) TYPE PARTICLES: protein

(xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 8:

Liz Leu Asp Tyr Liz Gln Met Leu

5 (2) INFORMATION FOR SEQUENCE ID NO .: 9:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 9:

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Cheese Leu Xaa Tyr Liz Gln Met Leu Leu Leu Cheese Leu Gli Tre Gli

10 (2) INFORMATION DRA SEQUENCE ID NO .:: 3.0:

(1) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: white (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 10:

Leu Asn Tyr Liz Gln Met Leu Leu Leu Cheese Leu Gli Cheese

Tre Gli

10 (2) INFORMATION FOR SEQUENCE ID NO .:: 11:

(i) CHARACTERISTICS OF THE SEQUENCES:

(AA) LENGTH: 1171 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) CCASTEK TYPE: cDNA

176 936 (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 11:

CCATGGCAAC

ACTACTAGTT

CAACATGTGC

GGTGGAATTA

AGGGAATCAT

GAAGTGGACA

ATAATAAAGA

AGTACAGGAG

GTTTATTGAC

GCTGCTGCCA

AAGATATTGT

AGTGGTTCAA

TTATTGGCCC

AAACTTGGAG

AAACTCGTGT

ATCAAAACAA

ATAAGCCAGT

GATGCTAAGA

TGTATGACAT

CATTACTTTA

TTACTCATAC

GGTGTTGCTA

CTGTTGGTGA

CAAGAGGATC

CAGCATTTTC

TCATTAGGCA

TACTAAATCT

TTTTTAATTT

TATTTTTTAT

TAAGTTGGAA

TCCAGCTATC

TGCAAGACTT

TGCTATGATA

ACCCTTTTAC

AATGTATGAT

GCATCAAGCT

AATATTCACT

ATGCTATTCC

TAGTAATGGT

TCCGGCGTTA

TTCAATTAAG

GAAATGGTGA

ATTCTCGAAT

GCAGATTACT

ACTACTCCAA

TTCGAACATG

GGAAAATATC

TTGACAGAAG

AAGTCAAATT

ACAGCAGCAG

GATATATG

TTATCCCTTA

TCATTGGATT

CTGTTCTTAG

TTCTTGAAGG

TTGATGTAAT

ATGAAAACAA

GCCCTCATAT

TTCTGCAAGT

TTGCCATCTC

TAGCAAAGTC

CTCCAATATA

AGTTCAATCT

GCGTTGCAAC

ACAAGCAAAT

GATATTAGCA

TATTGATGGA

120

ACAACTTCAG

180

TGGAGGAACA

240

TCGACCCTTT

300

TTTTAATTAT

360

TCTTCAAGAA

420

AAGCTTTGAC

480

TCCAGAATTG

540

TTTTCCTCCA

600

TGTTGATGGT

6660

GAGACTTGCA

7 ^ C

GTTGTTGCTC

780

176 936

CTGGCACTAA AAATGGGGTC TTCAGAGTTT GATAAAACAT ATACAGCACA AGAGGCAACT 880 CTCTACGATG GATGTTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900 ATTATTACAT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 900 AAGAAAATGC ATTAACAGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1000 AATTATTAGT ACAAGTTGGT GAAACATTAT TGAAGAAACC AGTTTCCAAA GACAGTCCTG H00 AAACCTATGA GGAAGCTCTA AAGAGGTTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1110 GAGCAAACAA AGCTTCTTAT TGATAGAATT C. 1111

(2) INFORMATION FOR SEQUENCE ID NO .: 12:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 37 base pairs (B) TYi: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 12:

CATGTGCTCT AGAAGATCTC CACCATGGCG TTGGAAG (2) INFORMATION FOR SEQUENCE ID NO .: 13:

176 936 (1) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: DNA (syityic) (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 13:

GCTTCTTATT GATAGAATTC AAGGTC 2 6 (2) INFORMATION FOR SEQUENCE ID NO .: 34:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 3305 bp (B) TYPE: Nucleic acid (C) CHAIN: Single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: NO.110: 13:

CCATGGCGTT ATTAAGGGAA GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA 66 TCATTCCAGC TATCATTCTC GAATTTCTTG aaggacaact TCAGGAAGTG GACAATAATA 120 AAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA

GGAGGTTTAT

130

176 936

TGACTGCTAT

GCCAAAGATA τaGTACccaa ^ AATTATTG

GCCCAATGTA

GGAGAAACTC

GTGTGCATCA

ACAAATAAGC

CAGTAATATT

AACATCTATG

ACATATGCTA ^ TATTACTC

ATACTAGTAA

GCTACTGTTG

GTGATCCGGC

GATCCAGCAT

TTTCTTCAAa

GGCACTGGCA

CT ^ ATTCAGA

GGTCCTCTAC

GAaGGATGaT

ACrGATTATT

ACATTTCTAC

GTTCAAGAAA

ATGCATTAAC

ATGGAATTAT

TAGTACAAGT

GATAACTACT

TTACTTCG ^ A

TGATGGAAAA

AGCTTTGACA

CACTAAGTCA

TTCCACAGCA aGGTGAaAaA

GTTATTATCC aAAGacAτaG

GTTTGATAAA

AGCTATACAG aGTTTTTCAA

AGGCACAACT

TGGTGAAACA

CCAAATGAAA

CATGCCCCTC

GAAGTTGCCA

AATTTAGCAA

GCAGCTCCAA

TATGAGTTCA

CaTAGCGaTG

GATTACAAGC

ACATATACAG

CAAATGACTA

GCTCGTCATa

ACTGAAATGG

TTATTGAAGA

CCTGAAACCT

ACAATCGACC

AaAaaτττAA

AACTTCTTCA

TCTCiAGCTT

AGTCTCCAGA aAaATττacc

ATcaaGaaGA

CAACGAGACT

AftATCTTCTT

CACAAGAGGC

ATGCAGCAAG

CACAAAACAA

ATGATGCG ^

AACCAGTTTC cττaGcaGCτ

240

TTATAGTGGT

300

AGAAAAACTT

360

TGACATCAAA

420

ATTGGATGCT

480

TCCACATTAC

540 aGGaGGTGTT

600

TGCACAAGAG

660

GCTCTCATTA

720

AGCTAAATGG

780

TTCTTACATG

840

TTACCTCAGC

0

TGAGGCTAAT

960

CAAAGACAGT

1020

176 936

ATGAGGAAGC TCTAAAGAGG TTTGCAAAAT TGCTCTCTGA TAGGAAGAAA

CTCCGAGCAA 1080

ACAAAGCTTC TTATTGATAG AATTC 1105 (2) INFORMATION FOR RR SECTION ID .: 15:

(i) CHARACTERISTICS OF THE SEKE / ERCJ! :

(A) LENGTH: 1106 bp (B) TYi: nucleic acid (C) CHAIN: single (D) TOiology: linear (ii) THESEi PARTICLES: cDRA (xi) OiIS SE ^^ OJI: RR SEQ ID .: 15:

CCATGGCGTT GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60 TCATTCCAGC TACCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAGTG GACAATAATA 120 AAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 110 TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC CTTTGCTGCT GCCAAAGATA 200 TTGTACCCTT TTACTTCGAA CATGGCCCTC ATATTTTTAA TTATAGTGGT TCAATTATTG 300 GCCCAATGTA TGATGGAAAA TATCTTCTGC AAGTTCTTCA AGAAAAACTT GGAGAAACTC 300) GTGTGCATCA AGCTTTGACA GAAGTTGCCA TCTCAAGCTT TGACATCAAA ACAAATAAGC 400

176 936

CAGTAATATT CACTAAGTCA AATTTAGCAA AGTCTCCAGA ATTGGATGCT AAGATGTATG 480 ACATATGCTA TTCCACAGCA GCAGCTCCAA TATATTTTCC TCCACATTAC TTTATTACTC 540 ATACTAGTAA TGGTGATATA TATGAGTTCA ATCTTGTTGA TGGTGGTGTT GCTACTGTTG 600 GTGATCCGGC GTTATTATCC CTTAGCGTTG CAACGAGACT TGCACAAGAG GATCCAGCAT 6650 TTTCTTCAAT TAAGTCATTG GATTACAAGC AAATGTTGTT GCTCTCATTA GGCACTGGCA 720 CTAATTCAGA GTTTGATAAA ACATATACAG CACAAGAGGC AGCTAAATGG GGTCCTCATC 780 GATGGATGTT AGCTATACAG CAAATGACTA ATGCAGCAAG TTCTTACATG ACTGATTATT 840 ACATTTCTAC TGTTTTTCAA GCTGGTCATT CACAAAACAA TTACCTCAGG GTTCAAGAAA 900 ATGCATTAAC AGGCACAACT ACTGAAATGG ATGATGCGTC TGAGGCTAAT ATGGAATTAT 960 TAGTACAAGT TGGTGAAAAA TTATTGAAGG AACCAGTTTC CAAAGACAGT CCTGAAACCT 1020 CTGAGGAAGC TCTAAAGAGG TTTGCAAAAT TGCTCTCTGA TAGAAAGAAA CTCCGAGCAA 1080 ACAAAGCTTC TTATTAATGA GAATTC 1106

(2) INFORMATION FOR SEQUENCE ID NO .: 16:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1172 base pairs (B) TYPE: Nucleic acid (C) CHAIN: single

176 936 (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA (ci) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 10:

CCATGGCAAC

ACTACTAGTT

CAACATGTGC

GGTGGAATTA

AGGGAATCAT

GAAGTGGACA

ATAATAAAGA

AGTACAGGAG

GTTTATTGAC

GCTGCTGCCA

AAGATATTGT

AGTGGTTCAA

TTTTTGGCCC

AAACTTGGAG

AAACTCGTGT

ATCAAAACAA

ATAAGCCAGT

GATGCTAAGA

TGAATGACAT

CATTACTTTG

TTACTCATAC

GCTGTTGCTA

CTGTTGGTGA

CAAGTGGATC

TACTAAATCT

GTTTTAGTTT

TATTTTTTAT

TACGTTGGGA

GAAATGGTGA

CTGTTCTTAG

TCCGGCTACC

TGCAAGACTT

TGCTATGATA

ACCTTTTTAC

AATGTATGAT

GCATCAAGCT

AATATTCACT

ATGCTATTCC

TAGTAATGGA

TCCGGCGTTA

ATTCTCGAAT

GCAGATTACT

ACTACTCCAA

TTCGAACATG

GGAAAATATT

TTGACAGAAG

AAGTCAAATT

ACAGCAGCAG

GATAAATATG

TTATCCCTTA

TTCTTGAAGG

TTGATGTAAT

ATGAAAACAA

GCCCTCATAT

TTCTGCAAGT

TTGCCATCTC

TAGCAAAGTC

CTCCAACATA

AGTTCAATCT

GCGTTCGAAC

GATATTAGCA

TATTGATGGA

120

ACAACTTCAG

180

TGGAGGAACA

240

TCGACCCTTT

30Ό

TTTTAATTCT

60

TCTTCAAGAA

020

AAGCTTTGAC

080

TCCAGAATTG

540

TTTTCCTCCA

000

TGTTGATGGT

060

GAAACTTGCA

720

176 936

CAAAATTTGC TCATTAGGCA TTCAATTAAG TCATTGAATT acaacgaaaa GTTGTTGCTC 7 70 CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCAGA AGAGGCAGCT AAATGGGGTC 840 CTCTACGATG GATATTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900 ATTATTACCT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 960 AAGAAAATGC ATTAACAGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1020 AATTATTAGT ACAAGTTGGT GAAAAATTAT TGAAGAAACC AGTTTCCAAA GACAGTCCTG 1080 AAACCTATGA GGAAGCTCTA AAGAGGTTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140 GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC 1172

(2) INFORMATION FOR SEQUENCE ID NO .: 17:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1175 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) CCASTEK TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 17:

CCATGGCAAC TACTAAATCT TTTTTAATTT TAATTGTTAT GATATTAGCA

ACTACTAGTT 60

176 936

CAACATTTGC

GGTGGAATTA

AGGGAATCAT

AAAATGGACA

ATAATGCAGA

AGTACAGGAG

GTTTATTGAC

GCTGCTGCCA

ATGAAATTGT

AGGTACTGGC

CAATTTTTTG

GAAAACCTTG

GAGAAACTCG

GACATCAAAA

CAAATAAGCC

TTGGATGCTA

AGATGTATGA

CCACATTACT

TTACTACTAA

GGTGCTGTTG

CTACTGTTGC

GCAGAAAAGG

ATCCAGCATT

CTCTCATTAG

GCACTGGCAC

GCTAAATGGG

GTGCTATACA

TCTTACATGA

TTCGTTGGAA

TCCGGGTACC

TGCAAGACTT

TTCTATGATA

ACCTTTTTAC

GCCAAAATAT

TGTGCATCAA

AGTAATATTC

CATATGTTAT

TACTATTAAT

TGATCCGGCG

TGCTTCAATT

TACTTCAGAG

ATGGATGTTG

GAAATGGTGA

ATTCTCGAAT

GCAGATTACT

ACTACTCCAA

TTCGAACATG

GATGGAAAAT

GCTTTGACTG

ACCAAGTCAA

TCCACAGCAG

GGAGATAAAT

TTATTATCCA

AGGTCATTGA

TTTGATAAAA

GTTATACAGC

CTGTTCTTAG

TTCTTGAAGG

TTGATGTAAT

ATGAAAACAA

GCCCTCATAT

ATCTTATGCA

AAGTTGCCAT

ATTTAGCAAA

CAGCTCCAAC

ATGAGTTCAA

TTAGCGTTGC

ATTACAAAAA

CATATACAGC

GAATGACTGA

TATTGATGGA

120

ACAACTTCAG

00

TGGAGGAACA

200

TCGACCCTTT

300

TTTTAATTCT

360

AGTTCTTCAA

420

CTCAAGCTTT

480

GTCTCCAGAA

540

ATATTTTCCT

600

TCTTGTTGAT

660

AACGAGACTT

720

AATGTTGTTG

780

AGAAGAGACA

040

TGCAGCAAGT

900

176 936

CTGATTATTA CCTTTCTACT GTTTTTCAAG CTCAAAATTC ACAAAAGAAT TACCTCAGGG 900 TTCAAGAAAA TGCGTTAACA GGCACAACTA CTGAAATGGA TGATGCTTCT GAGGCTAATA 1102 TGGAATCATT AGTACAAGTT GGTGAAAATT TATTGAAGAA ACCAGTTCCC AAAGACAATC 1100 CTGAAACCTA TGAGGAAGCT CTAAAGAGGT TTGCAAAATT GCTTTCTGAT AGGAAGAAAC 1110 TTCGAGCAAA CAAAGCTTCT TATTAATGAG AATTC 1117)

(2) INFORMATION FOR SECTION JR ID .: 10:

(i) CHARACTERISTICS OF THE SECTION:

(A) LENGTH: 1100 base pairs (B) TYi: nucleic acid (C) CHAIN: single (D) Topology: linear (ii) PARTICLE TYPE: cDJA (xi) SECTION DESCRIPTION: RR SECTION ID .: 10: CCATGGCGTT GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60 TCATTCCGGC TACCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAGTG GACAATAATA 120 AAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 180 TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC CTTTGCTGCT GCCAAAGATA 240

176 936

TTGTACCTTT

TCAATTTTTG

GCCCAATGTA

GGAGAAACTC

GTGTGCATCA

ACAAATAAGC

CAGTAATATT

AAGATGTATG

ACATATGTTA

TTTGTTACTC

ATACTAGTAA

GCTACTGTTG

GTGATCCGGC

GATCCAAAAT

TTGCTTCAAT

GGCACTGGCA

CTAATTCAGA

GGTCCTCTAC

GATGGATATT actgattatt

ACCTTTCTAC

GTTCAAGAAA

ATGCATTAAC

ATGGAATTAT

TAGTACAAGT

CCTGAAACCT

ATGAGGAAGC

CTCCGAGCAA

TTACTTCGAA

TGATGGAAAA

AGCTTTGACA

CACTAAGTCA

TTCCACAGCA

TGGAGATAAA

GTTATTATCC

TAAGTCATTG

GTTTGATAAA

AGCTATACAG

TGTTTTTCAA

AGGCATAACT

TGGTGAAAAA

TCTAAAGAGG

CATGGCCCTC

TATTTTCTGC

GAAGTTGCCA

AATTTAGCAA

GCAGCTCCAA

TATGAGTTCA

CTTAGCGTTG

AATTACAAGC

ACATATACAG

CAAATGACTA

GCTCGTCATT

ACTGAAATGG

TTATTGAAGA

TTTGCAAAAT

ATATTTTTAA.

AAGTTCTTCA

TCTCAAGCTT

AGTCTCCAGA

CATATTTTCC

ATCTTGTTGA

CAACGAAACT

AAATGTTGTT

CAGAAGAGGC

ATGCAGCAAG

CACAAAACAA

ATGATGCGTC

AACCAGTTTC

TGCTCTCTGA

TTCTAGTGGT

300

AGAAAAACTT

3300

TGACATCAAA

420

ATTGGATGCT

400

TCCACATTAC

540

TGGTGCTGTT

660

TGCACAAGTG

660

GCTCTCATTA

770

AGCTAAATGG

8 0

TTCTTACATG

88 ^ 0

TTACCTCAGG

900

TGAGGCTAAT

990

CAAAGACAGT

1100

TAGGAAGAAA

1180

176 936

ACAAAGCTTC TTATTAATGA GAATTC 1106 (2) INFORMATION FOR SEQUENCE ID NO .: 19:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1172 base pairs (B) TYPE: Nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 19:

CCATGGCAAC ACTACTAGTT TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA 60 CAACATTTGC TACATTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180 ATAATGCAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA GGTACAGGAG 240 gtttattgac TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCTTTT GCTGCTGCTA 300 AAGATATTAT ACCTTTTTAC TTCGAACACG GCCCTCATAT TTTTAATTAT AGTGGTTCAA 360 TTTTAGGCCC AATGTATGAT GGAAAATATC TTCTGCAAGT TCTTCAAGAA AAACTTGGAG 420 AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGCCATCTC AAGCTTTGAC ATCAAAACAA 480

176 936

ATAAGCCAGT

GATGCTAAGA

TGTATGACAT

CATCACTTTG

TTACTCATAC

GCTGTTGCTA

CTGTTGGTGA

CAAGAGGATC

CAGCATTTTC

TCATTAGGCA

CTGGCACTAA

AAATGGGGTC

CTCTACGATG

TACATGACTG

ATTATTACAT

CTCAGGGTTC

AAGAAAATGC

GCTAATATGG

AATTATTAGT

GACAGTCCTG

AAACCTATGA

AAGAAACTCC

GAGCAAACAA

AATATTCACT

ATGCTATTCC

TAGTAATGGT

TCCGGCGTTA

TTCAATTAAG

TTCAGAGTTT

GATGTTAGCT

TTCTACTGTT

ATTAAATGGC

ACAAGTTGGT

GGAAGCTCTA

AGCTTCTTAT

AAGTCAAATT

ACAGCAGCAG

GCTAGATATG

TTATCCCTTA

TCATTGGATT

GATAAAACAT

ATACAGCAAA

TTTCAAGCTC

ACAACTACTG

GAAACATTAT

AAGAGATTTG

TAATGAGAAT

TAGCAAAGTC

CTCCAATATA

AGTTCAATCT

GCGTTGCAAC

ACAAGCAAAT

ATACAGCAGA

TGACTAATGC

GTCATTCACA

AAATGGATGA

TGAAGAAACC

CAAAATTGCT

TC

TCCAGAATTG

540

TTTTCCTCCA

600

TGTTGATGGT

600

GAGACTTGCA

720

GTTGTTGCTC

780

AGAGGCAGCT

800

AGCAAGTTCT

900

AAACAATTAC

960

TGCGTCTGAG

1020

AGTCTCCAAA

1180

CTCTGATAGG

1140

1112 (2) INFORMATION FOR JR SECTION ID .: 20:

(i) CHARACTERISTICS OF THE SEEKING:

(A) LENGTH: 1100 base pairs

176 936 (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 20:

CCATGGCGTT

ATTAAGGGAA

TCATTCCGGC

GACAATAATG

CAGATGCAAG

GGAGGTTTAT

TGACTGCTAT

GCTAAAGATA

TTATACCTTT

TCAATTTTAG

GCCCAATGTA

GGAGAAACTC

GTGTGCATCA

ACAAATAAGC

CAGTAATATT

AAGATGTATG

ACATATGCTA

TTTGTTACTC

ATACTAGTAA

GCTACTGTTG

GTGATCCGGC

GATCCAGCAT

GGAAGAAATG

TACCATTCTC

ACTTGCAGAT

GATAACTACT

TTACTTCGAA

TGATGGAAAA

AGCTTTGACA

CACTAAGTCA

TTCCACAGCA

TGGTGCTAGA

GTTATTATCC

GTGACTGTTC

GAATTTCTTG

TACTTTGATG

CCAAATGAAA

CACGGCCCTC

TATCTTCTGC

GAAGTTGCCA

AATTTAGCAA

GCAGCTCCAA

TATGAGTTCA

CTTAGCGTTG

TTAGTATTGA

AAGGACAACT

TAATTGGAGG

ACAATCGACC

ATATTTTTAA

AAGTTCTTCA

TCTCAAGCTT

AGTCTCCAGA

TATATTTTCC

ATCTTGTTGA

CAACGAGACT

TGGAGGTGGA

TCAGGAAGTG

120

AACAGGTACA

180

TTTTGCTGCT

240

TTATAGTGGT

300

AGAAAAACTT

360

TGACATCAAA

020

ATTGGATGCT

080

TCCACATCAC

500

TGGTGCTGTT

000

TGCACAAGAG

000

176 936

aaacaτcAAa GGCACTGGCA TAAGTCATTG GATTACAAGC AAATGTaGTT GCTCTCATTA 723 ^ AATTCAGA GTaaGATAAA ACATATACAG CAGAAGAGGC AGCTAAATGG GGTCCTCTAC 780 GATGGATGTT AGCTATACAG CAAATGACTA ATGCAGCAAG TTCTTACATG ACTGATaAaT 840 ACATT ^ TAC TGaTTTTCAA GC ^ GTCATT CACAAAACAA TTACCTCAGC GTTCAAGAAA 9900 ATGCATTAAA TGGCACAACT ACTGAAAaGG AaGATGCGaC aGAGGCTAAT ATGGAATTAT 900 TAGTACAAGT TGGTGAAACA TTATTGAAGA AACCAGTCTC CAAAGACAGT CCTGAAACCT 1020 ATGAGGAAGC TCTAAAGAGA aaaGCAAAAa TGCTCTCaGA TAGGAAGAAA CTCCGAGCAA 1080 ACAAAGCTTC TTATTAATGA GAATTC 1106

(2) INFORMATION FOR SEQUENCE ID NO .: 21:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1109 bp (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 21:

CCAaGGCGTa GGAAGAAATG GaGGCTGTaC TTACTATTGA TGGAGGTGGA

ATTAAGGGAA

176 936

TCATTCCGGG

GACAATAATG

CAGATGCAAG

GGAGGTTTAT

TGACTGCTAT

GCCAATGAAA

TTGTACCTTT

TGGCCAATTT

TTTGGCCAAA

CTTGGAGAAA

CTCGTGTGCA

AAAACAAATA

AGCCAGTAAT

GCTAAGACGT

ATGACATATG

TACTTTGCTA

CTAATACTAT

GTTGCTACTG

TTGCTGATCC

GAGGATCCAG

CATTTGCTTC

TTAGGCACTG

GCACTACTTC

TGGGGTGCTC

TACAATGGAT

ATGACTGATT

ATTACCTTTC

AGGGTTCAAG

TACCATTCTC

ACTTGCAGAT

GATAACTACT

TTACTTCGAA

ATATGATGGA

TCAAGCTTTG

ATTCACTAAG

TTATTCGACA

TAATGGAGAT

GGCGTTATTA

AATTAGGTCA

AGAGTTTGAT

GTTGGTTATA

TACTGTTTTT

GAATTTCTTG

TACTTTGATG

CCAAATGAAA

CATGGCCCTC

AAATATCTTA

ACAGAAGTTG

TCAAATTTGG

GCAGCAGCTC

AAATATGAGT

TCCGTTAGCG

TTGAATTACA

AAAACACATA

CAGCAAATGA

CAAGATCTTC

AAGGACAACT

TAATTGGAGG

ACAATCGACC

ATATTTTTAA

TGCAAGTTCT

CCATCTCAAG

CAAAGTCTCC

CAACATATTT

TCAATCTTGT

TTGCAACGAG

AAAAAATGTT

CAGCAGAAGA

CTGAGGCAGC

ATTCACAAAA

TCAGAAAATG

120

AACAAGTACA Fr.

CTTTGCTGCT

240

TTCTAGGTAC

300

TCAAGAAAAA

360

CTTTGACATC

420

AGAATTGGAT

0

TCCTCCACAT

540

TGATGGTGCT

600

ACGTGCACAA

660

GTTGCTCTCA

720

GACAGCTAAA

780

AAGTTCTTAC

840

CAATTACCTC

600

176 936

AAAATGCATT AACAGGCACA ACTACTAAAG CGGATGATGC TTCTGAGGCT

AATATGGAAT 996

TATTAGCACA AGTTGGTGAA AATTTATTGA AGAAACCAGT TTCCAAAGAC

AATCCTGAAA 1322)

CCTATGAGGA AGCTCTAAAG AGGTTTGCAA AATTGCTTTC TGATAGGAAG

AAACTTCGAG 1300

CAAACAAAGC TTCTTATTAA TGAGAATTC 133 ^^ (2) INFORMATION FOR SEQUENCE ID NO .: 22:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 3,372 bp (B) TYPE: Nucleic acid (C) CHAIN: Single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 22:

CCATGGCAAC ACTACTAGTT TACTAAATCT TTTTTAATTT TATTTTTTAT GATATTAGCA 60 CAACATGTGC TAAGTTGGAA GAAATGGTTA CTGTTCTAAG TATTGATGGA GGTGGAATTA HO AGGGAATCAT TCCAGCTATC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 13 01 ATAATAAAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 220 GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 330

176 936

AAGATATTGT

AGTGGTTCAA

TTTTAGGCCC

AAACTTGGAG

AAACTCGTGT

ATCAAAACAA

ATAAGCCAGT

GATGCTAAGA

TGTATGACAlT

CATCACTTTG

TTACTCATAC

GCTGTTGCTA

CTGTTGGTGA

CAAGAGGATC

CAGCATTTTC

TCATTAGGCA

CTGGCACTAA

AAATGGGGTC

CTCTACGATG

TACATGACTG

ATTATTACAT

CTCAGGGTTC

AAGAAAATGC

GCTAATATGG

AATTATTAGT

GACAGTCCTG

AAACCTATGA

ACCCTTTTAC

AATGTATGAT

GCATCAAGCT

AATATTCACT

ATGCTATTCC

TAGTAATGGT

TCCGGCGTTA

TTCAATTAAG

TTCAGAGTTT

GATGTTAGCT

TTCTACTGTT

ATTAAATGGC

ACAAGTTGGT

GGAAGCTCTA

TTCGAACATG

GGAAAATATC

TTGACAGAAG

AAGTCAAATT

ACAGCAGCAG

GCTAGATATG

TTATCCCTTA

TCATTGGATT

GATAAAACAT

ATACAGCAAA

TTTCAAGCTC

ACAACTACTG

GAAACATTAT

AAGAGATTTG

GCCCTCATAT

TTCTGCAAGT

TTGCCATCTC

TAGCAAAGTC

CTCCAATATA

AGTTCAATCT

GCGTTGCAAC

ACAAGCAAAT

ATACAGCAGA

TGACTAATGC

GTCATTCACA

AAATGGATGA

TGAAGAAACC

CAAAATTGCT

TTTTAATTAT

360

TCTTCAAGAA

422

AAGCTTTGAC

420

TCCAGAATTG

540

TTTTCCTCCA

660

TGTTGATGGT

660

GAGACTTGCA

770

GTTGTTGCTC

770

AGAGGCAGCT

880

AGCAAGTTCT

990

AAACAATTAC

966

TGCGTCTGAG

102 0

AGTTTCCAAA

108 0

CTCTGATAGG

AAGAAACTCC

1120

176 936

GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC (2) INFORMATION FOR SEQUENCE ID NO .: 23:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1100 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 23:

CCATGGTTGG AAGAAATGGT GACTGTTCTA AGTATTGATG

TAAGGGAATC

ATTCCAGCTA TCATTCTCGA ATTTCTTGAA GGACAACTTC

CAATAATAAA

GATGCAAGAC TTGCAGATTA CTTTGATGTA ATTGGAGGAA

AGGTTTATTG

ACTGCTATGA TAACTACTCC AAATGAAAAC AATCGACCCT

CAAAGATATT

GTACCCTTTT ACTTCGAACA TGGCCCTCAT ATTTTTAATT

AATTTTAGGC

CCAATGTATG ATGGAAAATA TCTTCTGCAA GTTCTTCAAG

AGAAACTCGT

GTGCATCAAG CTTTGACGGA AGTTGCCATC TCAAGCTTTG

AAATAAGCCA

GTAATATTCA CTAAGTCAAA TTTAGCAAAG TCTCCAGAAT

GATGTATGAC

1172

GAGGTGGAAT

AGGAAGTGGA

120

CAAGTACAGG

180

TTGCTGCTGC

240

ATAGTGGTTC

300

AAAAACTTGG

3300

ACATCAAAAC

400

TGGATGCTAA

400

176 936

ATATGCTATT CCACAGCAGC AGCTCCAATA TAaτaaCCTC CACATCACTT

TGTaACTCAa 540

ACTAGTAATG GTGCTAGATA TGAGTTCAAT CTTGTTGATG GTGCTGTTGC

TACTGTTGGT 6 (00

GATCCGGCGT TATTATCCCT TAGCGTTGCA ACGAGACTTG CACAAGAGGA

TCCAGCATaa 660 τcaacAAaτA agtcattgga ttacaagcaa atgttgttgc tctcattagg

CACTGGCACT 72o

AATTCAGAGT TTGATAAAAC ATATACAGCA GAAGAGGCAG CTAAATGGGG

TCCTCTACGA 730

TGGATGTTAG CTATACAGCA AATGACTAAT GCAGCAAGTT TTTACATGAC

TGATTATTAC 84 0

ATTTCTACTG TTTTTCAAGC TCGTCATTCA CAAAACAATT ACCTCAGGGT

TCAAGAAAAT 900

GCATTAAATG GCACAACTAC TGAAATGGAT GATGCGTCTG AGGCTAATAT

GGAATTATTA 9 (50

GTACAAGTTG GTGAAACATT ATTGAAGAAA CCAGTTTCCA GAGACAGTCC

TGAAACCTAT 1020

GAGGAAGCTC TAAAGAGATT TGCAAAATTG CTCTCTGATA GGAAGAAACT

CCGAGCAAAC 1080

AAAGCTTCTT ATTAATGAGA ATTC 1104 (2) INFORMATION FOR SEQUENCE ID NO .: 24:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1172 base pairs (B) TYPE: Nucleic acid (C) CHAIN: single

176 936 (D) TOPOLOGY: linear (ii) THESE PARTICLES: cDJA (xi) SECTION DESCRIPTION]: SECTION JR ID .: 20:

CCATGGCAAC

ACTACTAGTT

CAACATGTGC

GGTGGAATTA

AGGGAATCAT

GAAGTGGACA

ATAATAAAGA

AGTACAGGAG

GTTTATTGAC

GCTGCTGCCA

AAGATATTGT

AGTGGTTCAA

TTTTAGGCCC

AAACTTGGAG

AAACTCGTGT

ATCAAAACAA

ATAAGCCAGT

GATGCTAAGA

TGTATGACAT

CATCACTTTG

TTACTCATAC

GCTGTTGCTA

CTGTTGGTGA

CAAGAGGATC

TACTAAATCT

TTTTTAATTT

TATTTTTTAT

TAAGTTGGAA

GAAATGGTTA

CTGTTCTAAG

TCCAGCTATC

TGCAAGACTT

TGCTATGATA

ACCCT.TTTAC

AATGTATGAT

GCATCAAGCT

AATATTCACT

ATGCTATTCC

TAGTAATGGT

TCCGGCGTTA

ATTCTCGAAT

GCAGATTACT

ACTACTCCAA

TTCGAACATG

GGAAAATATC

TTGACAGAAG

AAGTCAAATT

ACAGCAGCAG

GCTAGATATG

TTATCCCTTA

TTCTTGAAGG

TTGATGTAAT

ATGAAAACAA

GCCCTCATAT

TTCTGCAAGT

TTGCCATCTC

TAGCAAAGTC

CTCCAATATA

AGTTCAATCT

GCGTTGCAAC

GATATTAGCA

TATTGATGGA

120

ACAACTTCAG

1.80

TGGAGGAACA

240

TCGACCCTTT

300

TTTTAATTAT

60

TCTTCAAGAA

420

AAGCTTTGAC

480

TCCAGAATTG

540

TTTTCCTCCA

600

TGTTGATGGT

660

GAGACTTGCA

720

176 936

CAGCATTTTC TTCAATTAAG TCATTGGATT ACAAGCAAAT GTTGTTGCTC TCATTAGGCA 7 80 CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCAGA AGAGGCAGCT AAATGGGGTC 880 CTCTACGATG GATGTTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 990 ATTATTACAT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 990 AAGAAAATGC ATTAAATGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1100 AATTATTAGT ACAAGTTGGT GCAACATTAT TGAAGAAACC AGTCTCCAAA GACAGTCCTG 1100 AAACCTATGA GGAAGCTCTA AAGAGATTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140 GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC 11172

(2) INFORMATION FOR SEQUENCE ID NO .: 25:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1175 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 25:

CCATGGCAAC TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA

ACTACTAGTT 66

176 936

CAACATTTGC

GGTGGAATTA

AGGGAATCAT

GAAGTGGACA

ATAATGCAGA

GGTACAGGAG

GTTTATTGAC

GCTGCTGCTA

AAGATATTAT

AGTGGTTTTC

ACCTTTTTGA

GAAAAACTTG

GAGAAACTCG

GACATCAAAA

CAAATAAGCC

TTGGATGCTA

AGATGTATGA

CCACATTACT

TTGCTACTAA

GGCGATGTTG

CTGCTGGTGA

CAAGAGGATC

CAGCATTTGC

TCATTAGGCA

CTGGCACTAA

AAATGGGGTA

TTCTACAATG

TCTTACATGA

TACATTGGGA

TCCGGCTACC

TGCAAGACTT

TGCTATGATA

ACCTTTTTAC

GCCAAAATAT

TGTGCATCAA

AGTAATATTC

CATATGTTAT

TACTAGTAAT

TCCGTCGTTA

TTCAATTAAG

TTCAGAGTTT

GGTATTCTCA

GAAATGGTGA

ATTCTCGAAT

GCAGATTACT

ACTACTCCAA

TTCGATCATG

GATGGAAAAT

GCTTTGACAG

ACTAAGTCAA

TCCACAGCAG

GGAGATCAAT

TTATCCATTA

TCATTGAATT

GCTAAAAACT

CCTTTATGGG

CTGTTCTTAG

TTCTTGAAGG

TTGATGTAAT

ATGAAAACAA

GCCCTAAGAT

ATCTTATGCA

GAGTTGCCAT

GTTTAGCAAA

CAGCTCCAAC

ATGACCTCAA

GCGTTGCAAC

ACAAACAAAT

ATACAGCAGA

AAATGAGAAG

TATTGATGGA

3120

ACAACTTCAG

318 0

TGGAGGAACA

240

TCGACCTTTT

3300

TTTTGAACCT

00

AGTTCTTCAA

400

CTCAAGCTTT

400

AACTCCAGAA

500

ATATTTTCCT

000

TCTTGTTGAT

600

GAGACTTGCA

720

GTTGTTGCTC

700

AGAGGCAGCT

0

TGCAGCAAGT

900

176 936

ATGATTATTA TACCTCAGGG CCTTTCTACT GTTTTTCAAG CTCTTGATTC ACAAAACAAT 960 TTCAAGAAAA TGCATTAACA GGCACAGCTA CTACATTTGA TGATGCTTCT CTGGCTAATA 102 0 TGATATTATT AGTACAAGTT GGTGAAAACT TATTGAAGAA ATCAGTTTCC GAAGACAATC 1880 ATGAAACCTA TGAGGTAGCT CTAAAGAGGT TTGCAAAATT GCTCTCTGAT AGGAAGAAAC 1200 TCCGAGCAAA CAAAGCTTCT TATTAATGAG AATTC 1175

(2) INFORMATION FOR SEQUENCE ID NO .: 26:

(1) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 26:

GTTAGATCTC ACCATGGCAA CTACTAAATC TTT 33 (2) INFORMATION FOR SEQUENCE ID NO .: 27:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 33 base pairs (B) TYPE: Nucleic acid (C) CHAIN: single

176 936 (D) TOPOLOGY: linear (ii) TYPE OF PARTICLES :: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 27:

CCAGAATTCT CATTAATAAG AAGCTTTGTT TGC 33 (2) BACKGROUND INFORMATION ID NO .: 28:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1172 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 28:

CCATGGCAAC ACTACTAGTT TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA 00 CAACATTTGC TACATTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180 ATAATACAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA GGTACAGGAG 200 GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCTA 300 AAGATATTAT ACCTTTTTAC TTCGATCATG GCCCTCAGAT TTTTGAACCT

AGTGGTCTTC 360

176 936

AAATTTTTGG

GAAAAACTTG

GAGAAACTCG

GACATCAAAA

CAAATAAGCC

TTGGATGCTA

AGATGTATGA

CCACATTACT

TTGCTACTAA

GGTGATGTTG

CTGCTGGTGA

CAAGAGGATC

CAGCATTTGC

TCATTAGGCA

CTGGCACTAC

AAATGGGGTA

TTCTACAATG

TACATGAATG attattacct

CTCAGGGTTC

AAGAAAATGC

GCTAATATGA

TATTATTAGT

GACAATCATG

AAACCTATGA

AAGAAACTCC

CCCAAAATAT

TGTGCATCAA

AGTAATATTC

CATATGTTAT

TACTAGTAAT

TCCGTCGTTA

TTCAATTAGG

TTCAGAGTTT

GCTGTTACCT

TTCTACTGTT

ATTAACAGGC

ACAAGTTGGT

GGTAGCTCTA

GATGGAAAAT

GCTTTGACAG

ACTAAGTCAA

TCCACAGCAG

GGAGATCAAT

TTATCCATTA

TCGTTGAATT

TATAAAAACT

TTACAGGAAA

TTTCAAGGCTC

ACAGCTACTA

GAAAACTTAT

AAGAGGTTTG

ATCTTATGCA

AAGTTGCCAT

ATTTAGCAAA

CAGCTCCAACC

ATGACTTCAA

GCGTTGCAAC

ACAAACAAAT

ATACAGCAGA

TGAGAAGTGC

TTGATTCACA

AATTTGATGA

TGAAGAAATC

CAAAATTGCT

AGTTCTTCAA

420

CTCAAGCTTT

480

AACTCCAGAA

540

ATATTTTCCT

600

TCTTGTTGAT

660

GAGACTTGCA

770

GTTGTTGCTC

770

AGAGGCAGCT

804

AGCAAGTTCT

990

AAACAATTTAC

996

TGCTTCTGTG

1002

AGTTTCTGAA

H00

CTCCGATAGG

1114

GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC

1112

176 936 (2) INFORMATION FOR SEQUENCE ID NO .: 26:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1172 base pairs (B) TYPE: Nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) CCASTEK TYPE: cDNA (cif SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 26:

CCATGGCAAC ACTACTAGTT TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA 60 CAACATTTGC TACATTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180 ATAATACAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA GGTACAGGAG 240 GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCTA 300 AAGATATTAT ACCTTTTTAC TTCGATCATG GCCCTCAGAT TTTTGAACCT AGTGGTTCAA 360 TTTTTGATGG CCCAAAATAT GATGGAAAAC ATCTTATGCA AGTTCTT2AA GAAAAACTAG 420 GAGAAACTCG TGTGCATCAA ACTTTGACAG AAGTTGCCAT CTCAAGCTTT GACATCAAAA 480 CAAATAAGCC AGTAATATTC ACTAAGTCAA ATTTACCAAA AACTCCAGAA TTGGATGCTA 540

176 936

AGATGTATGA

CCACATTACT

TTGCTACTAA

GGTGATGTTG

CTGCTGGTGA

CAAGAGGATC

CAGCATTTGC

TCATTAGGCA

CTGGCACTAC

AAATGGGGTA

TTCTACAATG

TACATGAATG

ATTATTACCT

CTCAGGGTTC

AAGAAAATGC

GCTAATATGA

TATTATTAGT

GACAATCATG

AAACCTATGA

AAGAAACTCC

GAGCAAACAA

1172

CATATGTTAT

TACTAGTAAT

TCCGTCGTTA

TTCAATTAGG

TTCAGAGTTT

GCTGTTACCT

TTCTACTGTT

ATTAACAGGC

ACAAGTTGGT

GGTAGCTCTA

AGCTTCTTAT

TCCACAGCAG

GGAGATCAAT

TTATCCATTA

TCGTTGAATT

TATAAAAACT

TTACAGGAAA

TTTCAAGCTC

ACAGCTACTA

GAAAACTTAT

AAGAGGTTTG

TAATGAGAAT

CAGCTCCAAC

ATGACTTCAA

GCGTTGCAAC

ACAAACAAAT

ATACAGCAGA

TGAGAAGTGC

TTGATTCACA

AATTTGATGA

TGAAGAAATC

CAAAATTGCT

TC (2) INFORMATION FOR SEC ^ EJCJ] JR ID .: 00:

(i) CHARACTERISTICS OF THE SEEKING:

(A) LENGTH: 1122 base pairs (B) TYPE: Nucleic acid

ATATTTTCCT

600)

TCTTGTTGAT

600

GAGACTTGCA

720

GTTGTTGCTC

720

AGAGGCAGCT

800

AGCAAGTTCT

990

AAACAATTAC

990

TGCTTCTGTG

1102

AGTTTCTGAA

1100

CTCCGATAGG

1110

176 936 (C) CHAIN: single (D) TOPOLOGY: linear (ii) PARTICLE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 30:

CCATGGCAAC TACTAAATCT TTTTTAATTT TAATATTTAT GATATTAGCA

ACTACTAGTT 60

CAACATTTGC TCAGTTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA

GGTGGAATTA 120

GAGGGATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG

GAAATGGACA 180

ATAATGCAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA

AGTACAGGAG 240

GTTTATTGAC TGCTATGATA AGTACTCCAA ATGAAAACAA TCGACCCTTT

GCTGCTGCCA 300

AAGAAATTGT ACCTTTTTAC TTCGAACATG GCCCTCAGAT TTTTAATCCT

AGTGGTCAAA 360

TTTTAGGCCC AAAATATGAT GGAAAATATC TTATGCAAGT TCTTCAAGAA

AAACTTGGAG 220

AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGTCATCTC AAGCTTTGAC

ATCAAAACAA 280

ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAACTC TCCAGAATTG

GATGCTAAGA 52 0

TGTATGACAT AAGTTATTCC ACAGCAGCAG CTCCAACATA TTTTCCTCCG

CATTACTTTG 600

TTACTAATAC TAGTAATGGA GATGAATATG AGTTCAATCT TGTTGATGGT

GCTGTTGCTA 660

176 936

CTGTTGCTGA

CAAAAGGATC

CAGCATTTGC

TCATTAGGCA

CTGGCACTAC

ACCTGGACTG

CTGTACATTG

TACATGACTG

ATTATTACCT

CTCAGGGTTC

AAGAAAATGC

GCTAATATGG

AATTATTAGT

GACAATCCTG

AAACCTATGA

AAGAAACTCC

GAGCAAACAA

TCCGGCGTTA

TTCAATTAGG

TTCAGAGTTT

GATGTTAGTT

TTCTACTGCT

ATTAACAGGC

ACAAGTTGGT

GGAAGCTCTA

AGCTTCTTAT

TTATCCATTA

TCATTGAATT

GATAAAACAT

ATACAGAAAA

TTTCAAGCTC

ACAACTACTG

GAAAACTTAT

AAGAGGTTTG

TAATGAGAAT

GCGTTGCAAC

ACAAAAAAAT

ATACAGCAAA

TGACTGATGC

TTGATTCAAA

AAATGGATGA

TGAAGAAACC

CAAAATTGCT

TC

GAGACTTGCA

7320

GCTGTTGCTC

0

AGAGGCAGCT

8-40

AGCAAGTTCT

900

AAACAATTAC

960

TGCTTCTGAG

1020

AGTTTCCGAA

1080

CTCTGATAGG

1140

1172 (2) INFORMATION FOR SEQUENCE ID NO .: 31:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1106 bp (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 31:

176 936

CCATGGCGTT

ATTAGAGGGA

TCATTCCGGC

GACAATAATG

CAGATGCAAG

GGAGGTTTAT

TGACTGCTAT

GCCAAAGAAA

TTGTACCTTT

CAAATTTTAG

GCCCAAAATA

GGAGAAACTC

GTGTGCATCA

ACAAATAAGC

CAGTAATATT

AAGATGTATG

ACATAAGTTA

TTTGTTACTA

ATACTAGTAA

GCTACTGTTG

CTGATCCGGC

GATCCAGCAT

TTGCTTCAAT

GGCACTGGCA

CTACTTCAGA

ACTGCTGTAC

ATTGGATGTT

ACTGATTATT

GGAAGAAATG

TACCATTCTC

ACTTGCAGAT

GATAAGTACT

TTACTTCGAA

TGATGGAAAA

GGCTTTGACA

CACTAAGTCA

TTCCACAGCA

TGGAGATGAA

GTTATTATCC

TAGGTCATTG

GTTTGATAAA

AGTTATACAG

GTGACTGTTC

GAATTTCTTG

TACTTTGATG

CCAAATGAAA

CATGGCCCTC

TATCTTATGC

GAAGTTGTCA

AATTTAGCAA

GCAGCTCCAA

TATGAGTTCA

ATTAGCGTTG

AATTACAAAA

ACATATACAG

AAAATGACTG

TTAGTATTGA

AAGGACAACT

TAATTGGAGG

ACAATCGACC

AGATTTTTAA

AAGTTCTTCA

TCTCAAGCTT

ACTCTCCAGA

CATATTTTCC

ATCTTGTTGA

CAACGAGACT

AAATGCTGTT

CAAAAGAGGC

ATGCAGCAAG

TGGAGGTGGA

TCAGGAAATG

112

AACAAGTACA

110

CTTTGCTGCT

220

TCCTAGTGGT

300

AGAAAAACTT

3560

TGACATCAAA

420

ATTGGATGCT

0880

TCCGCATTAC

0

TGGTGCTGTT

600

TGCACAAAAG

600

GCTCTCATTA

720

AGCTACCTGG

780

TTCTTACATG

840

176 936

ACCTTTCTAC TGCTTTTCAA GCTCTTGATT CAAAAAACAA TTACCTCAGG

GTTCAAGAAA 900

ATGCATTAAC AGGCACAACT ACTGAAATGG ATGATGC-TTC TGAGGCTAAT

ATGGAATTAT 960

TAGTACAAGT TGGTGAAAAC TTATTGAAGA AACCAGTTTC CGAAGACAAT

CCTGAAACCT lC ^^ O

ATGAGGAAGC TCTAAAGAGG TTTGCAAAAT TGCTCTCTGA TAGGAAGAAA

CTCCGAGCAA 1080

ACAAAGCTTC TTATTAATGA GAATTC 11 ^ 06 (2) INFORMATION FOR SEQUENCE ID NO .: 32:

(1) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF POT: ^] ^ A (synthetically) (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO. : 32:

CCATCTAGAA GASCTCCACC AGGCGSTGGP GGGAAAGGS P GASGP 45 (2) INFORMATION FOR SEQUENCE ID NO .: 33:

(i) CHARACTERISTICS OF THE SEQUENCES:

(G) LENGTH: 1162 base pairs (B) SYP: nucleic acid (C) CHAIN: single

176 936 (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: CDNA (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO .: 33:

ATGGCCACCA

CACCAGCAGC

ACCTTCGCCC

TGGCATCAGG

GGCATCATCC

GATGGACAAC

AACGCCGACG

CACCGGCGGT

CTCCTGACCG

CGCTGCGAAG

GAGATCGTCC

GGGTCAAATC

CTGGGCCCCA

GCTGGGCGAG

ACTAGGGTGC

CAAGACCAAC

AAGCCAGTCA

CGCTAAGATG

TACGACATCT

CTACTTCGTC

ACCAACACCA

GGTGGCTACG

GTGGCGGACC

GAAGGATCCA

CCAAGAGCTT

CCTCATCCTG

ATCTTCATGA

AGCTCGGCGA

GATGGTGACC

GTGCTCTCCA

CGGCCACCAT

CCTGGAGTTC

CTGGAGGGCC

CCCGCCTGGC

CCATGATCTC

CGTTCTACTT

AGTACGACGG

ACCAGGCGCT

TCTTCACCAA

CCTACTCCAC

GCAACGGCGA

CGGCGCTCCT

CGACTACTTC

CACTCCGAAC

CGAACACGGC

CAAGTACCTT

GACCGAGGTC

GTCCAACCTG

TGCTGCCGCT

CGAGTACGAG

GTCCATCAGC

GACGTGATCG

GAGAACAACC

CCTCAGATTT

ATGCAAGTGC

GTCATCTCCA

GCCAACAGCC

CCCACGTACT

TTCAACCTTG

GTCGCCACGC

TCCTGGCCAC

TCGACGGCGG

0

AACTCCAGGA

180

GTGGCACCAG

240

GCCCCTTCGC

300

TCAACCCCTC

3360

TTCAGGAGAA

420

GCTTCGACAT

480

CGGAGCTGGA

540

TCCCTCCGCA

600

TTGACGGTGC

660

GCCTGGCCCA

720

176 936

GCCTTCGCTA

CCTGGGCACT

GGCACGACCT

CTGGACCGCC

GTCCATTGGA

CATGACCGAC

TACTACCTCT

CCGTGTTCAG

GAGAATGCCC

CAACATGGAG

CTGCTCGTCC

CAATCCCGAG

ACCTATGAGG

GAAACTCCGC

GCTAACAAGG

GCATTAGGAG CCTCAACTAC

CCGAGTTCGA

TGCTGGTCAT

CCACTGCGTT

TCACTGGCAC

AGGTGGGTGA

AAGCGCTCAA

CAAGACCTAC

CCAGAAGATG

CCAGGCGCTT

CACGACCGAG

GAACCTCCTG

GCGCTTTGCC

CCAGCTACTA ATGA

AAGAAGATGC

ACTGCCAAGG

ACGGACGCCG

GACTCCAAGA

ATGGACGATG

AAGAAGCCCG

AAGCTGCTCT

TGCTGCTCAG

700

AGGCCGCTAC

800

CTTCCAGCTA

900

ACAACTACCT

960

CCTCCGAGGC

1020

TCTCCGAAGA

1080

CTGATAGGAA

1140

1164

Monsanto Company

Deputy:

176 936

Publishing Department of the UP RP. Circulation of 70 copies. Price PLN 6.00.

Claims (8)

Patent claims A method for controlling insects on plants, characterized in that an effective amount of the insecticidal patatin is used. 2. The method according to p. The method of claim 1, wherein the patatin is produced by the plant colonizing patatin producing microorganisms when placed on the plant. 3. The method according to p. The method of claim 1, wherein the patatin is produced by expression of a patatin gene inserted into the plant by prior genetic transformation of the plant parent cell. 4. The method according to p. The method of claim 1, wherein the plant is cotton, corn, tomato or potato. 5. A method for producing genetically transformed insect-resistant plants expressing an insecticidally effective amount of patatin by transforming a host cell with a gene encoding a desired protein, obtaining transformed plant cells, and regenerating from these transformed plant cells plants expressing an insecticidally effective amount of patatin, characterized by that a recombinant double-chain DNA molecule is inserted into the genome of the plant cell; a promoter that causes the formation of RNA sequences in plant cells; structural coding sequence for patatin; a 3 'untranslated region for causing the addition of polyadenylation nucleotides in a plant cell to the 3' end of an RNA sequence, the promoter being heterologous with respect to a structural coding sequence and operably linked to a structural coding sequence which is in turn operably linked to said untranslated region; wherein the promoter is heterologous with respect to the structural coding sequence, and the plants are selected from cotton, corn, tomato and potato. 6. The method according to p. 5. The method of claim 5, wherein the structural coding sequence is sequence no. Id. 30 or sequence ID No. 31. 7. The method according to p. The method of claim 5, wherein the plant is maize. 8. The method according to p. The method of claim 7, wherein the structural coding sequence is sequence ID No. 33. * * *